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1. enviMass version 1 0 User Manual 6 Online Installation The screening workflow is embedded into Excel Visual Basic VBA 2003 2007 and 2010 32 bit versions and makes use of R code and packages via the RExcel software under Windows XP and Windows 32 bit OS Therefore RExcel and R need to be installed on a computer via the following steps 1 Make sure You have Microsoft Excel 2003 or a higher version installed on Your computer 2 Make sure You are online 3 Go to the download section of http rcom univie ac at Under RExcelAndFriend download run the online installation RAndFriendsSetupXXXXVX X X X latest version and let Your computer restart this will install the most up to date versions of R RExcel statconnDCOM rcom on Your computer The mentioned webpage also provides the individual tools for offline installation Further information on the statconn tools their installation and problem handling is provided by the named website and variuos RExcel statconnDCOM user platforms Open Excel Under Excel 2010 RExcel should appear automatically as add in Otherwise and for earlier releases of Excel go to Start gt All Programs gt statconn gt RExcel gt Activate RExcel as Add In Thereupon open Excel gt Add Ins gt RExcel gt Set R Server gt select Server type background The named homepage gives detailed advice
2. id a a a SEE SEE u lt D E Figure 31 Interface of the batch mode tool enviMass version 1 0 User Manual 58 Isotopic pattern spreadsheet Description This spreadsheet contains a stand alone tool for calculating the isotopic fine structure of a given molecular formula This aids at comprehending the settings used for Tool 3 of the enviMass workflow Spreadsheet inputs 1 Isotope list from spreadsheet isotopes 2 Electron mass from spreadsheet isotopes 3 Adducts and their masses are defined in the spreadsheet adducts Spreadsheet outputs A result list of isotopic pattern data containing the mass abundance and isotopic composition of each peak in the isotopic pattern of the compound For the profile mode additional lists with masses and abundances of sticks surviving peaks and centroid peaks are listed too Results are plotted Calculations amp parameter settings Parameter settings akin to those selectable in Tool 3 of the spreadsheet target_screening In contrast to Tool 3 however molecular formulas are not read from a spreadsheet but have to be inserted into the interface directly Elements in the molecular formula must always be followed by numbers atom counts of that element except for preceding numbers in square brackets indicating individual isotopes defined in the element name column of the isotope spreadsheet e g 14 C or 18 O For example
3. sample ID for standard screening sample spreadsheet of sample peak s to which the most abundant peak mostly the monoisotopic one of the target compound isotopic pattern could be matched to If no matches were found the entry value is set 0 2 Columns S and AB Isotopic hits list the ID number s from column R sample ID for standard screening sample spreadsheet of all those sample peak s to which the rescaled target compound isotopic pattern could be matched to In other words not only the most abundant peak as in point 1 is listed here but all those sample peaks referred to by score That is if score 1 states e g 4 of 6 6 count of expected internals standard peaks after rescaling four peaks are listed The first ID number in the string i e the most abundant one is identical to the one s listed under point 1 3 Columns T and AC delta m z ppm list the accuracy in Am z between matched sample peaks cp column A sample spreadsheet i e Am z measured expected and for all those peaks listed under point 2 4 Columns U and AD delta RT list the accuracy in RT between matched sample peaks cp column E sample spreadsheet and expected peaks cp column E targets spreadsheet i e RT measured expected for each of those peaks listed under point 2 5 Columns V and AE delta intens list the accuracy in intensity between matched s
4. to FALSE here the charge defined in the workflow interface set to 1 is used In contrast the double positively defined ionization state is established by setting the second row entry of charge not to FALSE but to a charge value namely 2 4 If resolution datasets from the spreadsheet resolution are utilized for defining resolving power and or stick discretization a preliminary check if compound masses fall within the range of the masses of the resolution data enviMass version 1 0 User Manual 13 sets is conducted For this masses for a targets spreadsheet targets column D and masses for b internal standards spreadsheet internal standards column D are utilized These masses have been calculated by Tool 2 Data Check 5 If selected resolution data sets spreadsheet resolution are used to define the resolving power and or the stick discretization width 6 Isotope data spreadsheet isotopes serves as input to the isotope pattern calculation 7 Electron mass from spreadsheet isotopes 8 Adducts and their masses are defined in the spreadsheet adducts Spreadsheet outputs 1 Masses of isotope peaks and 2 their abundances relative to that of the monoisotopic peak are written to spreadsheet targets columns N and O and to spreadsheet internal_standards columns L and M for a the targets and b the internal standards respectively Abundances
5. 0 33 Occasionally different sets of peaks from the sample peak list can be matched to one target isotopic pattern set of peaks In this case scores for all sets are calculated and only the results for the sets with the two best scores are printed The tool provides a detailed list of achieved accuracies in a Am z b RT and c intensity for the peaks matched for score 1 The tool furthermore stores which sample list peaks are matched to which target substance peak Spreadsheet inputs The screening tool compares peak information from the internal standards list with peak information from the sample peak list The following columns from spreadsheet targets are used 1 Column N Isotopic m z and column O Isotopic abundance of spreadsheet targets provide isotopic peak masses and abundances They are a result of Tool 3 2 Column A ID for the internal standard ID 3 retention time from spreadsheet targets column E 4 Column M use for screening allows to omit target compound entries rows in spreadsheet targets from being used in the screening 5 Column P omit peak states the index of which peaks of the isotopic pattern of a single target substance should be omitted from screening cp Tool 4 The following columns from spreadsheet sample are used enviMass version 1 0 User Manual 39 6 Centroid m z in column A of the sample
6. mass deviations cp Figure 17 for details In case these first two steps of the recalibration procedure indicate no matches or if no internal standards are provided in the internal standards spreadsheet the Tool can either be skipped or a data set from spreadsheet known can be used for recalibration Recalibration Skip recalibration Search internal standards in sample amp calculate deviation Tolerances 5 mz mmu ppm 1 RT min Humber of samples identified as standard including several sample matches for one standard Humber of samples identified as standard 83 excluding ambiguous matches Run recalibration s Humber of knots default 10 use known deviations for recalibration as listed in worksheet known basis choice enalized cubic regression spli B thin plate regression splines Ic Figure 16 User interface of the recalibration tool The upper part of the interface aids at detecting potential internal standards in the sample peak data set The lower part of the tool interface uses these matches to run the mass recalibration of the sample peak data set enviMass version 1 0 User Manual 29 Figure 17 First result plot from the recalibration tool Depicted are deviations in mass between theoretical values calculated from the molecular formula of internal standards and matches found for these internal standards in the sample peak list ordinate mmu unit
7. of the sample peak list have a a match column L with the blank blind peak list b a mark for being a potential spark column K and c another match column T from target screening for non monoisotopic target peaks Thus this sample peak has three possible match pairs namely a b a c and b c Therefore this peak would contribute to the counts in cells 8 1 8 2 and 2 1 of that third table Only sample peaks having one match will be counted in the green cells of that table e g a peak having only a blank match will be listed as a count in cell 2 2 Note Again the sum of the counts in this third table does not necessarily equal the Total number of samples since one sample peak may have several matches enviMass version 1 0 User Manual 54 Filter sample peak list sanple data Summalize amp filter sample peak list Internal standards monoisotopic Internal standards non monoisotopic and their adducts Targets monoisotopic Targets non monoisotopic Retention time frin Samples remaining after exclusion Thereof possible non monois peaks Thereof possible adducts Figure 27 Tool 14 for filtering of the sample peak list based on the results of Tools 5 to 13 Tables summarize the number distribution and overlap of matches in the sample peak list of sparks blanks blinds internal standards and targets The plot in the upper right corner locates these matches within the mass vs rete
8. should be set to a small value since isotopologues should elute with very similar RT Note Since sorting along decreasing intensities the routine assumes that non monoisotopic peaks i e those with an isotopic mass difference relative to the monoisotopic isotope composition have a lower abundance intensity than monoisotopic ones While this is correct for most organic molecules discrepancies may arise for molecules having e g more than four Cl atoms In the latter case isotopologue peaks would not be detected Search for other non monoisotopic peaks Peak search Skip peak search Number of peaks found 1014 Tolerances 5 Amiz mmu ppm 05 ART within scan min v userecalibrated masses D 115 108 D 79Br 81Br D 12c 13c oE O 40Ca 42Ca O 40Ca 48Ca O 40Ca 43Ca O 40Ca 46Ca O 3501 37cC1 D 56Fe 54Fe O 56Fe 57Fe D seFe 58Fe EH 39K 41K 39K 40K 7Li 6Li 24Mg 26Mg 24Mg 25Mg 14N 15N 150 180 Update isotope list Figure 25 Tool 12 for search of peaks within specific isotopic distances in mass from potential monoisotopic peak masses enviMass version 1 0 User Manual 50 Tool 13 Adduct search non targets non int stand Description Tool 13 searches for differences in m z a compound attains for having different adducts More precisely the tool screens all possible pairs of peaks from the sample peak list for having differences in m z possibly resulting from formation of different adducts Because t
9. 13 C2C35H67N1013 is the molecular formula of erythromycin labeled at two C positions with 13 C C37H67N1013 is the molecular formula of the unlabeled compound For further details refer to the section Isotopic pattern generation of Tool 3 enviMass version 1 0 User Manual 59 Data sheets Note that inappropriate sorting of spreadsheet information may lead to program malfunctioning what is contained in individual spreadsheets for which tool and how these contents may be sorted or manipulated is described below isotopic_pattern data sheet Contents VB user interface for calculating isotopic fine structures and profiling for a given molecular formula Results are printed onto the spreadsheet directly Permitted manipulations The user interface must not be altered target_screening data sheet Contents VB user interface of the enviMass workflow Permitted manipulations The user interface must not be altered targets data sheet Contents This spreadsheet contains a information on target compounds to be screened for columns A to M b isotopic pattern information columns N to Q and c results from the screening quantification and adduct search steps columns R onward Filling columns A to C and E to M for each compound row is obligatory for the user grey headers Column A ID unique ID of each compound character string eg 234 or 234B Column B compound name name of the target compound characte
10. 224712239217 0 112 0 1 0718817 20f4 20f4 20f4 5065 75052 1781274074 0 01 0 01 0145188 2of2 1of2 1of2 Only monoisotopic peak s Found 5011 4970 4394 4995 1 005 71 214927 2 83194 10 55072 0 137 0 19 0 237 0 23 0119382157552 1 230877 40f5 20f5 20f5 4960 74944 1 987 1 2 58036 0 12 70 12 0158427 20f2 20f2 20f2 4849 14818 14842 12041139854 3 38428 0 224 0 224 0 22 0107691788166 30f5 20f5 20f5 483274809 1 978 1 2 17476 0 12 10 12 01121021 2of4 2of4 20f4 No monoisotopic hit found no isotopic pattern fit conducted 4772 14753 147312 13084 0 37 10 37 0184229 20f3 2of3 2of3 Not used for screening 4666 74652 1 526 2 88975 0 33 10 33 0175238 20f3 1of3 1of3 Only monoisotopic peak s Found 4612145801 4537 13521124334 1245051 0 12 10 12 10 12 07111059 57975 30f6 30f6 30f6 No monoisotopic hit found no isotopic pattern fit conducted 45117 44881 4468 1 5014 2 474441 5 76227 0 18 70 18 70 18 0146726 49311 3of4 2of4 2of4 Figure 22 Subtable in the internal_standards spreadsheet listing the results for the screening of internal standards Tool 8 on the first monoisotopic match found in the sample peak data set enviMass version 1 0 User Manual 38 Tool 9 Target screening Description In close to analogy to Tool 8 Tool 9 screens the sample peak list for matches with the target compounds listed in the targets spreadsheet The underlying routine rescales the abundance of the expected peaks in the target isotopic patter
11. are automatically rescaled to that of the monoisotopic peak Additionally two more dummy columns are established for each a the targets spreadsheet targets columns P and Q and b the internal standards spreadsheet internal standards columns N and O 3 The first one omit peak 7 indicates which of the isotopic peaks shall be omitted from screening when overlap between peak patterns of target compounds and internal standards is detected in subsequent Tool 4 Match standards and target patterns 4 The second column peak for quantif sets the isotopic peak used for quantification cp Tool 10 Target quantification and can be modified by the user As a default the intensities of the first monoisotopic peaks of target and internal standard are used for quantification i e these values are set to 1 Calculations amp parameter settings Calculations are derived in three hierarchical steps that can be selected in the Output options of the workflow interface Figure 4 In a first step the isotopic pattern is calculated In a second optional step a Gaussian profile is fitted to the resulting peaks and surviving peaks extracted In a third optional step Gaussian profiles are converted to centroid peak data The peaks resulting from the last two steps can optionally be filtered by Recursive Base Peak Framing RBPF Hence depending on what the user selects a peak data of isotopic pattern OR b profile s
12. column Z isotopic hits 7 internal _ standards spreadsheet Two entries are made to spreadsheet targets 7 Column AK adducts for first monoisotopic hit pattern lists per target compound the adduct name and the sample peak ID contained in column U in spreadsheet sample of the concomitant matched peak in the sample peak list Here matching refers to adducts found for the screening results of column S isotopic hits targets spreadsheet 8 Column AL adducts for second monoisotopic hit pattern lists per target compound the adduct name and the sample peak ID contained in column U in spreadsheet sample of the concomitant matched peak in the sample peak enviMass version 1 0 User Manual 46 list Here matching refers to adducts found for the screening results of column AB isotopic hits targets spreadsheet Adduct search for targets internal standards Adduct search tr targets amp internal standards Skip adduct search Or targets Calculations finished Tolerances amiz C mmu ppm ART within scan min score threshold charge 1 charge 1 H charge 1 formiate charge 1 NH 4 charge 1 Update adduct list Figure 24 Tool 11 Adduct search for target compounds and internals standards Calculations amp parameter settings Screening Tools 8 and 9 have listed results for matching isotopic patterns of internal standards and targe
