Protocol 2 - In-gel digestion for cell/tissues (+/- fractionation)

Transcription

1 Protocols General information After you have made an experimental design for your proteomics experiment (please check Overview and Analysis options in the Proteomics services section for information) you can start your sample preparation using our protocols. Each protocol lists required reagents and consumables. For internal customers (Radboud University Medical Center, Radboud University) we can provide certain reagents and/or consumables (Digestion enzymes Trypsin and LysC, C18 OMIX tips, Detergent removal column). If you want to make use of this option please fill out a sample submission form and contact us via to pick up your order, of course you can also order them yourself. Below you find the 5 different sample preparation protocols to prepare your samples: Protocol 1 - In-solution digestion for cells/tissues Protocol 2 - In-gel digestion for cell/tissues (+/- fractionation) Protocol 3 - In-solution digestion for immunopurifications/affinity purifications/purified proteins Protocol 4 - In-gel digestion for immunopurifications/affinity purifications/purified proteins Protocol 5 - In-solution digestion protocol for human body fluids (plasma, serum, CSF) 1 V1.0 8/10/2016

2 Protocol 1 - In-solution digestion for cells/tissues Sample requirements and precautions Preferred amount of sample is 10 μg of total protein Samples are not radioactive Samples are detergent-free (e.g. Triton X-100, PEG-44) Salt concentrations should be less than 500 μm Glycerol concentration should be less than 2.5 % No organic solvents present Special care must be taken to avoid contamination with keratins from skin or hair (wear gloves, lab coat at all times and clean equipment vigorously) Equipment Recommended: 2D-Quant kit for protein concentration determination (# GE Healthcare) Recommended: Sample Grinding kit ( , GE Healthcare) Pierce detergent removal spin columns, 1 column/sample: article# Agilent Bond Elut C18 Omix tips, 1 tip/sample, article# A Micro centrifuge suitable for 1.5 ml reaction vials Incubator for digestion at 37 C Adjustable pipettes Speedvac Recommended Reagents Name Supplier Article number 2-chloroaetamide (CAA) Sigma G-F Urea GE Healthcare Dithiotreitol (DTT) Sigma D9163-5G Trishydroxymethylaminomethane (Tris) GE Healthcare Lysyl endopeptidase C (LysC) WAKO Chemicals Sequencing grade modified trypsin C=0.5μg/μl Promega V Water, Milli-Q grade - - Ammonium bicarbonate (ABC) Sigma A-6141 Trifluoroacetic acid (TFA) Pierce 9470 Acetonitrile HPLC-S grade (ACN) Biosolve Formic Acid (FA) Merck Solutions Adjust volumes if necessary mm Ammonium bicarbonate (ABC) Dissolve 200 mg in 50 ml Milli-Q. Prepare fresh, discard remaining solution mm dithiothreitol (reduction buffer) Dissolve 7.7 mg in 5 ml Milli-Q. Prepare fresh, discard remaining solution mm 2-chloroacetamide in 50 mm ABC (alkylation buffer) Dissolve mg in 5 ml solution 1. CAA is light sensitive so store the solution in the dark. Prepare fresh, discard remaining solution. 2 V1.0 8/10/2016

