Proteomics in ADME/Tox studies: Automated spot handling of
Contents
1. lication note Automated spot handling OGE approved Proteomics in ADME Tox studies Automated spot hand ling of peptides prior to MALDI and electrospray MS identification of proteins altered by drug treatment key words Ettan Spot Handling Workstation protein identification Ettan DIGE system sample preparation e MALDI ToF MS e LC MS MS This application describes the use of the Ettan proteomics platform to produce biologically significant proteomics data Samples from livers of mice either treated with a candidate drug or untreated were quantified labeled and subjected to 2 D gel electrophoresis Following 2 D gel scanning and imaging pick lists that included protein spots differentially regulated following treatment with the candidate drug were created for unattended reproducible spot handling on Ettan Spot Handling Workstation Subsequent mass spectrometry using MALDI ToF chemically assisted fragmentation MALDI and LC MS MS resulted in confident identification of 100 of the proteins showing at least 50 changed level after treatment with the drug candidate Introduction It is estimated that about 50 of drugs in development fail during clinical trials because of deficiencies in their ADME Tox absorption distribution metabolism elimination and toxicity properties 1 The cost of these failures so late in the drug development process is naturally very high In addition it has been sugge2. turning chemicals into drugs Nature Biotechnology 19 722 726 2001 overall investigation The picklist generated by DeCyder 2 Ettan DIGE User Manual Amersham Biosciences 18 1164 40 edition AA rat liver protocol p 299 2001 3 Ettan DIGE User Manual Amersham Biosciences 18 1164 40 edition AA p 39 40 Differential Analysis Software allowed automatic processing of all selected protein spots and made them ready for 2001 analysis by MS techniques All proteins regulated following 4 Ettan DIGE User Manual Amersham Biosciences 18 1164 40 edition AA p 94 treatment with the candidate drug were thus successfully 2001 C 5 Ettan DIGE User M Amersham Biosciences 18 1164 40 edition AA p 59 64 detected selected prepared and identified in a smooth pona Gia nvm eg te ES nel accurate and highly efficient manner For more information or to shop online visit www amershambiosciences com The identified proteins were grouped in different functional categories and included proteins that may be involved in the to order Asia Pacific Tel 852 2811 8693 Fax 852 2811 5251 Australia Tel 61 2 9899 0999 Fax 61 2 9899 7511 Austria Tel 01 57 606 16 19 Fax 01 57 606 16 27 Belgium Tel 0800 73 888 Fax 03 272 1637 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 Central East South East Europe Tel 43 1 982 3826 Fax 43 1 985 8327 Denmark Tel 45 16 2400 Fax 45 16 2424 Finland amp Baltics Tel 358 0 9 512 3940
3. in automatic mode with internal calibration using trypsin To further improve the identification rate chemically assisted fragmentation MALDI was used on proteins not successfully identified by PME This technique enables peptide sequence data to be acquired simply quickly and with good sensitivity by analyzing the CAF labeled peptides in post source decay PSD mode on Ettan MALDI ToF Pro Finally the few spots that were still not identified using MALDI MS were subjected to LC MS MS analysis The tandem mass spectrometric analysis was performed on Finnigan LTQ linear ion trap mass spectrometer Thermo Electron Corporation fitted with Biobasic C18 column 100 x 0 1 mm Thermo Electron Corporation running at 1 pl min flow rate A fast gradient profile enabled a total analysis time of 20 min during which approximately 5500 scans were acquired using the data dependent triple play routine In this mode peptides detected in a survey scan are selected for high resolution Zoom Scan followed by fragmentation by MS MS The spectra were then processed automatically by SEQUEST to get unambiguous identification based on peptide sequence contained in the product ion spectrum The built in features of data dependent acquisition and dynamic exclusion allows automated collection of useful data and enables efficient handling of coeluting peptides ensuring maximum sensitivity and sequence coverage for identified proteins Results