13. element e g 35 Cl and 37 Cl and 2 many atom counts for the latter e g hypothetical C1500 On the other hand too low values for the limit may lead to the unnecessary calculation of peaks with very little abundance The user is requested to find a trade off possibly by comparing peak lists derived from different limit settings using the isotopic pattern simulation tool in spreadsheet isotopic pattern enviMass version 1 0 User Manual 20 Tool 4 Match standards and target patterns Description A target substance can be linked to an internal standard with known concentration so as to quantify target concentrations via ratios of the peak intensities of both compounds Often these internal standards are isotopically labeled isotopologues of the target substance to be quantified As a consequence certain peaks in the calculated isotopic patterns of both substances may be identical leading to an increased intensity of the resulting net peak If this net peak is used for quantification or screening outcomes may be erroneous Therefore Tool 4 matches the isotopic pattern peaks of the target with those of the internal standard to which it is linked via an ID If an overlap between both peak patterns is detected the concerned peaks are marked so as to be omitted from screening and quantification Apart from such overlaps in peak patterns the tool also checks the consistency of a the isotopic patterns and b the ID linking of a target c
14. for target screening of spreadsheet sample gives an ID to each row peak of the sample peak list This unique ID is used to identify sample peaks from entries in the screening results written to the targets spreadsheet Calculations amp parameter settings The below steps are passed for each target compound listed in the targets spreadsheet 1 In a very first step a match between a the most abundant peak of each target compound isotopic pattern first entry in the strings of columns L and M spreadsheet targets and b a peak of the sample peak list is screened for within tolerance settings of Am z and ART Parameters for these tolerances have to be specified in the textboxes Am z and ART listed vs measured min respectively If recalibration results were accepted in Tool 7 recalibrated masses from column O instead of non recalibrated ones from column A of the sample spreadsheet are used Occasionally this first step can lead to several hits in the sample peak list depending on the tolerance settings In this case below steps 2 to 6 are passed for each of these hits leading to several result sets The routine outputs only the two best ones to the targets spreadsheet based on score 1 2 Thereupon target compound peaks with too low abundances are identified Provided that the above named single peak hits a vs b were found the abundances taken from column M spreadsheet
15. internal standards spreadsheet i e difference in intensity observed expected for each of those peaks listed under point 2 enviMass version 1 0 User Manual 35 6 Columns U and AD score 1 show score 1 for each internal standard 7 Columns V and AE score 2 show score 2 for each internal standard 8 Columns W and AF score 3 show score 3 for each internal standard 9 Columns X and AG sum score gives the weighted sum of scores 1 to 3 from columns U to W and AD to AF respectively 10 Columns Y and AH conc ng l are established but remain empty Cells will be filled with estimates of concentrations ng l by using the quantification Tool 10 after screening for internal standards and targets Three entries are made to spreadsheet sample 11 Columns P monoisotopic hit for standard of spreadsheet sample lists the ID of the internal standard column A for which a monoisotopic hit was matched to this peak of the sample peak list 12 Column Q isotopic hit for standard 7 of spreadsheet sample lists the ID of the internal standard column A for which a hit of any of its peaks from its isotopic pattern was matched to this peak of the sample peak list 13 Column R sample ID for standard screening of spreadsheet sample gives an ID to each row peak of the sample peak list This unique ID is used to identify sample peaks from entries in the
16. made If waiting longer than 10 minutes consult above point 8 I installed RExcel as described in the installation section Nontheless when trying to run RExcel I get the error message that the DCOM server is unavailable Make sure step 2 of the installation insstructions has installed the DCOM server In some cases Antivirus software spuriously blocks installation of the DCOM software or other parts of the RExcel installation Consider disabling the Antivirus software for the installation period I perpetually receive the message This should not have happened Connection between R and RExcel failed The error message most often and most sporadically encountered when using Excel with R RExcel The best is to ignore the message and to rerun the concerned tool However should the error message persist please inform the authors 19 I have installed RExcel and all components such as DCOM sucessfully 20 having been logged in as administrator Logged in as another user on the same computer and when opening the workflow I receive the message Could not start R server and thereupon There seems to be no R process connected to Excel Ensure You have Activated RExcel under Start gt All programms gt statconn gt RExcel before usage The automatic download of the R isopat package fails In this case isopat must be downloaded manually Online installation a open the R version installed during step 1 of
17. of 5 if recalibrated sample peak list masses are used for screening cp Tool 7 8 Retention time RT min 9 Intensity from column A 10 blank from column L Spreadsheet outputs The screening routine makes entries to both the sample and the internal standards spreadsheets Entries to the internal_standards spreadsheet are exemplified in Figure 22 Occasionally different sets of peaks from the sample peak list can be matched to one internal standard isotopic pattern set of peaks if several sample peaks match to the most abundant peak denoted as monoisotopic hit of the internal standard isotopic pattern In this case scores for all possible sets are calculated and only the results for the sets with the best two scores 1 are printed Thus the first block of results in spreadsheet internal standards i e nine columns Q to Y refers to the set of sample peaks with the best score 1 for a match with an internal standard isotopic pattern The second block in the nine columns Z to AH refers to results from the set with the second best score 1 Overall 1 2 x 9 19 new columns are assigned to spreadsheet internal standards 1 Column P Monoisotopic hits lists the ID number s from column R sample ID for standard screening sample spreadsheet of sample peak s to which the most abundant peak mostly the monoisotopic one of the internal standard isotopic pa
18. of the internal standard spreadsheets is set to FALSE so as to omit these compounds from screening Similarly if a a mismatch between the number of isotopic masses and isotopic abundances is detected for a single substance or b several internal standards have the same ID in column A of spreadsheet internal standards as referred to by column I of the target spreadsheet entries in column use for screening are set to FALSE and an error message is printed to columns peak for quantif too Calculations amp parameter settings The parameter A retention time target vs standard min cp Figure 7 states the difference in retention time between the target compound and its ID linked internal standard below which the isotopic pattern check appears necessary In other words even if the target and its internal standard would have a subset of identical isotopic peaks a difference in retention time equal or greater to the specified value would separate the peaks of both compounds so as to have no overlap between the concerned peaks of their isotopic patterns Match standards and target patterns Match standard vs target isotope peaks 2 A retention time target vs standard min Figure 11 User interface of Tool 4 enviMass version 1 0 User Manual 22 Tool 5 Spark removal Description Peaks with a particularly short chromatographic eluation period may be regarded as artifacts noise MS sparks and conse
19. problems when opening the enviMass 1 0 workflow in Excel Offline Installation For installing a computer without internet access follow the steps For installation on PC without internet connection the individual tools R RExcel Statcon have to be downloaded separately under the following links l 2 R RExcel StatconnDCOM Isopat http www r project org http rcom univie ac at see Download RExcel 3 2 0 in the download section http rcom univie ac at see Download statconnDCOM 3 1 2B7 in the download section http cran r project org Isopat must be downloaded manually a open your web browser internet explorer firefox and b browse to http cran r project org There c under CRAN click search and d search for isopat e From the search results select CRAN package isopat and the package source site opens This source site has a download section f there choose the download fitting your OS g unpack the download and h copy paste the unpacked folder isopat into your R library folder The R library folder usually resides under C Program Files R R X XX X library and contains the folders of all packages used in your R environment enviMass version 1 0 User Manual 8 Input data formats The enviMass workflow processes txt or dat text files with input data of 1 peak m z 2 peak intensities and 3 peak retention times from HRMS m
20. settings on the left side cp Figure 12 and Figure 13 1 The plotting functionality aids at determining values A RT or A scan number above or below which sample peaks may be regarded as noise data For this purpose command button Plot histogram depicted in Figure 13 plots histograms with a predefined number of histogram bars and up to a certain value for both A scan number right plot and A RT left plot for all sample peaks listed in spreadsheet sample Consult Figure 14 for an exemplification Optionally a preliminary scan through the sample peak list for potential target compounds is made and the A scan number and A RT of the resulting matches in the sample peak list of spreadsheet sample included in the plot red bars This optional operation is enabled with the check box include samples matching targets as reference values When enabled masses of monoisotopic peaks from target list column N are searched for in the sample peak list column A Centroid m z within certain mass and retention time tolerances specified via entries to the two text boxes below the named check box 2 Filtering for sparks among row entries in the sample peak list of spreadsheet sample is done either via values for A scan number or A RT Figure 12 i e unless check box use scan number is checked A RT is used for filtering The list box entries allow to filter for peaks above use only enviMass version 1 0 User Manual
21. spreadsheet 7 recalibrated m z in column O of the sample spreadsheet instead of 5 if recalibrated sample peak list masses are used for screening cp Tool 7 8 Retention time RT min from column E of the sample spreadsheet 9 Intensity from column A of the sample spreadsheet 10 blank from column L of the sample spreadsheet Spreadsheet outputs The screening routine makes entries to both the sample and the targets spreadsheets Entries to the targets spreadsheet are comparable to those generated in Tool 8 and depicted in Figure 22 Occasionally different sets of peaks from the sample peak list can be matched to one target compound isotopic pattern set of peaks if several sample peaks match to the most abundant peak denoted as monoisotopic hit of that target isotopic pattern In this case scores for all possible sets are calculated and only the results for the sets with the best two scores are printed Thus the first block of results in spreadsheet targets i e nine columns S to AA refers to the set of sample peaks with the best score 1 for a match with a target compound isotopic pattern The second block in the nine columns AB to AJ refers to results from the set with the second best score 1 Overall 1 2 x 9 19 new columns are assigned to spreadsheet targets 1 Column R Monoisotopic hits lists the ID number s from column R
22. the installation instructions b in the opened R GUI select packages gt install packages c a window with R mirrors pops up press OK which opens d a list of packages available at this mirror site e Within the list search for isopat select and click OK Offline installation a open your web browser internet explorer firefox and b browse to http cran r project org There c under CRAN click search and d search for isopat e From the search results select CRAN package isopat and the package source site opens This source site has a download section f there choose the download fitting your OS g unpack the download and h copy paste the unpacked folder isopat into your R library folder The R library folder usually resides under CV Program Files R R X XX X library and contains the folders of all packages used in your R environment enviMass version 1 0 User Manual 72 21 22 I get an error message after enable editing on the downloaded Excel2010 enviMass file Please ensure that all settings for the protected view in Excel 2010 are disabled before opening file gt options gt trust center gt trust center settings gt protected view After you saved the opened Excel file you can re enable the protected view settings in Excel if you like The problem is caused by Excel that run only a part of the startup scripts after pressing enable editing
23. used for screening the other peaks of the rescaled isotopic pattern set of peaks specified in text box ART within scan min The reason being if a peak of the sample peak list indeed represents a monoisotopic peak of an internal standard all other sample peaks representing the rescaled internal standard isotopic pattern should elute at the very same retention time 4 Given the matched set of sample peaks the above scores 1 to 3 are calculated The parameter for tolerance in intensity has to be specified in text box Intensity 5 An overall score is calculated as weighted sum of the sores 1 to 3 The weights have to be assigned by the user through the three lowermost textboxes of Figure 21 6 Accuracies in Am z ART and intensity are calculated and plotted They may help to identify outliers from screening of the internal standard list 7 Note two cases exist for which NOT the monoisotopic peak is used in step 1 Firstly the routine omits internal standard peaks overlapping with those of targets cp Tool 4 In that case the most abundant internal standard peak not omitted is utilized for point 1 Similarly all other peaks of the internal standard not omitted are used in points 2 to 5 Secondly rare cases exist under which not the monoisotopic peak but another isotopologue has highest abundance for an internal standard e g C6Cl6 with maximum abundance rescaled to monoisotopic peak 1 9 In these c
24. 000 205 002 205 004 205 006 205 008 205 010 m z Figure 7 Two isotopic pattern peaks separated by resolving power Am In a next step the resulting overall profile is converted to stick representations Figure 10 with sticks having a defined distance of Am to each other as defined in the Stick discretization section of the Profile Centroid settings Figure 6 In analogy to the resolving power Am can be set in three ways 1 as a fixed value mmu 2 as function of mass ppm or 3 as a function of Resolution R under differing masses Again the latter uses the data sets from spreadsheet resolution and fits a spline regression model to establish R f mass At a given mass m Am for stick discretization is then given by Am m R or to derive Am in ppm units Am ppm I1E6 R Often the Am for stick discretization has a fixed relationship to the Am of the resolving power More precisely to adequately depict two adjacent profiles the Am for stick discretization must be smaller than the Am for resolving power Therefore Am for stick discretization may be defined as fraction of the Am chosen for the resolving power The value for factor in the Stick discretization settings allows for such a relationship Given that the Am for the resolving power is set with Resolution f mass on a data set x the same data set x can be chosen for the stick discretization and a factor z specified Thus the Am separatin