3 4. 10 mm Tris ph 8.0 Dissolve mg Tris in 45 ml Milli-Q. Adjust to ph 8.0 with HCl. Adjust volume to 50 ml with Milli-Q. Store 10 ml aliquots in -20 C up to 1 year. 5. 8M Urea in 10 mm Tris ph 8.0 Dissolve gram urea in 50 ml solution 4. Prepare fresh, discard remaining solution. 6. 2% Trifluoroacetic acid Dilute 1 ml TFA with 49 ml Milli-Q. Store in glass, shelf life 12 months μg/μl Lysyl endopeptidase C (LysC) If no aliquots are available, make aliquots: Dissolve the stock-vial of Lys-C to a final concentration of 0.5μg/μl in buffer A The amount of Lys-C in the vial can be found in the certificate of analysis. If not available, this can be ordered via mail from Wako (see Wako website for contact details). After diluting, prepare aliquots of 50 or 100 μl and store these in -80 C. Using an aliquot: after first use, store in -20 C for further usage (maximally: 1 year). 8. Buffer A: 0.1% FA in H 2 O (Milli-Q) 50 μl FA filled up till 50ml with Milli-Q. 9. Buffer B: 0.1% FA in ACN 50 μl FA filled up till 50ml with ACN. Procedure Digestion 1. Lyse cells using the grinding kit, sonication or snap freezing. (In our experience the grinding kit gives the best results using 8M urea/10mm Tris as lysis buffer) 2. Determine protein concentration (In our experience the 2D-Quant kit gives the best results) 3. We recommend to use 10 μg of total protein for digestion. 4. Dilute samples 1:1 with 8 M urea/10 mm Tris ph 8.0 (solution 5). For optimal digestion of the sample, the final urea concentration should be 4 M and the ph should be near ph Add 1 μl reduction buffer (solution 2) for every 50 μg sample protein and incubate 30 min at room temperature. ( In this procedure all steps prior to digestion are done at room temperature to reduce unwanted derivatization of amino acid side-chains by the denaturants) 6. Add 1 μl alkylation buffer (solution 3) for every 50 μg sample protein and incubate 20 min at room temperature in the dark. 7. Add 1 μg LysC/50 μg total protein and incubate for at least 3 hours at room temperature. 8. Dilute sample 4x with 50 mm ABC (solution 1). 9. Add 1 μg trypsin/50 μg total protein and incubate overnight at 37 C. 10. After incubation, spin down the water droplets condensed inside the lid of the test tube. 11. Proceed to Sample cleanup. Sample cleanup For more detailed information see the Pierce Detergent Removal Column manual 1. Remove bottom closure from Pierce Detergent Removal Column and loosen cap (do not remove cap). 2. Place column into a collection tube. 3. Centrifuge at 1500 x g for 1 minute to remove storage solution, do not exceed the indicated speed! 3 V1.0 8/10/2016

4 Note: When using fixed-angle rotors, place a mark on the side of the column where the compacted resin is slanted upward. Place column in centrifuge with the mark facing outward in all subsequent centrifugation steps. Improper orientation will result in reduced detergent removal efficiency. 4. Add 400 μl 50 mm ABC (solution 1) and centrifuge for 1 minute at 1500 x g. Discard the buffer and repeat this step two additional times. 5. Place column in a new 1.5 ml collection tube. Slowly apply sample (max 100 μl) to the top of the compact resin bed and incubate for 2 minutes at room temperature. (If sample volume exceeds 100 μl use a speedvac to concentrate or apply sample to more columns and pool afterwards) 6. Centrifuge at 1500 x g for 2 minutes to collect the detergent-free sample. Discard the used column. 7. Proceed to Sample desalting and concentration. Sample desalting and concentration For more detailed information see the OMIX tips manual. Maximum loading capacity is 10 μg! Divide sample over more tips if necessary. 1. Dilute sample 1:1 with 2% TFA (solution 6). 2. Prepare Omix tip (1 tip/sample): Aspirate 100 μl buffer B and discard solvent, repeat 1x Aspirate 100 μl buffer A and discard solvent, repeat 1x 3. Aspirate up to 100 μl sample, dispense and aspirate 5 times then discard liquid. If the sample volume is more than 100 μl: repeat until all sample has been passed through the tip. 4. Aspirate 100 μl buffer A and discard solvent, repeat 1x. 5. Aspirate 100 μl buffer B and dispense in a new collection tube. Discard the OMIX tip. 6. Speedvac sample to a volume of 2 μl. 7. Add buffer A to obtain a total volume of 20 μl. 8. Store sample in -20 C until shipment. Related literature Kinter, M., and Sherman, N. E. 2000, Protein sequencing and identification using tandem mass spectrometry. JohnWiley & Sons, Inc. pp Nielsen ML, Vermeulen M, Bonaldi T, Cox J, Moroder L & Mann M, Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nature Methods 2008, 5 Pierce Detergent Removal Column manual, available from the Pierce website or in the package containing the columns Agilent Bond Elut Omix C18 manual, available from the Agilent website or in the package containing the tips 4 V1.0 8/10/2016