4. 8 6 90 x 1019 5 86 72638 12 PMF 1 68 Liver carboxylesterase precursor 2494382 3 10x 108 5 88 60406 13 PMF 1 90 T complex protein 1 22654291 3 40 x106 5 97 57477 14 PMF 1 56 3 hydroxy 3 m ethylgl utaryl Coenzyme A synthase 1 20988709 4 70x10 5 65 57569 15 PMF 2 25 selenium binding protein 2 9507079 3 40 x 1010 5 78 52628 16 PMF 1 59 3 hydroxy 3 m ethylgl utaryl Coenzyme A synthase 2 12836439 0 0025 8 65 56823 17 PMF 1 66 3 hydroxy 3 m ethylgl utaryl Coenzyme A synthase 2 20965433 5 30 x 106 8 65 56823 18 PMF 1 61 methionine adenoyyl transferase 19526790 1 70 10 5 51 43509 19 PMF 2 02 methionine adenosyltransferase 19526790 8 10 x 101 5 51 43509 20 CAF 2 06 3 hydroxy 3 methyl glutaryl Coenzyme A synthase 2 12836371 2 80 x108 7 56 56221 21 CAF 1 87 gamm a actin 20885782 0 064 5 14 50041 22 PMF 1 68 cathepsin D 26354406 3 80 x106 6 85 48374 23 PMF 1 64 farnesyl diphosphate synthetase 19882207 3 00 x 10 5 48 40582 24 MS MS 2 09 hepatoma derived growth factor 31560691 0 1 4 52 40202 25 PMF 2 22 nudix 12847124 9 80 x 10 5 97 24603 26 PMF 2 02 thioether S methyltransferase 6678281 1 60x 1079 6 29460 27 PMF M S MS 1 75 thioether S methyltransferase IPP isomerase 1 6678281 13878548 2 40x10 5 82 23239 28 CA F M S MS 2 21 peroxiredoxin 4 1 PP isomerase 1 7948999 13878548 7 00x 109 6 34 22078 29 PMF 1 58 Major urinary proteins 11 and 8 127531 5 40 x 107 4 85 17560 30 PMF 1 54 Major urinary proteins 11 and 8 127531 0 0022 4 85 17560 V
5. Fax 358 0 9 512 39439 France Tel 0169 35 67 OO Fax 0169 41 9677 Germany Tel 0761 4903 490 Fax 0761 4903 405 Italy Tel 02 27322 1 Fax 02 27302 212 Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 Middle East Tel 30 2 10 96 00 687 Fax 30 2 10 96 00 693 Netherlands Tel 0165 580 410 Fax 0165 580 401 Norway Tel 2318 5800 Fax 2318 6800 Portugal Tel 21 417 7035 Fax 21 417 3184 Russia amp other C 1 S amp N I S Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 South East Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 Spain Tel 93 594 49 50 Fax 93 594 49 55 Sweden Tel 018 612 19 00 Fax 018 612 19 10 Switzerland Tel 0848 8028 12 Fax 0848 8028 13 UK Tel 0800 616 928 Fax 0800 616 927 USA Tel 1 800 526 3593 Fax 1 877 295 8102 CAF Cy CyDye DeCyder DeStreak Ettan Immobiline and Typhoon are trademarks of Amersham Biosciences Limited Amersham and Amersham Biosciences are trademarks of Amersham plc 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE technology is covered by US patent numbers US6 043 025 and US6 127 134 and foreign equivalents and exclusively licensed to Amersham Biosciences CyDye This product or portions thereof is manufactured under licence from Carnegie Mellon University under patent number 5268486 and other patents pending Some of these products may only be available to collaborators and customers within certain of our technology access programmes The p
6. alue above p 0 01 The identity was however confirmed by CAF labeling and MS MS respectively an 11 0004 19 AA 2004 03 p5 Automated spot handling Conclusions We have investigated the suitability of a highly automated proteomics approach to identify differential expression levels xenobiotic metabolism of drugs as well as proteins that might be of importance in toxicity Further work using narrower and more basic IPG strips to of proteins indicated in ADME Tox processes Such resolve more proteins on 2 D gels is under way In addition information should lead to the discovery of useable a focused study on proteins obtained by subcellular biomarkers signaling drug toxicity which would exclude the fractionation could prove to be very fruitful for further candidate drug from further development identification of proteins involved in metabolism and This study showed the value of combining products from toxicity in the liver following administration of drugs the Ettan range for sample preparation differential gel Greater understanding of these processes might significantly electrophoresis by 2 D DIGE automatic spot handling and improve ADME Tox investigations during drug discovery finally the identification of proteins by a combination of campaigns MALDI ToF PMF MALDI ToF PSD of CAF labeled peptides and LC MS MS References Ettan Spot Handling Workstation played 4 central role in the 1 Hodgson J ADMET