25. 1 ID Intensity RT min ID in 86 87 845019 i 597 40Ca 44Ca 253 01253 308 Nat monoisot 271 00062 87 88 842814 329 Na1 monoisot 341 05479 145423 88 88 836568 89 90 831154 377 4Na1 monoisot 276 04073 116246 90 91 823200 91 92 811716 i 468 Na1 monoisot 278 04083 76672 92 93 810083 93 94 796057 94 95 795215 226 Na1 monoisot 433 08844 261301 95 96 769280 i 96 97 767114 311 Na1 monoisot 158030 i 97 98 766055 E 311 40Ca 44Ca 158030 324 4Nal monoisot 415 18779 98 98 755312 99 100 758520 100 101 758420 101 102 748144 102 103 747076 103 104 744939 542 Na1 monoisot Figure 32 Filtered and grouped sample listing of spreadsheet non targets The orange column proposes masses of potential monoisotopic peaks sorted by decreasing intensity The columns E onward to the right suggest potential adducts and M X peaks Limitations enviMass version 1 0 was tested on large data inputs up to a 1500 targets b 600 internal standards c 15 000 entries in the blank blind peak list and d 30 000 entries in the sample peaks without malfunctions We expect enviMass to deal with datasets exceeding these sizes but have not run any tests yet The tools 12 and 13 are the most time consuming ones and may take several minutes for large data sets to complete Computer requirements OS Windows XP with Excel 2003 2007 or Windows 7 with Excel 2010 Internet access for a installing RExcel and b the R pa
26. 23 upper below use only lower or above AND below use both bounds the values specified in the text boxes The textboxes require entries for A RT and A scan number for values below which sparks are expected remove lt lower bound or above which sparks are expected remove gt upper bound ON En A EEE EEE EEE EEE En Spark removal use only lower retention time scan number bound use only upper retention time scan number bound use both bounds Figure 12 Parameter interface for spark removal section of Tool 5 enviMass version 1 0 User Manual 24 Plot settings 05 maximum ART in histogram min 150 maximum Ascan number in histogram Pict histogams m 100 number of histogram breaks 40 number of histogram breaks include samples matching targets as reference values Tolerances 5 mz ppm 3 ART min 2000 2000 1500 1500 Frequency 1000 Frequency 1000 500 5i r T T 1 0 50 100 150 J 8 a 4 Cee Ba pna Milla mr r r r r ij D 1 02 03 deta scannumber deta retention time RT Figure 13 Histogram plots of A scan number and A retention time from Tool gt 3 c 3 c m ention time RT Figure 14 Histogram from the p
27. I have installed RExcel and all components such as DCOM sucessfully having been logged in as administrator Logged in as another user on the same computer the RExcel addin is not shown in Excel In WindowsXP and Windows7 32bit navigate with the Windows Explorer to ProgramFiles gt RExcel gt xls and run the file RExcel2007AddinAutolnstall xlsm by double click After a restart of Excel RExcel should appear under the ribbon Add Ins Under Windows7 64bit the RExcel2007AddinAutoInstall xlsm can be found under ProgramFiles X86 gt RExcel gt xls enviMass version 1 0 User Manual 73 References Baier T Neuwirth E 2007 Excel COM R Computational Statistics 22 1 pp 91 108 De Laeter J B hlke K De Bievre P Hidaka H Peiser H Rosman K Taylor P 2003 Atomic Weights of the Elements Review 2000 IUPAC Technical Report Pure and Applied Chemistry Vol 75 No 6 pp 683 800 www iupac org publications pac 2003 pdf 7506x0683 pdf Generalized Additive Models An Introduction with R Wood 8 2006 Chapman amp Hall Boca Raton USA IUPAC Compendium of Chemical Terminology 2nd ed the Gold Book 1997 Kirchner M 2008 amsmercury R package for mercury 7 algorithm http hei iwr uni heidelberg de Staff mkirchner proteomics Li L Kresh J Karabacak N Cobb J Agar J and Hong P 2008 A hierarchical algorithm for calculating the isotopic fine structures of molecules Journal of the America
28. Loos M 2011 Calculation of isotopic fine structures isopat R package enviMass version 1 0 User Manual 69 FAQs 1 Q 3 4 5 6 7 8 Can I screen blank blind data without uploading a sample data set Yes You can upload Your blank or blind data as sample data set and skip the Tool for blank subtraction Can I screen a sample data set without uploading a blank blind data set Yes Just skip the blank blind upload and Tool 6 on blank subtraction Once I have run a tool can I go backward in the workflow to return to a tool upstream of the workflow No The workflow keeps track of the data generated at each Tool Since data from one tool often serves as input to other tools further downstream backward shifts in the workflow are prevented Can I skip a tool within the workflow Yes Just follow the workflow and press the skip button of the concerned tool Can I use data other than Formulator as data input Yes Read the section on input data formats I keep receiving a VBA Missing Reference error message when trying to open or run the workflow In that case a reference to a type library that does not exist is set and should be removed To do so open the Visual Basic for Application Editor Excel Tab Developer gt Visual Basic open Tools gt References and uncheck the concerned Available References Make sure the Reference to RExcelVBAlib remains checked I receive a Microsoft Visual Basic r
29. ample peaks cp column B sample spreadsheet and expected peaks after rescaling of column O targets spreadsheet ie intensity differences measured expected for each of those peaks listed under point 2 6 Columns W and AF score 1 show score 1 for each internal standard 7 Columns X and AG score 2 show score 2 for each internal standard enviMass version 1 0 User Manual 40 8 Columns Y and AH score 3 show score 3 for each internal standard 9 Columns Z and AI sum score gives the weighted sum of scores 1 to 3 from columns W to Y and AF to AH respectively 10 Columns AA and AJ conc ng l are established but remain empty Cells will be filled with estimates of concentrations ng l by using the quantification Tool 10 after screening for both the internal standards and the target compounds Three entries are made to spreadsheet sample 11 Columns S monoisotopic hit for target of spreadsheet sample lists the ID of the target substance column A from spreadsheet targets for which a monoisotopic hit was matched to this peak of the sample peak list 12 Column T isotopic hit for target 7 of spreadsheet sample lists the ID of the target substance column A from spreadsheet targets for which a hit of any of its expected peaks from its isotopic pattern was matched to this peak of the sample peak list 13 Column U sample ID
30. ank pesk list Skip load of blank peak list File name My Network SS Places Files of type Textdateien Figure 2 Data upload in the screening tool workflow enviMass version 1 0 User Manual 11 Tool 2 Data Check Description The Tool checks the list of a the internal standards and b the targets for consistency and missing values in columns A to O and A to Q respectively Should inappropriate non numeric values or gaps exists in these columns an error message is printed If missing isotopic pattern entries are detected the Tool subsequently redirects the workflow to Tool 3 for calculation of the isotopic patterns If names or IDs in columns A and B are not unique an error message is printed and the errors have to be corrected before running the tool again Moreover chemical formulas in columns C are checked for consistency and monoisotopic molecular masses are written to columns D Mon mass of the target and internal standard spreadsheets Spreadsheet inputs Columns A to O and A to Q of the internal standards and target spreadsheets respectively Spreadsheet outputs Monoisotopic molecular masses to columns D Mon mass of the target and internal standard spreadsheets Calculations amp parameter settings Monoisotopic molecular mass calculation No parameters to be set Data check Check list of internal standards I Check target list Figure 3 Data check tool redirecting to To
31. ases the most abundant peak used for point 1 is simply not the monoisotopic one enviMass version 1 0 User Manual 37 Internal standard screening mass acouracy retention time accuracy Internal standard screening Skip internal standard s creening Progress Calculations finished standards with monoisotopic hits Foe re ues Zt hc wm or v use recalibrated masses set step Tolerances 200 300 400 soo 7 300 400 soo 10 miz C mmu ppm 3 ART listed vs measured min intensity accuracy Ds A RT within scan min 50 Intensity Intensity cutoff default 5000 weights for summary score 07 Score 1 hits after rescaling to monoisotopic peak 03 Score 2 score 1 tolerance in intensity o Score 3 score 2 after subtraction of blank candidates soo Figure 21 Interface of the screening tool for internal standards in the sample peak list Monoisotopic hits Isotopic hits delta miz ppm delta intens scorel score2 score 3 sum score Only monoisotopic peak s Found Only monoisotopic peak s Found delta RT Not used for screening No monoisotopic hit found no isotopic pattern fit conducted 5285 15273 2 062 2 11318 0 16 70 16 0137555 2of4 20f4 20f4 5280 75256 3 075 4 20117 0 011 0 01 0131053 20f2 10f2 10f2 0 No monoisotopic hit Found no isotopic pattern fit conducted i 5121 5115
32. aximum abundance rescaled to monoisotopic peak 1 9 In these cases the most abundant peak used for point 1 is simply not the monoisotopic one enviMass version 1 0 User Manual 42 Tool 10 Target quantification Description Tool 10 allows for quantification of target compounds For this purpose any target compound a to be quantified is linked via an ID to a specific internal standard b Based on results of the screening Tools 8 and 9 the intensity a of the sample peak to which a match with the most abundant peak of the isotopic pattern of target compound a is found and the intensity b of the sample peak to which a match with the most abundant peak of the isotopic pattern of internal standard b isotopic is assigned are set in relation Q a I b From values of slope s and intercept q often g 0 from spreadsheet targets the ratio C of concentrations c a c b ng l of target vs internal standard are then approximated via the simple linear relation Q q s C Spreadsheet inputs From the targets spreadsheet 1 Column G intercept gives values of q for each target compound 2 Column H slope gives values of s for each target compound 3 Column I ID internal standard links each target a to the internal standard b used for its quantification The ID values of this column I refer to those IDs listed in column A of the internal_standards spreadsh
33. cerned isotope pair and b the associated non monoisotopic peak via the ID established in column AA of point 2 2 Column AA ID non monoisotopic peak ID established for this routine Calculations amp parameter settings 1 The underlying routine sorts all peaks in the sample peak list by decreasing peak intensity resulting in sorted peaks 1 2 3 n 2 Starting with the one most intensive peak 1 the routine checks all other peaks 2 3 n for isotopic mass differences within tolerances for m z and RT to peak 1 It is assumed that the one most intensive peak 1 is a monoisotopic peak 3 If the check detects a peak m from sample peak sublist 2 3 n then jim m is marked and omitted from the sublist resulting in sublist 2 3 n m 4 Next points 2 and 3 are repeated k times along peaks k of decreasing intensity i e in a next step the second most intensive peak 2 and the sublist Sc coni m is checked for isotopic mass differences within tolerances for m z and RT to peak 2 and so on 5 The routine stops when k n i e when all peaks were each either screened or omitted enviMass version 1 0 User Manual 49 The utilized isotopic mass differences must be chosen from the list box of the tool interface the tolerances in Am z and A RT within scan min for this check have to be specified in the two interface text boxes Figure 25 A RT within scan min
34. ckage isopat RExcel comes with an installation of the R statistical environment R foundation for statistical computing Licenses enviMass version 1 0 is a non commercial software workflow distributed by Eawag D bendorf enviMass version 1 0 is used at own risk Neither the authors nor the distributor 1s liable to any hard or software damages data losses and false inferences caused by using enviMass version 1 0 or any associated software parts Redistribution of enviMass version 1 0 is not permitted All warranties concerning the use of this software are disclaimed Technical support for the program usage is not mandatory Publications using enviMass are obliged to cite enviMass correctly We try but do not warrant that the enviMass files available are or will be free of infections or viruses enviMass version 1 0 User Manual 68 160673 152403 9 31 worms Trojan horses or other code that manifest contaminating or destructive properties The user is responsible for implementing sufficient procedures and checkpoints to satisfy the particular requirements for accuracy of data and data input and output R and isopat adhere to the GPL 2 license RExcel is distributed under the REXCEL PUBLIC LICENSE Baier and Neuwirth Citing For citation of enviMass version 1 0 use Loos M Ruff M Singer H 2011 enviMass version 1 0 target screening software Eawag D bendorf Switzerland For citation of R package isopat 1 0 use
35. d Column K spark Does this peak correspond to a spark i e TRUE Result of Tool 5 Column L blank Does this peak match with a peak in the blank blind peak list i e TRUE Result of Tool 6 Column M standard Does this peak match with an internal standard monoisotopic peak used for mass recalibration Name of that internal standard otherwise FALSE Result of Tool 7 Column N ppm deviation Mass deviation ppm between Column M internal standard and this peak Otherwise set to 0 Result of Tool 7 Column O recalibrated m z Recalibrated mass m z result of Tool 7 Column P monoisotopic hit for internal standard Result of internal standard screening cp section on Tool 8 Spreadsheet outputs Column Q isotopic hit for internal standard Result of internal standard screening cp section on Tool 8 Spreadsheet outputs Column R sample ID for internal standard screening Result of internal standard screening cp section on Tool 8 Spreadsheet outputs Column S monoisotopic hit for target Result of target screening cp section on Tool 9 Spreadsheet outputs Column T isotopic hit for target Result of target screening cp section on Tool 9 Spreadsheet outputs Column U sample ID for target screening Result of target screening cp section on Tool 9 Spreadsheet outputs Column V Target adducts monoisot 1 Results from screening for adduct peaks in li
36. d numeric enviMass version 1 0 User Manual 66 Column D m z expected expected m z of the compound numeric Permitted manipulations The user may add his her own data set to columns A2 to D2 Calculation inputs None samples_filtered Contents Copy of the sample spreadsheet with peaks rows removed from filtering with Tool 14 Permitted manipulations Do what you want This spreadsheet does not feed into any calculations Calculation inputs The spreadsheet is a result of Tool 14 for filtering the sample peak list non targets Contents Result of the filtering Tool 14 Figure 32 Based on the filtered sample peak list from spreadsheet samples filtered see above Tool 14 proposes possible non target peak groups Each row refers to one candidate group A group consists of one monoisotopic peak columns A to D and associated adduct and or isotope peaks identified with Tools 11 and 12 columns E onward The list is sorted by decreasing intensities of the monoisotopic peak Column C IDs e g Columns A E or J refer to those IDs listed in the samples_filtered spreadsheet column A Permitted manipulations Do what you want This spreadsheet does not feed into any calculations Calculation inputs The spreadsheet is a result of Tool 14 for filtering the sample peak list enviMass version 1 0 User Manual 67 A Jj ie BE IF G Its db K
37. d in the molecular formula E g 15 N in the table corresponds to the 15 N isotope given in the molecular formula C6H10CI1 15 N2N3 Thus note that 15 N2 is the correct entry into the formula whereas N 15 2 or N2 15 result in errors Beware if an element is contained in a molecular formula but not in the list to the left the monoisotopic weight and the isotopic pattern is calculated omitting this element No error message is printed in this case Calculation inputs None resolution Contents Permitted manipulations To add another table ensure that its first two cells contain a specification eg Res7500 which is loaded into the concerned selection list of the parameter spreadsheet Other than that stick to the shown format i e masses in a first column resolution in a second one two headers Beware adding a table specification without table contents can result in serious errors Calculation inputs None known Contents One data set listing expected known and HRMS measured masses of compounds These deviations between expected and measured masses can be used to derive a spline model for mass recalibration Check Tool 8 for details Each row refers to one compound Column A ID ID of the compound for which a deviation between measured and expected masses exists character string Column B compound name name of that compound character string Column C m z measured measured m z of the compoun