5 Protocol 2 - In-gel digestion for cell/tissues (+/- fractionation) Sample requirements and precautions Load 20 μg (protein) sample on gel for fractionation Samples are not radioactive Special care must be taken to avoid contamination with keratins from skin or hair (wear gloves, lab coat at all times and clean equipment vigorously) Do NOT use silverstaining! Only coomassie! Only cut gels on a clean glass plate Use a new scalpel blade Equipment Recommended: 2D-Quant kit for protein concentration determination (# GE Healthcare) Recommended: Sample Grinding kit ( , GE Healthcare) Recommended: BioRad Laemmli Sample Buffer 2x or 4x ( , , BioRad) Recommended: Mini-Protean 12% TGX pre-cast gel ( , Bio-Rad) Recommended: Colloidal Coomassie Blue stain ( , Severn Biotech) Agilent Bond Elut C18 Omix tips, 1 tip/sample, (article# A ) Micro centrifuge suitable for 1.5 ml reaction vials Incubator for digestion at 37 C Adjustable pipets Speedvac Reagents Name Supplier Article number 2-chloroaetamide (CAA) Sigma G-F Dithiotreitol (DTT) Sigma D9163-5G Sequencing grade modified trypsin C=0.5μg/μl Promega V Water, Milli-Q - - Ammonium bicarbonate (ABC) Sigma A-6141 Trifluoroacetic acid (TFA) Pierce 9470 Acetonitrile HPLC-S grade (ACN) Biosolve Formic Acid (FA) Merck Solutions Adjust volumes if necessary mm Ammonium bicarbonate (ABC) Dissolve 200 mg in 50 ml Milli-Q. Prepare fresh, discard remaining solution mm dithiothreitol (reduction buffer) Dissolve 7.7 mg in 5 ml Milli-Q. Prepare fresh, discard remaining solution mm 2-chloroacetamide in 50 mm ABC (alkylation buffer) Dissolve mg in 5 ml solution 1. CAA is light sensitive so store the solution in the dark. Prepare fresh, discard remaining solution. 4. 2% Trifluoroacetic acid Dilute 1 ml TFA with 49 ml Milli-Q. Store in glass, shelf life 12 months. 5 V1.0 8/10/2016