7. and discussion As expected when preparing samples from an organ like the liver the protein concentration in the four lots was about 10 mg ml treated 1 10 02 mg ml treated 2 9 67 mg ml treated 3 10 23 mg ml pooled untreated control 10 40 mg ml i e 1 100 of the prepared sample was protein This points to a very high recovery since about 1 10 of organs should consist of protein and the 0 5 g samples were diluted 10 times in 5 ml lysis buffer After 2 D gel electrophoresis and image analysis DeCyder software fully matched 1450 spots between the three different images for each gel Figure 2 shows the matched spots Automated spot handling Fig 2 Protein spots 1450 matched on the 2 D gel by DeCyder Differential Analysis Software A picklist of 552 spots was generated and automatically transferred to Ettan Spot Handling Workstation Of these 1450 protein spots 30 spots were found to be differentially regulated A total of 552 spots including the 30 diferentially regulated spots were selected as a reasonable test for automatic spot handling on the workstation which can handle a maximum of 1152 samples from up to twelve gels per batch The picklist was thus generated and automatically transferred to Ettan Spot Handling Workstation which used it to pick digest and spot all 552 proteins again with full automation and no manual intervention All 552 spotted proteins gave PMF spectra following preparation in Ettan Spo
8. iiis Fig 5 Product ion spectrum of peptide AFSVFLFNTENK isopentenyldiphosphate delta isomerase from mouse liver abbreviated to IPP isomerase in Table 1 on rows 27 and 28 with highlighted b and y ion series fragments detected in the Table 1 Protein identification results listing ID method protein name ID number NCBI database and relevant biochemical information The protein whose spectrum is shown in Figure 4 is found at position 8 in this list Average ratio shows extent of regulation negative values indicate down regulation positive values indicate up regulation ID method Average Name Protein ID Student s pl theo Mol Ratio NCBI nr db t test p retical weight 1 PMF 1 59 carba moyl phophate synthetase 1 8393186 3 70 x 107 6 33 164580 2 PMF 2 85 carba moyl phosphate synthetase 1 23621369 9 00 x 108 6 03 116816 3 PMF 2 65 carbamoyl phogphate synthetase 1 8393186 1 30x 108 6 19 116273 4 PMF 1 55 pyruvate carboxyl ase 7438124 1 10x 108 6 34 129777 5 PMF 1 74 pyruvate carboxylase 7438124 1 40x 106 6 34 129777 6 MS MS 1 54 ornithine transcarbamyl ase 762985 9 50 x 108 7 25 108595 7 PMF 1 69 gmilarto elongation factor 2 26328763 1 20 10 6 31 95257 8 PMF 1 81 Formyl transferase 25050159 9 40 x 10 5 64 99064 9 PMF 1 61 Formyl transferase 25050159 2 50x 101 5 64 99064 10 PMF MSMS 2 75 ubiquilin 1 serine proteinase inhibitor 20072434 15079234 0 0073 4 73 74813 11 CAF PMF 1 84 esterase 31 carboxylesterase 2 20886287 1952717
9. n Spot Handling Workstation A method that included spot picking digestion and spotting on Ettan MALDI target slides was selected in the software and the whole procedure was run automatically overnight without any manual intervention either within or between steps In this automated procedure gel plugs were cut by a 2 mm picking head and washed twice in 50 methanol and 50 mM ammonium bicarbonate and once in 75 acetonitrile before drying For digestion 10 pl trypsin solution 0 2 pg Trypsin Sequencing Grade Ettan Chemicals was added before incubation at 37 C for 2 h Extraction was performed in two steps by addition of 50 acetonitrile and 0 1 Trifluoroacetic Acid Ettan Chemicals The pooled extract was finally dried prior the two step spotting procedure where the matrix solution