38. d in the screening process TRUE or FALSE Permitted manipulations Sorting of rows is permitted Changing order of columns is NOT permitted Calculation inputs Isotopic pattern information columns N to Q Results from screening quantification and adduct search steps columns R onward internal standards data sheet Contents This spreadsheet contains a information on internal standards to be screened for columns A to K b isotopic pattern information columns L to O and c results from the screening and adduct search steps columns P onward Filling columns A to C and E to K for each compound row is obligatory for the user grey headers Column A ID unique ID of each compound character string e g 234 or 234B Column B compound name name of the target compound character string e g caffeine enviMass version 1 0 User Manual 61 Column C chemical formula Molecular formula of the target compound character string e g C8H7N402D3 The elements contained in a formula must be listed in the spreadsheet isotopes Column D mon mass Monoisotopic mass of the molecular formula numeric Calculated automatically within the workflow i e needs NOT to be inserted by the user Column E retention time chromatographic retention time of the target compound minutes numeric e g 5 3 Column F tolerance retention time not implemented yet Set to FALSE Column G use for reca
39. dducts for internal standard and target compound sample peak matches enviMass version 1 0 User Manual 53 Target monoisotopic and Internal standard monoisotopic refer to the number of sample peaks matched to the most abundant mostly monoisotopic peaks in each of the isotopic pattern for the targets and internal standards respectively In contrast non monoisotopic refers to the remaining peaks of the internal standard and target isotopic patterns Moreover and their adducts refers to sample peak matches derived with Tool 11 The matches of this first table are depicted in a first scatterplot over m z abscissa and RT ordinate with color of data points referring to the cell colors of this first table Figure 27 Note The Sum of these matches may be larger than the Total number of sample peaks if single peaks i e rows in the sample peak list have each attained several matches with sparks blank blind etc data A 2 The second table Number of sample peaks with matches shows how many peaks of the sample peak list have made either no 0 one 1 two 2 or more than four 74 matches within any of the steps from the named Tools 5 6 8 9 and 11 Note The sum of these numbers must be equal to the Sum of matches in the first table A 3 The third table Number of peaks in sample list with matches for details all dual match entries in the sample peak list For example let a peak row
40. directly in the spreadsheets targets and internal standards prior to using Tool 3 see point 2 under spreadsheet inputs in section Tool 3 Isotopic pattern generation Spreadsheet inputs From spreadsheet sample 1 Column A Centroid m z 2 Column E RT min 3 Column U sample ID From spreadsheet internal standards 4 Column A for IDs of internal standards 5 Column K use for screening Internal standards not used for screening FALSE are ab initio excluded from the calculations of Tool 11 6 Column Q Isotopic hits Lists sample peak IDs of column U in spreadsheet sample 7 Column X sum score Lists the screening Tool 9 sum score for the best monoisotopic hit 8 Column Z Isotopic hits Lists sample peak IDs of column U in spreadsheet sample 9 Column AG sum score Lists the screening Tool 9 sum score for the second best monoisotopic hit From spreadsheet targets 10 Column A for IDs of internal standards 11 Column M use for screening Target compounds not used for screening FALSE are ab initio excluded from the calculations of Tool 11 12 Column S Isotopic hits Lists sample peak IDs of column U in spreadsheet sample 13 Column Z sum score Lists the screening Tool 9 sum score for the best monoisotopic hit 14 Column AB Isotopic hits Lists sample peak ID
41. e matched to one internal standard isotopic pattern set of peaks In this case scores for all sets are calculated and only the results for the sets with the two best scores are printed The tool provides a detailed list of achieved accuracies in a Am z b RT and c intensity for the peaks matched for score 1 The tool furthermore stores which sample list peaks are matched to which internal standards peak Spreadsheet inputs The screening tool compares peak information from the internal standards list with peak information from the sample peak list The following columns from spreadsheet internal_standards are used 1 Column L Isotopic m z and column M Isotopic abundance provide isotopic peak masses and abundances They are a result of Tool 3 2 Column A ID for the internal standard ID 3 retention time from spreadsheet internal_ standards column E 4 Column K use for screening allows to omit internal standard entries rows in spreadsheet internal_ standards from being used in the screening 5 Column N omit peak 7 states the index of which peaks of the isotopic pattern of a single internal standard should be omitted from screening cp Tool 4 The following columns from spreadsheet sample are used enviMass version 1 0 User Manual 34 6 Centroid m z in column A 7 recalibrated m z in column O of the sample spreadsheet instead
42. easurements Two data sets can be loaded firstly a list of peaks for a measured sample must be loaded Secondly a list of peaks for blank or blind measurements can be loaded The first list is obligatory to the enviMass workflow the second is not To reduce the data size of the raw HRMS measurements processing with a filtering software is commonly conducted The resulting text files of sample and blank blind data peak lists can be loaded via Tool I data upload see below with each line corresponding to one peak of the HRMS scan Filtering can be based on the Thermo Scientific Formulator software tool which can be downloaded from the Thermo Electron Corporation homepage http sjsupport thermofinnigan com public detail asp id 450 Formulator requires 1 Thermo data files raw as input and 2 Xcalibur 2 0 or higher to be installed on your system The enviMass data upload is adapted to the Formulator data output format i e a tab delimited text file with 10 columns containing numeric values only Three of these columns contain data essential for the workflow column 1 centroid m z column 2 peak intensity and column 5 retention time Another four columns aid at filtering noise data from the data set namely columns 7 and 9 start and end retention time and or columns 6 and 8 start and end scan number However in case that filtering is skipped cp Tool 5 spark removal these latter four columns are not essential to run t
43. ed and extended by the user all data are handled in simple spreadsheet formats The enclosed isopat R package allows calculation of the isotopic fine structures indispensable for HRMS target screening enviMass version 1 0 User Manual 3 Getting started enviMass provides a full target screening framework based on convenient user interfaces A number of consecutive Tools 1 14 support HR MS data upload data fits to isotopic patterns of internal standard and target compounds noise removal mass recalibration screening quantification assemblage of candidate non target patterns and data filtering The steps comprising this workflow are depicted in Figure 1 The workflow is implemented in the target_screening spreadsheet of the enviMass Excel file The spreadsheet isotopic_pattern computes isotopic fine structures for a given molecular formula independently of the target screening workflow and may be used as stand alone tool Two basic input data sets are required 1 A list of sample peaks and optionally a list of blind or blank data peaks are needed These lists must at least contain information on a intensities b mass to charge ratios m z and c retention times for the individual peaks These lists are loaded into the workflow and are then stored in the spreadsheets sample and blank 2 Compound lists for targets and or internal standards are needed These lists have to be entered manually into the s
44. eet 4 Column Q peak for quantif gives the index of the expected isotopic pattern peak of columns N and O to be used for quantification The intensity of the sample peak matched to this single expected isotopic pattern peak is then used as a 5 The under point 4 indexed entry in each string of columns S and AB Isotopic hits give the IDs of the matched sample peaks In turn these IDs of the matched sample peaks are entries in column U sample ID for target screening of spreadsheet sample From the internal standards spreadsheet 6 Column A of the internal_ standards spreadsheet for internal standard IDs 7 Column O peak for quantif gives the index of the expected isotopic pattern peak of columns L and OM to be used for quantification The intensity of the sample peak matched to this single expected isotopic pattern peak is then used as b 8 The under point 7 indexed entry in the string of columns Q Isotopic hits gives the ID of the matched sample peak In turn this ID of the matched sample peak is an entry in column R sample ID for standard screening of spreadsheet sample From the sample spreadsheet 9 IDs from column U sample ID for target screening enviMass version 1 0 User Manual 43 10 IDs from column R sample ID for standard screening 11 Values from column B Intensity referenced with IDs from column U sample ID fo
45. efautt charge 1 Figure 26 Interface for adduct peak search of entries in the sample peak list not X identified as target or internal standard peaks Tool 13 enviMass version 1 0 User Manual 52 Tool 14 Filter sample peak list Description Tool 14 merges the results from the upstream Tools to 13 on spark removal blank subtraction internal standard screening target compound screening adduct searches and search for non monoisotopic peaks The tool numbers a the peaks affected by the different screening steps of the workflow it b counts the screening entries per peak row of the sample peak list and it c tabulates all possible dual interferences between screening entries Thereafter d the tool allows to filter the sample peak list so as to omit peaks which have been affected by any of the screening steps in the workflow As a result two non target lists are assembled The first list is a subset of the original sample peak list of spreadsheet samples The second list suggests non target components these are sets of 1 candidate monoisotopic peaks 2 their non monoisotopic peaks of isotopic mass differences and 3 candidate adduct peaks A number of plots aid at illustrating the sample peak list filtering and the intensity distribution of the remaining sample peaks Figure 28 and Figure 29 Spreadsheet inputs 1 Spreadsheet sample Information stored in columns K L and P to Y is used f
46. en calculating the isotopic patterns of target and internal standard compounds Permitted manipulations New adducts may be added to the list as new rows Column order MUST NOT be altered Calculation inputs None isotopes data sheet Contents Isotope masses and abundances given in accordance to the values specified in De Laeter et al 2003 Cell J2 contains the mass of a single electron Column A element Name of an element character Column B isotope One isotope of the element of Column A element character Column C weight Atom weight of that isotope numeric Column D abundance Relative abundance of an isotope numeric Relative abundance of all isotopes of one element must sum to 1 Column E use Use that element for calculations TRUE FALSE Permitted manipulations The isotope list may be freely extended or manipulated by the user as long as column order and placement in the spreadsheet is not changed enviMass version 1 0 User Manual 65 To NOT use an isotope of one element set its abundance to 0 and rescale that of the remaining isotopes In contrast the TRUE FALSE setting only aids to specify if ALL isotopes of a SINGLE element shall be used or not In other words to exclude an element from calculation set ALL its isotopes to FALSE For labeled compounds For deuterium the abbreviation D may be used For all other isotopes the notation isotope X must be use
47. enviMass 1 0 target screening workflow Martin Loos Matthias Ruff Heinz Singer 2011 User manual Contact Martin Loos Eawag D bendorf P O Box 611 Switzerland Telephone 41 0 58 765 5118 Fax 41 0 58 765 5311 martin loos eawag ch enviMass version 1 0 User Manual Contents Contents noe eoites iced oun vau pa ue eund ca uc e oo pa ee acne codecs doseseseoacoescdsdessudses sess ca ou Da eo eee oreta oa oaot Getting started Offline Installation esee cH Input data FOUN AES nenn eeehlienternnseehhsekeete Save settings and calculation results for later USAagGe cscccssssccssssccsessccssscssssrceses LO 7001 12 Data Upls3d unuten cei B Fool 2 Data Check nn aha 2 Tool 3 Isotopic pattern generation csssssssossesssssennsssnnnsssnnnsssnsnnsensnssensnssnssnssessnneessne 13 Tool 4 Match standards and target patterns ccccccssssscccsssssscccssssscccssssssccssssssres 2L Tool 5 Spark removal ecce Rear LO 2 4 Online Installation ua tg eec eS Co EVEN ERR vasceandenctiaageeecasuadoas dave aetiaeadee T 8 9 Tool 6 Blank subtraction asssendseeske eu 20 Tool 7 Reealibration s eise eode hae evt rar etse rn reno re ts cvs purasensnabaedsscavadesgivesecinecsaxesysetes LO Tool 8 Internal standard screening 4 eee eere eee e ee ee eee eee eene ee ee eo see se sees ssseeee OF Took 9 Target SCr enin MET t Tool 10 Targ
48. eratively changed towards less abundant isotopes For a first iteration generation i 1 12 C2 1 H6 would hence be changed to both 12 C1 13 C1 1 H6 and 12 C2 1 H5 2 H1 Similarly the latter two isotopologues then again lead to two exhanges each for C and H at second generation i 2 At each generation level i a number i of isotopes contained in the monoisotopic peak have been exchanged for less abundant isotopes over all possible permutations of size 1 Abundances are checked for lt the limit argument If below limit the concerned isotopologues are not changed forward to the next generation 1 1 The methodology used for updating abundances and masses when progressing from generation i to i resembles that of Li et al 2008 However the updating does not strictly follow increasing nucleon numbers Instead generations of isotopic compositions are derived from an initial monoisotopic peak 17 0 via progressing to less abundant isotopes Furthermore different combination orders carried from one generation to the next can eventually lead to the same isotopic composition at a given generation causing double occurrences for some peaks in a generation Therefore peaks are checked against double isotopologues at each generation level Finally the isotopic peak list is sorted by increasing masses Too high values e g 1E 5 for the limit may prevent the calculation of isotopologues for molecules containing both 1 several abundant isotopes of one
49. es some abundance measure 100 150 200 N N l 50 o T T T T T T 205 000 205 002 205 004 205 006 205 008 205 010 miz Figure 8 Two isotopic peaks with profiles overlapping at FWHM enviMass version 1 0 User Manual 18 some abundance measure 100 150 50 l 271 T T T T T T 205 000 205 002 205 004 205 006 205 008 205 010 miz Figure 9 Two peaks with indiviual green red and overall profiles black 300 N some abundance measure 100 150 50 l T T T T T T 205 000 205 002 205 004 205 006 205 008 205 010 miz Figure 10 Two peaks red green the resulting overall profile thick black and the stick representation black lines Algorithm for isotopic pattern calculation The algorithm for deriving the isotopic pattern for a given molecular formula is provided in the package isopat which has been included during installation In contrast to other algorithms and package implementations cp Rockwood et al 2006 Kirchner enviMass version 1 0 User Manual 19 2008 the provided algorithm allows calculation of isotopic fine structures for isotopologues with identical nucleon number Starting from a monoisotopic peak of a given molecule e g C2H6 with each elements set to those isotopes with highest abundance the isotopic compositions and the concomitant abundances of isotopologues are it
50. es 1 to 3 are derived A weighted sum of all three scores leads to a final overall score for each of the internal standards the weights must be set by the user Mind that score 1 is less restrictive than score 2 and score 2 less restrictive than score 3 i e score 1 gt score 2 gt score 3 score 1 Firstly the number of internal standard peaks expected vs the number of peaks found in the sample list within tolerance settings of retention time RT and Am z is evaluated for each internal standard compound For example if 6 peaks are expected but only 4 found in the sample peak list score 1 would result in 4 of 6 peaks i e score 1 4 6 0 67 score 2 Based on the peaks found the agreement of expected rescaled abundance versus observed peak intensities is evaluated for a second score For example from the above 4 peaks only 3 peaks are within the expected intensity range the range being set by an intensity tolerance of x of the expected intensity Thus score 2 3 6 0 50 score 3 is even more restrictive it also checks for interference of the remaining peaks with those of the blank blind peak list of spreadsheet blank For example of the above 3 peaks found in the sample peak list one peak already has been matched in Tool 6 with a peak of the blind blank peak list i e 2 of 6 peaks remain Therefore score 3 2 6 0 33 Occasionally different sets of peaks from the sample peak list can b
51. es a threshold to filter peaks with low abundance from the peak list after any of the steps a to c with default 1E 3 Form adducts specifies if adduct masses should be added to the isotopologue masses If selected and no adduct is chosen from the associated list box an hydrogen atom H default will be used as adduct Profile Centroid settings Given the above isotopic pattern a Gaussian profile is fitted to each peak of the pattern The settings therefor have to be specified in the interfaces for Resolving power FWHM Figure 6 enviMass version 1 0 User Manual 15 Profile Centroid Settings Resolving power FWHM Stick discretization C fixed 5 mmu fixed 10 mmu as function of mass 5 ppm as function of mass 2 ppm as function of mass with ppm 1E6 resolution factor with resolution as function of mass Resolution f mass data set factor 4 O Res7500 Res15000 Resolution f mass data set Res7500 O Res60000 Res15000 E Resiooooo Res30000 O Ress0000 E Res 00000 Update resolution data sets Update resolution data sets sdfactor default 1 Figure 6 Profile and centroid settings for the isotopic pattern calculation The resolving power Am defines the mass difference two peaks of same intensity and with mass m and m2 must have to be separable by HR MS Figure 7 The resolution R is thus defined as R m1
52. et quantification 1 ecce esee eere ee eren ee eren eee s eee ensseesnseeeesseeesse 43 Tool 11 Adduct search for targets internal standards 45 Tool 12 Search for other non monoisotopic peaks eeeeee eere eee ecce e 4D Tool 13 Adduct search non targets non int stand c eeeeeeeeeeeeeeeeeeees DL Tool 14 Filter sample peak List ssssscsssssssssssssssssssessesssssesssesesssssesssssscssesscsscssssses ID Batchmoder sea eiue ra coa rasen IC os Ru eeose sola pe oorsese aneii O Isotopic pattern spreadsheet ash nase reihen DO isotopic_pattern data sheet tee ie eet red esee atl e Rd s 60 target Sereening data sheet aea ds et eiaude ee 60 jargets datasheet ee nes 60 internal standards data sheet ur eins 61 Sample datasheet 2 ee eisen un 62 blank data SNCS ann nn N ER 64 adducts data SDR N a E BR SE FeO DU MED I M UN Pe 64 isotopes datasheet ann 65 1650 UNION ee ee ee 66 samples filtered sss cuo ou een Passo tes emt t LEE 67 HOD AAR SOLS e e e Rest toe Saat adda aa e tea neq aps 67 LXI a TOHS aeo de eere iE A teo ER VERIS EUR ENSUR PP ER Leo Mob pe arca TFT NR Da Computer requirements sosscsvesecevesseccnsseceosereecessaseveensevsonssseennavesuresvnsntesnanesduecyseonseeceesesvees OO LE Todi rer S TU m TIN OO Referelnces 2 reiceoe Sese Poss eoe oao sae caes EEE Dena o sa Deososos dose eas Seo sara
53. g two sticks is z times smaller than the Am of the resolving power ensuring accurate valley stick detection between the two peaks Such a parameter setting is exemplified for z 4 in Figure 6 Next surviving peaks or centroids are calculated for either the Profile or the Centroid setting respectively In general two isotopic pattern peaks can be separated if the sum of their profiles allows for a valley which in turn is represented by one stick encompassed by two adjacent sticks of higher intensity Figure 10 The surviving peaks of the Profile setting designate enviMass version 1 0 User Manual 17 those peaks that can be separated by a stick valley representation for those that cannot be separated by a valley only the one most intensive peaks survives In contrast the centroid peaks of the Centroid setting are intensity weighted sums of those sticks that are not separated by a valley Finally the isotopic pattern surviving centroid peaks can be optionally filtered by Recursive Base Peak Framing RBPF Herein the most intensive peak of the data set is selected and all other peaks close enough to this peak within a mass tolerance frame width in the Recursive Base Peak Framing RBPF settings are discarded From this reduced data set the second most intensive peak is selected and again other peaks in its vicinity discarded Thus RBPF is recursively applied over all peaks along decreasing intensiti
54. he workflow Columns 3 4 and 5 correspond to the peak signal to noise ratio the scan number and the mass chromatogram signal to noise ratio respectively Alternatively the user may work with peak data filtering tools other than Formulator such as MZmine http mzmine sourceforge net If doing so the user must reformat the text file to be loaded into the workflow to adapt to the above described Formulator output format i e ten columns of numeric values no characters no empty line or column positions dummy variables for columns 3 4 and 10 should be set to a 9999 values not more than ten columns with a a first column of peak centroid m z values b a second one with peak intensities c a fifth one with peak retention times and optionally the above columns for d start and end peak retention times and or e start and end peak scan numbers The enviMass example folder on the source website provides exemplary text files produces from Formulator peak picking enviMass version 1 0 User Manual 9 Save settings and calculation results for later usage Once the parameters in the workflow interfaces are chosen and input data sets defined and loaded the workflow can be saved and reused when being reopened Mind that the Start workflow button of Tool 1 Data upload DOES NOT REMOVE the parameters typed into the textboxes of the user interface However it DOES REMOVE 1 the sample and blind data sets in the
55. his screening is conducted for all peaks of the sample peak list and not only for peaks identified as internal standard or target peaks cp Tool 11 on results from Tools 8 and 9 the Tool 13 is termed Adduct search non targets non int stand Spreadsheet inputs Two columns are used from spreadsheet sample 1 Column A Centroid m z 2 Column E RT min From spreadsheet adducts 3 Column B Adduct name 4 Column C Adduct mass 5 Column G Charge for adduct search Spreadsheet outputs Two columns are inserted into spreadsheet sample 1 Column AB Non target adduct This column lists the adduct hits a adduct or deduct b adduct name from column E of the adducts spreadsheet and c ID from column AC of the associated sample peak For example the entry K1 127 indicates that the peak listed in this row is a candidate potassium adduct of the peak with ID 127 2 Column AC ID for non target adduct ID generated for this tool and used in column AB Calculations amp parameter settings The tool compares sample peaks assuming they are the result of different adducts formed during HR MS ionization For this pairwise comparison the routine has to subtract add 1 the mass of default adduct deduct a and 2 the electron mass es of the default charge from a given peak A and then subtracts adds 3 another adduct deduc
56. iMass version 1 0 User Manual 48 Tool 12 Search for other non monoisotopic peaks Description Tool 12 searches the sample peak list for peaks having a differences in m z equal to a specified difference in mass between two isotopes a and b of an element henceforth called isotopic mass difference with a being the most abundant isotope of that element For example any organic molecule with more than ten carbon atoms has besides its monoisotopic peak a with abundance 1 another isotopologue peak b with abundance gt 0 1 resulting from the substitution of one 12 C atom by a 13 C atom Tool 12 thus marks that sample peak b is most likely associated with sample peak a for a given isotopic mass difference i e b is an isotopologue of a In other words the tool screens for sample peaks resulting from substitution of one most abundant monoisotopic isotope by a less abundant one Spreadsheet inputs From spreadsheet sample 1 Column A Centroid m z 2 Column B Intensity 3 Column E RT min From spreadsheet isotopes 4 Column A element 5 Column B isotope 6 Column C weight u 7 Column D abundance Spreadsheet outputs Two entries are made to spreadsheet sample 1 Column Z non monoisotopic peak If no isotopologue match has been found for this sample peak the cell entry is set to 0 Otherwise the cell entry names a the con
57. ing an estimate of a 90 percentile data range enviMass version 1 0 User Manual 31 recalibrated standards E a E 3 o E E E Figure 19 Fourth result plot from the recalibration tool Depicted are deviations in mass between theoretical values calculated from the molecular formula of internal standards and matches found for these internal standards in the recalibrated sample peak list ordinate ppm units plotted against the mass of the internal standards abscissa Da unit for mass m z 1 Upper and lower dashed lines separate the highest and lowest 5 of the data enviMass version 1 0 User Manual 32 Recalibration of samples NS E ke 9 s E 8 j N E 3 e fo Q Q Figure 20 Last result plot of the recalibration tool plotting the modeled absolute deviation between model predicted and HRMS measured masses of the peaks in the sample list ordinate against mass of the peaks in the sample list abscissa Da unit for mass m z 1 enviMass version 1 0 User Manual 33 Tool 8 Internal standard screening Description Tool 8 screens the sample peak list for matches with the isotopic patterns of the internal standards listed in the internal standards spreadsheet The underlying routine rescales the abundance of the expected peaks in the internal standard isotopic pattern so as to exclude peaks of too low intensity from further consideration Thereupon three scor
58. ith a rescaled abundance i e an expected intensity lower than the value specified in text box Intensity cutoff default 5000 are then omitted from further considerations This step avoids to falsely screen for expected internal standard peaks which cannot surmount the detection noise thresholds intrinsic to the sample peak list data set For example consider a an internal standard isotopic pattern with abundances 1 0 5 0 25 0 1 and b a matched sample peak with intensity column B spreadsheet sample 50000 Rescaling the internal standard enviMass version 1 0 User Manual 36 abundances to the sample peak intensity leads to the expected intensities 50000 25000 12500 5000 Thus for an exemplary intensity cutoff 15000 only the first two internal standard peaks with expected intensities 50000 25000 would be screened for 3 The rescaled i e reduced isotopic pattern peak set of an internal standard is screened for in a third step While the tolerance parameter for Am z is still taken from the textbox Am z a different tolerance parameter for ART then the one of point 1 is now used namely the one from text box A RT within scan min The former tolerance in RT determined the search for the most abundant peak of an internal standard under point 1 based on the RT listed for each internal standard column E spreadsheet internal standards However a second preferably narrower RT is now
59. le peak list enviMass version 1 0 User Manual 56 ee Da BUE Intensity RT min ID 86 87 845019 597 40Ca 44Ca 253 0 51420 87 88 842814 329 Na1 monoisot 341 05479 145423 88 89 836568 89 90 831154 377 Na1 monoisot 276 04073 116246 90 9 823200 Eu 32 811716 468 Na1 monoisot 278 04083 76672 Im 3 810083 93 84 796057 94 95 795215 226 Na1 monoisot 33 08844 261301 95 96 769280 96 97 767114 311 Na1 monoisot 7 1975 158030 97 98 766055 311 40Ca 44Ca E 158030 98 99 765312 99 100 758520 100 101 758420 1101 102 748144 102 103 747076 103 104 744939 542 Na1 monoisot Figure 30 Screenshot of the filtered sample list results assembled in spreadsheet non targets The orange column proposes masses of potential monoisotopic peaks sorted by decreasing intensity The table fields to the right propose potential adducts and M X peaks of these monoisotopic peaks enviMass version 1 0 User Manual 57 152403 9 31 Batch mode Description The batch mode allows to run all or a subset of tools at once Spreadsheet inputs As used in Tools 5 to 14 cp the respective sections Spreadsheet outputs As results from Tools 5 to 14 cp the respective sections Calculations amp parameter settings To include a tool in the batch processing mark its Run checkbox The parameter settings are taken from the interfaces of the individual Tools 5 to 14
60. libration should this internal standard be used for mass recalibration Tool 7 TRUE or FALSE Column H remark open for remarks character strings e g pesticide If no remarks set to FALSE Column I build adduct Should this target compound have the default adduct for calculation of its isotopic pattern TRUE or FALSE cp Tool 3 Column J charge Should this target compound have the default charge for calculation of its isotopic pattern FALSE or charge other than default e g 1 cp Tool 3 Column K use for screening Should this target compound be included in the screening process TRUE or FALSE Permitted manipulations Sorting of rows is permitted Changing order of columns is NOT permitted Calculation inputs Isotopic pattern information columns L to O Results from screening and adduct search tools columns P onward sample data sheet Contents List of sample peaks each row refers to one peak The list columns A to J is loaded from a text file Tool 1 Columns K onward contain calculation results Columns A B and E contain peak nvz intensity and retention times respectively For more information please refer to section Input data formats enviMass version 1 0 User Manual 62 Permitted manipulations Sorting permitted the tools will resort the list for sparks and m z for calculations DO NOT change column orders DO NOT change data Calculation inputs From column K onwar
61. lind data set and the sample data set In the first case this substance is introduced to the HR MS analytics during laboratory processing only In the second case the substance exists in the sample data already before processing AND is additionally introduced during laboratory procedures As a result the concentration and thus the peak signal will be higher in the second case enviMass version 1 0 User Manual 26 relative to the first case Thus comparing relative intensities allows to distinguish both cases Blank subtraction Run blank s ubtraction Skip blark s ubtraction Tolerances subtracted data Amiz Retentiontime frin 05 ART min Intensity of blank times smaller than sample Ll Humber of samples removed Figure 15 User interface of the blank blind subtraction Tool 6 On the right circles mark subtraction matches between the sample and the blind blank data sets in the centroid m z vs retention time plot enviMass version 1 0 User Manual 27 Tool 7 Recalibration Description High Resolution Mass Spectrometers sometimes produce measurements with m z being systematically lower or higher than the expected m z of the measured substances Often such deviations are themselves a function of m z and it may be unclear whether they stem from shortcomings in either spectrometer hardware or spectrometer software In any case Tool 7 allows for a recalibration of m z to increase the accuracy
62. lotting functionality of the Spark removal Tool 5 with settings maximum ART in histogram min 1 and number of histogram breaks 60 The histogram suggests that a large number of entries in the sample peak list have very low delta retention times of 0 02 minutes These entries may be regarded as sparks or noise data and can therefore be removed with Tool 5 enviMass version 1 0 User Manual 25 Tool 6 Blank subtraction Description Tool 6 allows to compare the list of peaks stored in spreadsheet blank i e blank or blind data with that of spreadsheet sample In this way matrix peaks or any other background peaks which may be related to the laboratory processing of the sample data set can be subtracted from the sample peak list aiming at reducing the size of the sample peak data set If matches between peaks of both lists are detected the concerned peaks row entries are marked in the sample spread sheet These marks subsequently serve as input to the target and internal standard screening scores and the filtering routine of Tool 15 Spreadsheet inputs 1 Data on centroid masses from the blank peak list spreadsheet blank column A Centroid m z and the sample peak list spreadsheet sample column A 2 Data on retention times from the blank peak list spreadsheet blank column E RT min and the sample peak list spreadsheet sample column E 3 Data on