of 5 mg ml solution of recrystallized a cyano 4 hydroxy cinnamic acid LaserBio Labs in extraction liquid was deposited on the target In the final step before MALDI ToF a tenth of the dissolved sample was mixed with the matrix an 11 0004 19 AA 2004 03 p3 layer on the target leaving the rest for other analyses e g chemically assisted fragmentation MALDI and LC MS MS Protein identification using MALDI ToF chemically assisted fragmentation MALDI and LC MS MS Protein identification by peptide mass fingerprinting PMF was performed on Ettan MALDI ToF Pro Data acquisition spectrum processing and database searches were performed
10. ne pooled control each of three untreated livers were rinsed in PBS and homogenized in 10 volumes 5 ml lysis buffer 10 mM Tris HCl pH 8 3 7 M urea 2 M thiourea 5 mM magnesium acetate 4 CHAPS according to reference 2 To remove interfering nonprotein material 2 D Clean Up Kit was used on a 10 x 100 pl homogenate according to the kit instructions 2 D Quant Kit was used to quantitate the prepared samples also according to the kit instructions Sample labeling Samples were thawed and their pH determined by pipetting 1 pl of each onto pH paper The pH was adjusted to between pH 8 0 and pH 8 5 by adding 2 pl of 50 mM NaOH to each 100 pl aliquot of homogenate One hundred microgram of each sample was labeled according to reference 3 Individual samples treated and untreated livers to be analyzed were labeled with CyDye DIGE Fluor Cy 5 minimal dye and CyDye DIGE Fluor Cy3 minimal dye respectively In addition a pooled standard containing all samples included in the experiment was prepared and labeled with CyDye DIGE Fluor Cy2 minimal dye The differently labeled individual samples and the pooled standard were mixed prior to 2 D electrophoresis 2 D gel electrophoresis First dimension Cup loading was selected for analytical electrophoresis One hundred and fifty microgram of the mixed sample 50 pg of each Cy2 Cy3 and Cy5 labeled sample on each gel was applied to each 24 cm Immobiline DryStrip pH 3 10 NL For prepa
11. osciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences KK Sanken Building 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them A copy of these terms and conditions is available on request Amersham Biosciences AB 2004 All rights reserved Amersham Biosciences an 11 0004 19 AA 2004 03 p6 Produced by Wikstro ms Sweden 1040029 03 2004 Printed matter Licence 341 051 WY UL
12. our 67 were successfully identified spectra not shown For the two remaining proteins an LC MS MS method using the Finnigan ProteomeX Workstation and LCQ Deca XP Plus Ion Trap Mass Spectrometer proved successful example shown in Fig 5 The LC MS MS approach was also used to confirm three identifications made by MALDI ToF and chemically assisted fragmentation MALDI Table 1 shows the identities of all 30 proteins up or down regulated by more than 50 following treatment with the candidate drug Picking accuracy was high The backing of the gels stabilizes their dimensions and a self adhesive label attached prior to scanning links the electronic image with the actual gel In addition the design and movement of the picked head ensured a clean and precise cut that also promotes high Automated spot handling picking efficiency The clean cut combined with hydrophobic coatings on the picking head and needles minimized cross contamination Carry over was not detected Fig 4 Spectrum of a protein analyzed by MALDI ToF following automated handling and preparation on Ettan Spot Handling Workstation The protein was identified as formyl transferase see Table 1 row 8 eee j LIT Liga hd mr 1 da 1300 a iy E r F 2 a a a f L al 0 oe ae l E sind Dea i a CaF ILESE Mea 2c 4 el AFORE spectrum CS SPM EES tits FP 2 yiip mpar bi ti