63. m z measured and column D m z expected from spreadsheet known for a list of measured and theoretical m z values Spreadsheet outputs The recalibration procedure adds three new columns to the peak list in the sample spreadsheet 1 Column M standard refers to the name of the internal standard from column B internal standard name that has been matched to this peak of the sample peak list If no match could be assigned these cells are set to FALSE 2 Column N ppm deviation shows the Am z between the m z peak of the internal standard named in Column M and the m z peak of that sample peak in ppm units 3 Column O recalibrated m Z lists the recalibrated masses Calculations amp parameter settings 1 The first two steps a and b of the recalibration procedure are activated via command button search internal standards in sample amp calculated enviMass version 1 0 User Manual 28 deviation Hereby a internal standards are matched to peaks in the sample peak list and b deviations in mass are calculated corresponding entries to columns M and N of the sample spreadsheet are made The tolerances in m z and retention time RT between internal standard and potential match in the sample peak list are set in the text boxes below that button Figure 16 Since masses are not yet recalibrated at this step a relatively wide mass tolerance should be used Finally two plots are generated to help clarifying any trend in
64. m2 2 Am IUPAC 1997 Three options are provided to define the resolving power Am 1 as a fixed value mmu 2 as function of mass ppm or 3 based on a selected data sets of resolution R as function of mass provided in the spreadsheet resolution In the latter case a generalized additive model based on regression splines is fitted to predict R f mass Woods 2006 The model then interpolates R for a given mass m and the resolving power is derived from Am m R Given a value for the resolving power Am from any of the above options 1 to 3 a standard deviation o must be calculated for the two Gaussian profiles of the two peaks so as to have both profiles overlap at their Full Width at Half Maximum FWHM Figure 8 Implicitly this specific overlap property is henceforth assumed to make two peaks separable For two symmetrical distributions of two peaks of same intensity FWHM Am The standard deviation can then be calculated from the FWHM via the relationship FWHM 2N 21n2 c The parameter sd factor allows to multiply o with a factor i e to de and increase the standard deviation of the profiles default is sd factor 1 namely no de or increase Once Gaussian profiles are calculated for each peak they are summed so as to yield an overall profile of the m z spectrum Figure 9 enviMass version 1 0 User Manual 16 Am some abundance measure 100 150 l 50 l o 4 T T T T T T 205
65. n Society for Mass Spectrometry 19 1867 1874 Rockwood A Haimi P 2006 Efficient calculation of accurate masses of isotopic peaks Journal of the American Society of Mass Spectrometry 17 415 419 R version 2 12 0 2011 The R foundation for statistical computing Vienna Austria http www R project org enviMass version 1 0 User Manual 74
66. n so as to exclude peaks of too low intensity from further consideration Thereupon three scores 1 to 3 are derived A weighted sum of all three scores leads to a final overall score for each of the target compounds the weights must be set by the user Mind that score 1 is less restrictive than score 2 and score 2 less restrictive than score 3 1 e score 1 gt score 2 gt score 3 score 1 Firstly the number of target compound peaks expected vs the number of peaks found in the sample list within tolerance settings of retention time RT and Am z is evaluated for each target compound For example if 6 peaks are expected but only 4 found in the sample peak list score 1 would result in 4 of 6 peaks i e score 1 4 6 0 67 score 2 Based on the peaks found the agreement of expected rescaled abundance versus observed peak intensities is evaluated for a second score For example from the above 4 peaks only 3 peaks are within the expected intensity range the range being set by an intensity tolerance of x of the expected intensity Thus score 2 3 6 0 50 score 3 is even more restrictive it also checks for interference of the remaining peaks with those of the blank blind peak list of spreadsheet blank For example of the above 3 peaks found in the sample peak list one peak already has been matched in Tool 6 with a peak of the blind blank peak list i e 2 of 6 peaks remain Therefore score 3 2 6
67. ndards in spreadsheet internal standards column I build adduct To do so instead of setting the entry for a compound to TRUE using the adduct specified in the workflow interface set it to FALSE and include the adduct in the chemical formula directly see above point 1 For example let the compound Cytarabin have two adducts namely H and Na adducts To include both adducts for screening have two entries rows in the target or internal standard spreadsheet list for Cytarabin For the H adduct first row use the molecular formula of Cytarabin C9H13N305 set build adduct to TRUE and chose Form adducts H default in the workflow interface For the Na adduct however second row set build adduct to FALSE and extend the molecular formula to contain Nal i e C9H13N305Nal 3 Charges other than the one chosen from the workflow interface can be defined for a the target compounds in spreadsheet targets column L and b the internal standards in spreadsheet internal_ standards column J charge To do so do not set the column entry to FALSE but enter a value for the charge For example let the compound Cytarabin have two ionization states a single positively charged and a double positively charged To include both charges for screening have two entries rows in the target or internal standard spreadsheet list for Cytarabin For the single charge state set the one row entry charge
68. ne with the target screening results Cp section on Tool 11 Spreadsheet outputs Column W Target adducts monoisot 2 Results from screening for adduct peaks in line with the target screening results Cp section on Tool 11 Spreadsheet outputs enviMass version 1 0 User Manual 63 Column X Standard adducts monoisot 1 Results from screening for adduct peaks in line with the internal standard screening results Cp section on Tool 11 Spreadsheet outputs Column Y Standard adducts monoisot 2 Results from screening for adduct peaks in line with the internal standard screening results Cp section on Tool 11 Spreadsheet outputs Column Z non monoisotopic peak Result from search for non monoisotopic peaks Cp section on Tool 12 Spreadsheet outputs Column AA ID non monoisotopic peak ID on results from search for non monoisotopic peaks Cp section on Tool 12 Spreadsheet outputs Column AB Non target adduct Result from search for possible non target adduct peaks Cp section on Tool 13 Spreadsheet outputs Column AC ID for non target adduct ID on results from search for possible non target adduct peaks Cp section on Tool 13 Spreadsheet outputs blank data sheet Contents List of blank and blind peaks each row refers to one peak The list columns A to J is loaded from a text file Tool 1 Columns A B and E contain peak m z intensity and retention times respectively For mo
69. ngs once you have reached it going downstream the workflow Beware rerunning implies that the outcomes of the previous run are overwritten I intend to recalibrate my sample peak list masses from known deviations not from a match between internal standards and the peak list What can I do Read the section about Tool 7 copy Your data set to the spreadsheet known and mark the checkbox calculate deviation from the data set listed in I receive the error message Error 2147220203 in Module RExcel Arrays File name or sheet name too long more than 65 characters total The name of your Excel file including the path is too long Rename it to the most shortest one You can think of and choose a shorter path for your stored Excel file I receive the error message Run time error 6 buffer overflow May occur when Your sample peak list has more than 30 000 entries rows Please report the affected tool in the workflow and the size of the sample peak list to the authors thank You Workaround use only the 30 000 most intensive peaks of Your workflow for screening enviMass version 1 0 User Manual 71 16 17 18 The Tool 12 Search for other non monoisotopic peaks and or the Tool 13 Adduct search non targets non int stand seem to freeze while running Eventually they are not froozen but calculating these two tools take the longest time in calculating as extensive searches in the sample peak list have to be
70. ntion time relation of the sample peaks B Filtering of sample peak list The checkmarks Exclude to the right side of the first table Number of matches within sample list for Figure 27 allow to filter matched peaks from the sample peak list and to have the filtered sublist written to spreadsheet samples filtered The two textboxes allow to exclude internal standard and target matches in the sample peak list from filtering if the concomitant sum score of screening in Tools 8 and 9 lies below the specified text box values The thus filtered sample peak sublist is automatically sorted for decreasing intensities and values for Centroid m z and RT min of the columns A and E are plotted in a second graph into the interface Figure 28 The numbers in that plot localize the ten most intensive peaks In addition intensities are plotted along the row index of the filtered and sorted sample peak list of spreadsheet samples filtered in a third plot Figure 29 enviMass version 1 0 User Manual 55 sample data T E o E c 2 c 2 o a Centroid m z Figure 28 The second plot being part of the results of Tool 14 numbers and locates the ten most intensive peaks within the filtered sample peak list Abscisse centroid masses Da Ordinate retention time min Intensity of non excluded samples intensity Figure 29 Distribution of intensities in the filtered samp
71. of the measurements The mass recalibration involves four steps Step a aims at detecting the mass deviations For this purpose the sample peak list is screened for internal standard monoisotopic peaks Having assigned potential matches between sample and internal standard peaks the mass differences between both are calculated in a second step b Thereupon step c builds a nonlinear model to relate these mass differences Am z to the mass m z of the internal standards matched Finally d this model is utilized to correct all masses listed in the sample peak list for the observed mass differences The thus recalibrated masses of the sample peak list can then optionally be utilized for all downstream workflow tools Alternatively to step a and b a list of known and measured m z values from spreadsheet known can be used for model definition of step c Spreadsheet inputs 1 Centroid m Z in column A of the sample spreadsheet 2 Retention time RT min from column E of the sample spreadsheet 3 Names of internal standard substances from column B of the sample spreadsheet 4 The first monoisotopic mass entry of the string in Isotopic m Z in column L of the spreadsheet internal standards 5 Column E retention time from spreadsheet internal standards 6 Column G Use for recalibration If set to FALSE the corresponding internal standard 1s not included in the recalibration routine 7 Columns C
72. ol3 isotopic pattern calculation enviMass version 1 0 User Manual 12 Tool 3 Isotopic pattern generation Description The tool calculates the isotopic patterns and fine structures for the molecular formulas of all listed a target compounds and b internal standards It furthermore allows for Gaussian profiling of these patterns via representation by stick data and conversion to surviving peaks and centroid peaks Subsequently these latter isotope peaks may then be filtered by Recursive Base Peak Framing RBPF The isotopic peaks are stored in the spreadsheets lists of the targets and internal standards and later used as input to the screening Tools 8 and 9 Spreadsheet inputs 1 Molecular formulas for a the target compounds spreadsheet targets column C and b the internal standards spreadsheet internal standards column D Element names must be followed by numbers atom counts of that element except for preceding numbers in square brackets indicating individual isotopes defined in the element name column of the isotope spreadsheet e g 14 C or 18 O For example 13 C2C35H67N1013 is the molecular formula of erythromycin labeled at two C positions with 13 C C37H67N1013 is the molecular formula of the unlabeled compound 2 Individual adducts other than the one chosen from the workflow interface can be defined for a the target compounds in spreadsheet targets column K and b the internal sta
73. ompound to an internal standard Spreadsheet inputs 1 Isotopic masses from column N Isotopic m z of the target spreadsheet and column L Isotopic m z of the internal standards spreadsheet 2 The column I ID internal standard in the targets spreadsheet specifies the ID of the column A ID in spreadsheet internal_ standards to link a target compound to a specific internal standard for a quantification purposes Tool 10 or b if an isotopically labeled compound of a target is listed in the internal standards spreadsheet making a match of isotope patterns necessary 3 Retention times from columns E of both spreadsheets are utilized Spreadsheet outputs 1 If an identicalness between isotopic peaks of a target compound and its internal standard is detected the indices of the concerned peaks are each written to the omit peak 7 columns P and N for the target and the internal standard respectively For the internal standard the string of indices is preceded by the row number e g 21 of the target compound entry in the spreadsheet targets in turn the relation target to internal standard is manifested by the ID value As a consequence the concerned peaks are omitted at the downstream screening steps 2 Moreover modifications in the column peak for quantif are made to the default value 1 columns Q and O for the target and the internal standard spreadshee
74. or filtering assembling of non target peak groups Spreadsheet outputs 1 Spreadsheet samples filtered Subset of the sample spreadsheet with peaks rows removed from filtering and a new ID attached in column A Put differently peaks rows not contained in this filtered list may be sparks matches with blank blind list peaks matches with target and internal standard peaks etc depending on which filter options the user selects in the tool interface 2 Spreadsheet non targets Based on the filtered sample peak list from spreadsheet samples filtered see point 1 this list proposes possible non target peak groups Each row refers to one candidate group A group consists of one monoisotopic peak columns A to D and associated adduct and or isotope peaks identified with Tools 11 and 12 columns E onward The list is sorted by decreasing intensities of the monoisotopic peak Column C IDs e g columns A E or J refer to those IDs listed in the samples filtered spreadsheet column A Calculations amp parameter settings A Summarizing results of Tools 1 to 13 Tool 14 uses screening results stored in columns K L and P to Y of the sample spreadsheet to summarize and tabulate the following cp Figure 27 A 1 First table Number of matches within sample list for lists the number of sample peaks with matches from Tools 5 sparks 6 blank blind peaks 8 internal standards 9 targets and 11 additional a
75. peak intensities from the blank peak list spreadsheet blank column B Intensity and the sample peak list spreadsheet sample column B Spreadsheet outputs 1 Potential blank matches in the sample peak list are marked by insertion of a new column in spreadsheet sample This new column L with header blank lists potential sample peaks being blanks in the sample list as TRUE cell entries are set to FALSE otherwise Calculations amp parameter settings Figure 15 shows the user interface of the discussed blank subtraction tool Three tolerance settings specify the precision accuracy with which the matching between blank and sample peaks is conducted 1 A tolerance in mass Am z must be specified in the first text box a match between target and blank is only accepted if the difference in centroid mass between target and internal standard is lower than this mass tolerance 2 Akin the tolerance in retention time ART in the second text box 3 Thirdly and once the tolerances for Am z and ART are complied to the peak intensities of a potential matches between blank blind and sample data are compared Namely such a potential match is discarded if the intensities of the blank peak is X times smaller than that of the concerned sample peak to be specified in text box Intensity of blank times smaller than sample For example it may well be the case that a substance indeed occurs in both the blank b
76. preadsheets targets and internal standards cp the Data sheets section and contain the molecular formulas and retention times of individual target compounds and internal standard substances Note Isotopic patterns of targets and internal standards are calculated and stored in the spreadsheets targets and internal standards for each HRMS ionization mode If the user wants to switch between ionization modes i e between positive and negative ionization he has to maintain two separate enviMass worksheets One worksheet contains isotopic patterns calculated for positive ionization and another one contains patterns for negative ionization Most tools allow to be skipped if underlying input data are missing Thus separate parts of the workflow can be combined and others omitted tailored to the needs of the user For example the screening uses blind data if provided if not provided dummy variables are used instead All separate steps of the workflow are sequenced so as to guide the user from one tool to the next in a self explaining manner Stepping back to a tool further upstream from a downstream tool is disabled the workflow automatically manages the actions that can be conducted for the various spreadsheets and the addition of information therein Once all tools in the workflow have been adjusted parameterized to the specific needs they can be run in a batch mode We recommend the user to start with the below installa
77. precisely the sample peak was matched to one of the entries in the string contained in the internal standard cell of column Q spreadsheet internal standards i e generated in Tool 10 after matching to the first monoisotopic target peak 4 Column Y Standard adducts monoisot 2 lists the adduct name and the ID column A spreadsheet internal standards of that internal standard compound to which the sample peak was matched as a potential adduct More precisely the sample peak was matched to one of the entries in the string contained in the internal standard cell of column Z spreadsheet internal standards i e generated in Tool 10 after matching to the second monoisotopic target peak Two entries are made to spreadsheet internal_standards 5 Column AI adducts for first monoisotopic hit pattern lists per internal standard the adduct name and the sample peak ID contained in column U in spreadsheet sample of the concomitant matched peak in the sample peak list Here matching refers to adducts found for the screening results of column Q isotopic hits 7 internal_standards spreadsheet 6 Column AJ adducts for second monoisotopic hit pattern lists per internal standard the adduct name and the sample peak ID contained in column U in spreadsheet sample of the concomitant matched peak in the sample peak list Here matching refers to adducts found for the screening results of
78. quently filtered for to reduce the size of the peak list Therefore based on differences in either 1 start and end retention times A RT min or 2 start and end scan number A scan number entries in the sample peak list spreadsheet samples are marked as noise data and are omitted from all downstream steps of the workflow including those of the screening tools Spreadsheet inputs 1 For calculation of A RT the differences in End RT min and Start RT time min is derived from columns I and G of spreadsheet sample 2 For calculation of A scan number the differences in End Scan Number and Start Scan Number is derived from columns H and F of spreadsheet sample 3 For including potential target candidates in the plotting functionality entries in column N Isotopic m z and column E retention time of the target list spreadsheet targets are used Spreadsheet outputs 1 Potential sparks in the sample peak list are marked by insertion of a new column in spreadsheet sample This new column K with header spark lists sparks as TRUE cell entries are set to FALSE otherwise All peaks i e rows in the sample peak list with spark TRUE are subsequently omitted from all workflow processes downstream of Tool 5 Calculations amp parameter settings The tool provides an auxiliary plotting function on the right side of the dashed line and the spark removal
79. r string e g caffeine Column C chemical formula Molecular formula of the target compound character string e g C8H7N402D3 The elements contained in a formula must be listed in the spreadsheet isotopes Column D mon mass Monoisotopic mass of the molecular formula numeric Calculated automatically within the workflow i e needs NOT to be inserted by the user Column E retention time chromatographic retention time of the target compound minutes numeric e g 5 3 enviMass version 1 0 User Manual 60 Column F tolerance retention time not implemented yet set to FALSE Column G intercept intercept for quantification numeric e g 0 cp Tool 10 Column H slope slope for quantification positive numeric e g 0 8 cp Tool 10 Column I ID internal standard ID of internal standard column A of internal standards spreadsheet character string e g 234 or 234B Column J remark open for remarks character strings e g pesticide If no remarks set to FALSE Column K build adduct Should this target compound have the default adduct for calculation of its isotopic pattern TRUE or FALSE cp Tool 3 Column L charge Should this target compound have the default charge for calculation of its isotopic pattern FALSE or charge other than default e g 1 cp Tool 3 Column M use for screening Should this target compound be include
80. r target screening and column R sample ID for standard screening serve as I a and I b respectively Spreadsheet outputs Two entries are made to spreadsheet targets 1 Concentration ratio C is written to column AA conc ratio It is calculated with the sample peak intensity a referenced to via an entry in the ID string of column S Isotopic hits The entry of this string is in turn indexed via information from column Q peak for quantif 2 Concentration ratio C is written to column AJ conc ratio It is calculated with the sample peak intensity a referenced to via an entry in the ID string of column AB Isotopic hits 7 The entry of this string is in turn indexed via information from column Q peak for quantif Target quantification Target quantification Sk ip target quantification Calculation skipped Figure 23 Target quantification Tool 10 Calculations amp parameter settings No parameters have to specified via text boxes the tool solely uses results from the preceding screening Tools 8 and 9 and information stored in the spreadsheets targets internal standards and sample Note If columns Q or O peak for quantif of spreadsheet targets or spreadsheet internal standards index outside of the number of Isotopic hits 7 contained in columns S AB spreadsheet targets and column Q spreadsheet internal
81. re information please refer to section Input data formats Permitted manipulations Sorting permitted the tools will resort the list for sparks and m z for calculations DO NOT change column orders DO NOT change data Calculation inputs None adducts data sheet Contents Specifies information on adducts used in Tools Column A ID ID of the adduct any character Column B Adduct Name of the adduct used in the list boxes in the workflow character string enviMass version 1 0 User Manual 64 Column C Mass Mass of the adduct numeric Can be calculated with the isotopic_pattern spreadsheet by using the formula from column E setting charge to 0 and not choosing Form adducts This entry will be used in different calculation steps Column D comment comment character string Column E formula Molecular formula of the adduct This entry is used in different calculation steps Column F removed from molecule Is the adduct added to the molecule set to TRUE or is it a fragment removed from the molecule set to FALSE essentially making the adduct a deduct This entry is used in different calculation steps Column G charge for adduct search Used only by tool 13 for non target adduct search Specifies the charge of a molecule when being associated with that adduct In contrast all other tools concerned with the ionization of a molecule utilize the charge specified in Tool 3 wh
82. s plotted against the mass of these internal standards abscissa Da unit for mass m z 1 2 Subsequent c model fitting and d mass recalibration of peaks in the sample peak list are triggered by command button Run recalibration Figure 16 The model relates the under point 1 determined deviations in Am z to the m z listed in spreadsheets sample in columns ppm deviation and Centroid m Z respectively An additive nonlinear model is used the user can select from a list box if thin plate regression splines or penalized cubic regression splines shall be used Wood 2006 The difference between both spline types is mostly negligible for the recalibration outcomes Furthermore the user can also specify the number of knots used in the nonlinear model via another text box The knot number controls the wiggliness of the model i e how easily the model fits to local nonlinearities in the relation Am z versus m z The default knot number is set to 10 However we recommend the user may rerun the Run recalibration button under different knot numbers to infer the requested wiggliness of the model from the concomitant plots see Figure 18 to Figure 20 To use a known data set with measured versus expected masses from spreadsheet known instead of matches from steps a and b of point 1 checkmark checkbox calculate deviation from the data set listed in spreadsheet known located above the list box for spline selection Fig
83. s of column U in spreadsheet sample 15 Column AI sum score Lists the screening Tool 9 sum score for the second best monoisotopic hit From spreadsheet adducts 16 Column B Adduct name enviMass version 1 0 User Manual 45 17 Column C Adduct mass Spreadsheet outputs Four entries are made to spreadsheet sample 1 Column V Target adducts monoisot 1 lists the adduct name and the ID column A spreadsheet targets of that target compound to which the sample peak was matched as a potential adduct More precisely the sample peak was matched to one of the entries in the string contained in the target cell of column S spreadsheet targets i e generated in Tool 9 after matching to the first monoisotopic target peak 2 Column W Target adducts monoisot 2 lists the adduct name and the ID of column A spreadsheet targets of that target compound to which the sample peak was matched as a potential adduct More precisely the sample peak was matched to one of the entries in the string contained in the target cell of column AB spreadsheet targets i e generated in Tool 9 after matching to the second monoisotopic target peak 3 Column X Standard adducts monoisot 1 lists the adduct name and the ID column A spreadsheet internal standards of that internal standard compound to which the sample peak was matched as a potential adduct More
84. s on the installation and debugging of RExcel RExcel should run the workflow under the default settings These are set under Excel in the toolbar via RExcel gt Options Missing values Loose Dataframe name Workbook select DFVarnames as Rnames and Warning before overwrite and Activate Dataframe in Rcommander and Rcommander gets focus with output 4 Download the enviMass workflow from the resource webpage Open the enviMass 2010 xlms or enviMass 2003 amp 2007 xls file to run the target screening workflow under Excel 2010 or Excel2007 Excel2003 respectively The workflow is operated via the spreadsheet target_screening The user must at no point disable any Excel Visual Basic Macro functionalities i e the user must enable all spreadsheet contens 5 Calculation of isotopic patterns in enviMass is based on the R package isopat If being online while using the enviMass worksheet this package while be downloaded automatically at the appropriate workflow step enviMass version 1 0 User Manual 7 Alternatively isopat can be downloaded manually To do so a open the R version installed during step 3 b in the opened R GUI select packages gt install packages c a window with R mirrors pops up press OK which opens d a list of packages available at this mirror site e Within the list search for isopat select and click OK Check the FAQ section for further problem handling It also refers to startup
85. saccos TA ZA enviMass version 1 0 User Manual 2 Abstract The enviMass workflow supports screening high resolution mass spectrometry HRMS data for internal standards and target compounds and subsequent grouping of the remaining non target data Based on sample and a blank or blind HRMS peak lists enviMass provides tools for a the removal of noise data b blank blind data subtraction c mass recalibration screening for isotopic patterns of d internal standards and e target compounds f target quantification and g search for additional adducts of targets internal standards Subsequent steps incorporates h search for non target isotopic peak patterns and i search for potential non target adduct peaks Finally data are j summarized filtered and a non target candidate list is compiled Tools a to j can be conveniently run in a batch mode An additional tool allows simulation of profile isotopic fine structures for molecular formulas The procedure is implemented in an Excel VisualBasic setting that utilizes RExcel to make use of the R statistical environment and its packages Thus speed of calculation is strongly increased as compared to using Excel VisualBasic alone R and RExcel can be downloaded and installed free of charge All calculation steps are controlled via user interfaces and are embedded in a convenient and selfexplaining workflow Parameters workflow settings and underlying input data can be modifi
86. screening results written to the internal standards spreadsheet Calculations amp parameter settings The below steps are passed for each internal standard listed in the internal standards spreadsheet 1 In a very first step a match between a the most abundant peak of each internal standard isotopic pattern first entry in the strings of columns L and M spreadsheet internal standards and b a peak of the sample peak list is screened for within tolerance settings of Am z and ART Parameters for these tolerances have to be specified in the textboxes Am z and ART listed vs measured min respectively If recalibration results were accepted in Tool 7 recalibrated masses from column O instead of non recalibrated ones from column A of the sample spreadsheet are used Occasionally this first step can lead to several hits in the sample peak list depending on the tolerance settings In this case below steps 2 to 6 are passed for each of these hits leading to several result sets The routine outputs only the two best ones to the internal standards spreadsheet based on score 1 2 Thereupon internal standard peaks with too low abundances are identified Provided that the above named single peak hits a vs b were found the abundances taken from column M spreadsheet internal standards are rescaled to the intensity of the sample peak b Isotopic pattern peaks of the internal standard w
87. spreadsheets sample and blank and 2 the results in the spreadsheets samples filtered and non targets and 3 all graphs pasted into the workflow enviMass version 1 0 User Manual 10 Tool 1 Data Upload Description Command button Start workflow resets the workflow so that it can be started anew i e all downstream Tools are reset to operation mode This embraces the deletion of 1 all graphs and tables contained in the workflow and 2 the data contained in the blank blind input and all output spreadsheets Command Load sample peak list allows upload of a text file containing peak lists of a sample data set to be screened Similarly command button Load blank peak list allows upload of a text file with a peak list of blank or blind data In case no blank blind data are available the latter step can be skipped For input formats of these text files check above section Input data formats Spreadsheet inputs None Spreadsheet outputs Spreadsheets sample blank samples filtered and non targets are cleared New lists of sample data and blind blank data are written to spreadsheets sample and blank Calculations amp parameter settings None Data upload E matze al X uu gt Tos ER B 110124new_RUS_pos_15_blind 2 110124new_RUS_pos_57_Momentanprobe My Recent 3 110124new_RUS_pos_60_Momentanprobe 50 l Documents Desktop IN Load bl