13. rative electrophoresis 1 mg samples were loaded onto 24 cm Immobiline Automated spot handling DryStrip gels pH 3 10 NL by in gel rehydration in DeStreak Rehydration solution 2 IPG buffer pH 3 10 The rehydration step was performed according to the user manual Second dimension Electrophoresis 2 W gel was performed overnight using lab cast gels 4 on Ettan DALTiwelve electrophoresis system according to the protocols in reference 5 The preparative gels were poststained by SYPRO Ruby according to the manufacturer s instructions Scanning Each gel was scanned at 100 um resolution on Typhoon 9410 Variable Mode Imager using 520BP40 Cy2 S580BP30 Cy3 and 670BP30 Cy5 emission filters Image analysis Images were analyzed using DeCyder Differential Analysis Software v5 0 in both the DIA and the BVA modules For statistical analysis two way ANOVA was used with the Student s t test value set to 0 01 1 5 times intensity Individual images were created for the different Cy2 Cy3 and Cy5 labeled gels and matched using DeCyder software Two preparative gels were added to the experimental study and parallel pick lists that included all spots of interest i e those differentially regulated following treatment with the candidate drug were created for subsequent spot handling prior to mass spectrometry Automated spot handling Selected proteins were subjected to fully automated spot handling in Etta
14. sted that over 5 of hospitalized patients still suffer serious adverse reactions to drugs that have successfully completed their development and come onto the market 1 Improved means of gathering ADME Tox information earlier in drug development should thus benefit pharmaceutical manufacturers and of course patients We have investigated whether a proteomics approach would identify differential levels of mouse liver proteins after treatment of mice with a candidate drug Such information could help elucidate which proteins are involved in metabolism and toxicity and thus increase the value of ADME Tox studies in drug discovery an 11 0004 19 AA 2004 03 pl Untreated sample Sample treated with candidate drug Label with CyDye DIGE Fluor Cy5 minimal dye y na A ar Perr y mix Vv v h y vy Yy y y y y v Ettan Spot Handling y Yy v Label with CyDye DIGE Fluor Cy3 minimal dye 2 D gel electrophoresis 7S a a a a A Da Da a Ba Ba Workstation Spot picking Addad a Digestion Chemically assisted MALDI ToF fragmentation MALDI PMF Fig 1 The experimental workflow used for the successful 100 identification of proteins selected by the criterion of displaying at least a 50 change in abundance after F LC MS MS treatment with the candidate drug In the experimental workflow a second gel was run where the labelling was reversed i e treated sample was labeled with Cy3 and
15. t Handling Workstation When expectation values lt 0 05 were collected 298 of these spots 54 were automatically identified Manual editing should increase this number significantly Figure 3 illustrates the results workflow and shows the methods and outcome of the protein identification an 11 0004 19 AA 2004 03 p4 Number of protein spots matched on the 2 D gel 1450 Yy Protein spots processed through Ettan Spot Handling Workstation 552 4a Proteins identified in automatic mode 299 52 hA hA Proteins found to be at least 50 up or down regulated 30 552 A Proteins identified by peptide mass fingerprinting 24 30 Yy Proteins identified by chemically assisted fragmentation 4 6 y Proteins identified by LC MS MS 212 iS Fig 3 Workflow from the initial matching of 1450 spots on the 2 D gels to the successful identification of all 30 mouse liver proteins up or down regulated by more than 50 following treatment with a candidate drug LC MS MS was used to confirm three identities made by the other two methods At this stage we decided to focus on identifying the regulated 30 proteins MALDI ToF analyses identified 24 of the 30 regulated proteins 80 directly by peptide mass fingerprinting Figure 4 shows the spectrum of one of these 24 proteins later identified as formyl transferase position 8 in Table 1 Six spots were analyzed by chemically assisted fragmentation MALDI of which f