88. standards no quantification is possible and an error message is printed to columns S AB spreadsheet targets or column Q spreadsheet internal_ standards For example let peak for quantif be set to 3 If an Isotopic hits ID string such as 2036 2040 only refers to two peaks of the sample peak list no third peak would be available for quantification enviMass version 1 0 User Manual 44 Tool 11 Adduct search for targets internal standards Description The internal standard and target adducts which are screened for in Tools 8 and 9 are appointed in Tool 3 during the isotopic pattern generation For example let Tool 3 use the adduct H default hydrogen to calculate the isotopic patterns of the target compounds and internal standards As a consequence the screening Tools 8 and 9 using these isotopic patterns screen for hydrogen adducts However other potential adducts than the one appointed in Tool 3 may be expected Based on the Tool 8 and 9 screening results Tool 11 allows to search for such potential adducts in the sample peak list Note This tool screens for adducts other than the one s defined via Tool 3 for isotopic pattern generation over ALL targets and internal standards listed in the spreadsheets targets and internal standards having a sufficiently high screening sum score If you only want to screen for adducts for PARTICULAR targets and internal standards include them
89. t first be installed on R server when running the isotopic pattern simulators While runing the isotopic pattern simulator R could not find the package isopat Either the package was simply not installed in that case redo point 6 of the installation instructions Eventually You have several R versions on the computer and You have simply installed the package to the wrong version To find out which R version is connected to RExcel go to Excel gt RExcel gt About RExcel I do not have a list of internal standards but only one for target substances Can I still use the workflow for screening purposes of these targets Sure Fill in a dummy data set of at least three internal standards in spreadsheet internal standards Then simply skip those tools whit internal standards being involved i e recalibration internal standard screening and target quantification Alternatively and if You want to use potential target compounds for recalibration which may be misleading unless You can be sure that the majority of targets can indeed be found in Your sample peak data set insert the list of target compounds not in spreadsheet targets but in spreadsheet internal standards Put differently use Your targets as if they were internal standards I have run a tool using specific parameter settings and want to rerun it now for comparison under other settings Can I do that Yes You can rerun most tolls several times under different setti
90. t mass b and 4 electron mass es of the charge associated with adduct b to search for a peak B This is repeated for all adduct masses b and associated charges selected in the adduct list box of the Tool interface Figure 26 over all peaks A listed in the sample peak list Candidate peaks B are then marked in the sample peak list cp above paragraph on spreadsheet outputs Defaults 1 and 2 constitute the parent adduct composition This parent adduct corresponds to those settings used for isotopic pattern generation for internal standards in Tool 3 For example if Tool 3 calculates internal standard isotopic patterns using the adduct H default and charge 1 i e positively ionized 1 the mass of a hydrogen atom and 2 the mass of an electron is added to peak A before calculating the mass of any peak B via above steps 3 and 4 enviMass version 1 0 User Manual 51 Moreover the electron mass es to be added or subtracted for step 4 have to be defined in column G of the adducts spreadsheet and are listed in the adducts list box of Tool 13 Tolerances in Am z and RT for pairing sample peaks A and candidate B have to be specified in the interface text boxes Am z and ART within scan min respectively Figure 26 Adduct search non targets non int stand Finished Number of adducts found 675 charge 1 charge 1 H charge 1 formiate charge 1 NH_4 charge 1 H d
91. targets are rescaled to the intensity of the sample peak b Isotopic pattern peaks of the target compound with a rescaled abundance i e an expected intensity lower than the value specified in text box Intensity cutoff default 5000 are then omitted from further considerations This step avoids to falsely screen for expected target compound peaks which cannot surmount the detection noise thresholds intrinsic to the sample peak list data set For example consider a a target compound isotopic pattern with abundances 1 0 5 0 25 0 1 and b a matched sample peak with intensity column B spreadsheet sample 50000 Rescaling the target compound abundances to the sample peak intensity leads to the expected intensities enviMass version 1 0 User Manual 41 50000 25000 12500 5000 Thus for an exemplary intensity cutoff 15000 only the first two target compound peaks with expected intensities 50000 25000 would be screened for 3 The rescaled i e reduced isotopic pattern peak set of a target compound is screened for in a third step While the tolerance parameter for Am z is still taken from the textbox Am z a different tolerance parameter for ART then the one of point 1 is now used namely the one from text box A RT within scan min The former tolerance in RT determined the search for the most abundant peak of a target compound under point 1 based on the RT listed for each target compound col
92. tion section Subsequently reading the section about data input formats and data upload via Tool 1 seems apposite Afterwards the user may step from tool to tool reading the tool sections of this manual Each tool section has a tool description and gives clarification about in and outputs as well as the required recommended parameter settings Alternatively the user may continue after installation with the example workflow which is based on the contents of the example folder found on the source website The example folder already includes example lists of sample and blank peaks and a enviMass version 1 0 User Manual 4 workflow with target and internal standard compounds and exemplary parameter settings Figure 1 next page flowchart representation of the screening workflow Green input data stored in spreadsheets Blue input data read from text files Yellow output spreadsheet lists enviMass version 1 0 User Manual 5 sample peak list u data upload HE blank peak list blind peak list i isotope data isotopic pattern generation for internal standards targets data upload match internal standards targets resolution data batch mode data modification o NC list of internal standards e Screening E E filtered sample peak lit preparation of non target list with candidate non target peaks list filteri ng
93. ts respectively For example consider a target compound and an internal standard the latter being represented by an isotopically labeled molecule identical to that of the target The default peak to be used for quantification for both the target and its internal standard is the most abundant mostly monoisotopic one listed in the cells of columns N and O targets and columns L and M internal standards i e peak index 1 From labeling with heavier isotopes the internal standard isotopic peaks are shifted towards higher masses relative to those of the target Thus there is no overlap for the target monoisotopic peak with any of those isotopic peaks of the internal standard pattern For the internal standard however the most intensive peak may indeed overlap with the target pattern enviMass version 1 0 User Manual 21 Hence column O in the internal standard list does not list peak for quantif 1 but the error message Target monoisotopic peak overlaps with standard The user is hence requested a to check for error messages in these columns b to set the index for the peak for quantif to another value and c to rerun the tool 3 If no peaks remain after the match e g for two identical substances 1 an error message string is printed to the concerned cells in columns omit peak and peak for quantif and 2 the cell of columns use for screening column M of the target and column K
94. ts with peaks rows of the sample peak list The results can be found in columns S and AB of the target spreadsheet and in columns Q and Z of the internal_ standards spreadsheet they contain the IDs of the sample peaks matched Adduct search is now conducted via these sample peaks matched For example the routine uses the ID string 3849 3818 3787 target column S to extract three peaks from the sample spreadsheet via the therein listed ID of column U sample ID provided the sum score in column Z is higher or equal to the value specified in Tool 11 text box score threshold The routine then a subtracts the adduct mass used for calculating the target isotopic pattern from each of these sample peaks and b enviMass version 1 0 User Manual 47 adds masses for the adducts searched for in Tool 11 The resulting masses are then used for screening the sample peak list for additional adduct peaks The adducts to be used in Tool 11 have to be selected from the interface list box cp Figure 24 Furthermore the adduct screening requires tolerance settings from two interface text boxes for Am z and A RT within scan min Text box score threshold allows to exclude targets and internal standards from the adduct search if their screening sum score columns Al or Z of spreadsheet targets columns X or AG of spreadsheet internal_ standards is below the value defined in that text box env
95. ttern could be matched to If no matches were found the entry value is set 0 2 Columns Q and Z Isotopic hits 7 list the ID number s from column R sample ID for standard screening sample spreadsheet of all those sample peak s to which the rescaled internal standard isotopic pattern could be matched to In other words not only the most abundant peak as in point 1 is listed here but all those sample peaks referred to by score That is if score 1 states e g 4 of 6 6 count of expected internals standard peaks after rescaling four peaks are listed The first ID number in the string i e the most abundant one is identical to the one s listed under point 1 3 Columns R and AA delta m z ppm list the accuracy in Am z between matched sample peaks cp column A sample spreadsheet and expected peaks cp column L internal_ standards spreadsheet i e Am z measured expected for all those peaks listed under point 2 4 Columns S and AB delta RT list the accuracy in RT between matched sample peaks cp column E sample spreadsheet and expected peaks cp column E internal standards spreadsheet i e ART measured expected for each of those peaks listed under point 2 5 Columns T and AC delta intens list the accuracy in intensity between matched sample peaks cp column B sample spreadsheet and expected peaks after rescaling of column M
96. umn E spreadsheet targets However a second preferably narrower RT is now used for screening the other peaks of the rescaled isotopic pattern set of peaks specified in text box A RT within scan min The reason being if a peak of the sample peak list indeed represents a monoisotopic peak of a target compound all other sample peaks representing the rescaled target compound isotopic pattern should eluate at the very same retention time 4 Given the matched set of sample peaks the above scores 1 to 3 are calculated The parameter for tolerance in intensity has to be specified in text box Intensity 5 An overall score is calculated as weighted sum of the sores 1 to 3 The weights have to be assigned by the user through the three lowermost textboxes of Figure 21 6 Accuracies in Am z ART and intensity are calculated and plotted They may help to identify outliers from screening of the target compound list 7 Note two cases exist for which NOT the monoisotopic peak is used in step 1 Firstly the routine omits target compound peaks overlapping with those of targets cp Tool 4 In that case the most abundant target compound peak not omitted is utilized for point 1 Similarly all other peaks of the target compound not omitted are used in points 2 to 5 Secondly rare cases exist under which not the monoisotopic peak but another isotopologue has highest abundance for a target compound e g C6Cl6 with m
97. un time error 424 Object required When pressing End the calculation stops when pressing Debug a section of the VBA code referring to a R connected process e g RInterface RRun is highlighted Go to Visual Basic for Application Editor Excel Tab Developer gt Visual Basic open Tools gt References and check the Available References to RExcelVBAlib Should there be no such reference re install RExcel If there are two such references prefer the one pointing to a x a file over that pointing to a xlma file under Excel lower than 20 0 and vice versa for Excel 2010 Excel freezes while running a tool of the workflow Wait three minutes Excel and RExcel might still be in the process of calculation and thus do not react If that does not help open the task manager press control alt delete therein open the Processes sheet highlight StatConnectorSrv exe and stop the latter via End Process Resume Your calculation in the Exce workflow and retry running the concerned tool enviMass version 1 0 User Manual 70 9 10 11 12 13 14 15 When opening the workflow an error messages appears indicationg that no connection to R could be established Have any of the Rexcel components been modified Any of the Rexcel settings In any case de and reinstall all RExcel components and try again I have followed the above installation instructions Nonetheless I receive the error message Package isopat mus
98. ure enviMass version 1 0 User Manual 30 16 In this case mass differences are derived from the differences of the values in columns C and D of the named spreadsheet Beware If the matches do not cover the m z range of the sample peak list masses or too few matches have been found an error message is printed and the recalibration procedure must either be skipped via command button Skip recalibration or a rerun with other tolerance settings may be attempted 3 Aided by the graphical outputs the user must finally decide whether to accept or reject the recalibration results using the command buttons Accept recalibration results or Reject recalibration results If accepted all subsequent calculations of the workflow will be based on recalibrated masses non recalibrated standards T Q amp S E 3 8 g H S E 3 E g Q o spline fit T T 700 Figure 18 Third result plot from the recalibration tool Depicted are deviations in mass between theoretical values calculated from the molecular formula of internal standards and matches found for these internal standards in the sample peak list ordinate ppm units plotted against the mass of these internal standards abscissa Da unit for mass m z 1 The green line shows the model predictions for the relation deviation as function of mass which is used for mass recalibration Upper and lower dashed lines separate the highest and lowest 5 of the data giv
99. urviving peaks OR c profile centroid peaks are written to the target or internal standard lists for the settings Pattern Profile or Centroid respectively these peaks are optionally RBPF filtered for cases b and c General settings For the first mandatory step settings have to be specified in the General Settings interface Figure 5 enviMass version 1 0 User Manual 14 Output options Pattern L Profile Filter by RBPF L e Filter by RBPF I Figure 4 Output options for the isotopic pattern calculation Centroid um General Settings I Form adducts Charge 1 i Abundance limit 1E 10 LH default 1E 10 oO D Abundance cutoff 1E 3 O formiate default 1E 3 O NH4 Update adduct list Figure 5 General settings for the isotopic pattern calculation Charge defines the ionization state of the compounds cp section Spreadsheet inputs Point 3 For no charge set to 0 Use a minus sign for negative charge Abundance limit defines the abundance threshold below which isotopic combinations in the molecule are not further permuted towards othercombinations within the isotopic pattern calculation algorithm The defaults is set to 1E 10 but much lower values should be used for molecules with elements having several isotopes of high abundance cp below section Algorithm for isotopic pattern calculation In contrast Abundance cutoff giv

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