16. the untreated sample labeled with Cy5 CyDye DIGE Fluor minimal dye respectively Amersham Biosciences Automated spot handling The Ettan design platform offers a complete solution of reagents kits and systems for proteomics based research at a stage before biological validation is feasible Central to the whole proteomics workflow is the Ettan Spot Handling Workstation a highly flexible and robust system that allows fully automated unattended low and or high throughput handling of protein spots separated by 2 D gel electrophoresis A key feature of the workstation is that it avoids the manual errors that normally have a negative impact on obtaining reproducible results Avoiding such errors is of critical importance since downstream biological validation of the data obtained is very labor intensive Every effort made to gather reproducible and accurate data at the proteomics experimental stage will pay dividends later In other words it is essential that the right spots are chosen and picked for downstream validation studies Figure 1 illustrates the full experimental workflow and key techniques used in the investigation which are described in more detail in the Methods section Products used Amersham Biosciences products used Ettan sample preparation kits and reagents 2 D Clean Up Kit 2 D Quant Kit DeStreak Reagent 2 D electrophoresis systems and consumables CyDye DIGE Fl
17. uor Cy2 minimal dye 25 nmol CyDye DIGE Fluor Cy3 minimal dye 25 nmol CyDye DIGE Fluor Cy5 minimal dye 25 nmol Immobiline DryStrip pH 3 10 NL 24 cm Ettan IPGphor II IEF System Ettan DALT twe ve Large Vertical Electrophoresis System Typhoon 9410 Variable Mode Imager DeCyder Differential Analysis Software Spot handling and mass spectrometry Ettan Chemicals Trypsin Sequencing Grade Ettan Chemicals Trifluoroacetic Acid Ettan Spot Handling Workstation Ettan MALDI ToF Pro Ettan CAF MALDI Sequencing Kit an 11 0004 19 AA 2004 03 p2 80 6484 51 80 6483 56 17 6003 19 RPKO272 RPKO273 RPKO275 17 6003 77 80 6505 03 80 6466 27 60 0055 81 18 1163 05 17 6002 75 17 6002 76 18 1164 05 18 1156 53 17 6002 97 Other materials required SYPRO Ruby stain Molecular Probes Alpha cyano 4 hydroxycinnamic acid LaserBio Labs Biobasic C18 column Thermo Hypersil Finnigan ProteomeX Workstation Thermo Electron Corporation Finnigan LTQ Linear lon Trap Mass Spectrometer Thermo Electron Corporation Finnigan LCQ Deca XP Plus lon Trap Mass Spectrometer Thermo Electron Corporation Methods Treatment of mice with candidate drug The selected candidate drug was administered orally to inbred C57BL 6 mice over five days Livers from treated mice and untreated littermates were removed dissected and snap frozen in liquid nitrogen Sample preparation and quantitation Sample 0 5 g of each liver three treated livers and o
18. urchase of CyDye Fluors includes a limited license to use the CyDye Fluors for internal research and development but not for any commercial purposes A license to use CyDye Fluors for commercial purposes is subject to a separate license agreement with Amersham Biosciences Finnigan LCQ LTQ ProteomeX SEQUEST data dependent acquistion and dynamic exclusion are trademarks of the Thermo Electron Corporation SYPRO is a trademark of Molecular Probes Inc Patent information Chemically Assisted Fragmentation CAF is protected by patent application WO 00 43792 with foreign equivalents as licensed from the Procter amp Gamble Company to Amersham Biosciences AB as well as WO 02 095412 and WO 02 095419 both co owned by the Procter amp Gamble Company and Amersham Biosciences AB The purchase of Ettan CAF MALDI Sequencing Kit includes a limited license to use the technology for research internal research and development but not for any commercial purposes No right to perform or offer commercial services or products of any kind using the Sequencing Kit is hereby granted A license to use the technology for commercial purposes is subject to a separate license agreement with Amersham Biosciences AB Amersham Biosciences AB Bj rkgatan 30 SE 751 84 Uppsala Sweden Amersham Biosciences UK limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA England Amersham Biosciences Corporation 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Bi
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