6 5. 12,5 ng/μl trypsin in 50 mm ABC (Digestion buffer) Dilute 1 μl trypsin with 39 μl 50 mm ABC. Alternatively, for concentrated samples in a small total gel volume, 100 ng trypsin/sample in 50 mm ABC can be used. Use immediately. 6. Buffer A: 0.1% FA in H 2 O (Milli-Q) 50 μl FA filled up till 50 ml with water. 7. Buffer B: 0.1% FA in ACN 50 μl FA filled up till 50 ml with ACN. Procedure Digestion All volumes are for reference purposes only; please adjust the volumes so that all gel particles are covered with liquid. 1. Lyse cells by use of the grinding kit, sonication or snap freezing. (In our experience the grinding kit gives the best results using 8M urea/10mm Tris as lysis buffer) 2. Determine protein concentration. (In our experience, the 2D-Quant kit gives the best results) 3. We recommend to load 20μg sample for fractionation, or 10μg if no fractionation is needed. Add Laemmli sample buffer to the samples and heat samples 5 min at 95 C. 4. Run samples on 12% SDS gel: (We recommend to use pre-cast gels like the 12% TGX gel from Bio-Rad) For fractionation: run the gel until front reaches the end of the gel. If no fractionation is needed: (SDS-PAGE used as sample cleanup only): run sample approx. 1-2 cm into the gel. 5. Stain and destain gel (Only Coomassie stainings!). (We recommend the ready to use Colloidal Coomassie Blue from Severn Biotech) 6. Fractionate the gel in the desired amount of fractions (Fraction sizes may vary, try to keep abundant bands (e.g. albumin), if present, in 1 separate fraction). In case no fractionation is performed, cut out the lane until the sample front. 7. Chop the excised fractions in pieces of approximately 1 x 1 mm with use of a clean scalpel. Transfer the gel pieces into clean microcentrifuge tubes. Any excess gel will lead to background noise, so cut precisely. 8. Add 100 μl 50 mm ABC. Incubate for 5 minutes at room temperature. Discard all liquid. 9. Add 100 μl 50% ACN/50% H2O Incubate 5 minutes at room temperature Discard all liquid. 10. Add 100 μl acetonitrile. Incubate for 5 minutes at room temperature. Discard all liquid. 11. Repeat steps 7to 9 twice. 12. Add 100 μl reduction buffer (solution 2) and incubate for 20 minutes at 56 C, discard all liquid. 13. Add 100 μl acetonitrile. Incubate for 5 minutes at room temperature. Discard all liquid. 14. Add 100 μl alkylation buffer (solution 3) and incubate 20 min at room temperature in the dark. 15. Add 100 μl acetonitrile. Incubate for 5 minutes at room temperature. Discard all liquid. 16. Add 100 μl 50 mm ABC. Incubate for 5 minutes at room temperature. Discard all liquid. 17. Add 100 μl acetonitrile. Incubate for 5 minutes at room temperature. Discard all liquid. 18. Repeat steps 16 and Add digestion buffer (solution 5) until all gel pieces are covered. Incubate for minutes at room temperature. Add more digestion buffer if the digestion buffer is completely absorbed. Incubate for 30 minutes at room temperature. Add μl 50 mm ABC to cover all gel pieces and keep them wet during digestion. 20. Incubate overnight at 37 C. 6 V1.0 8/10/2016

7 21. After incubation, spin down the water droplets condensed inside the lid of the test tube. 22. Add 2% TFA (solution 4) 1:1 and incubate 20 minutes, shaking at room temperature. 23. Transfer supernatant to a new tube. 24. Repeat steps 22 and 23 with buffer B (solution 7) and pool supernatants after incubation. 25. Speedvac samples to ensure all ACN has been evaporated. (tip: set a mark on the new tube after transferring the first supernatant. When during speedvac the level of sample reaches the mark, as the second supernatant consists of ACN, almost all ACN has been evaporated) 26. Dilute sample 1:1 with 2% TFA (solution 4). 27. Proceed to Sample desalting and concentration. Sample desalting and concentration For more detailed information see the OMIX tips manual. Maximum loading capacity is 10 μg! Divide sample over more tips if necessary. 1. Prepare Omix tip (1 tip/sample): Aspirate 100 μl buffer B and discard solvent, repeat 1x Aspirate 100 μl buffer A and discard solvent, repeat 1x 2. Aspirate up to 100μl sample, dispense and aspirate 5 times then discard liquid. If the sample volume is more than 100 μl: repeat until all sample has been passed through the tip. 3. Aspirate 100 μl buffer A and discard solvent, repeat 1x. 4. Aspirate 100 μl buffer B and dispense in a new collection tube. Discard the OMIX tip. 5. Speedvac sample to a volume of 2 μl. 6. Add buffer A to obtain a total volume of 20 μl. 7. Store sample in -20 C until shipment. Literature Nielsen ML, Vermeulen M, Bonaldi T, Cox J, Moroder L & Mann M, Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nature Methods 2008, 5 Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S. et al., Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 1996, 379, Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes Nature Protocols (2006), 1(6), Agilent Bond Elut Omix C18 manual, available from the Agilent website or in the package containing the tips 7 V1.0 8/10/2016

8 Protocol 3 - In-solution digestion for immunopurifications/affinity purifications/purified proteins Sample requirements and precautions Preferred amount of sample (protein) is 10 μg Samples are not radioactive Samples are detergent-free (e.g. Triton X-100, PEG-44) Salt concentrations should be less than 500 μm No organic solvents present Special care must be taken to avoid contamination with keratins from skin or hair (wear gloves, lab coat at all times and clean equipment vigorously) Equipment Pierce detergent removal spin columns, 1 column/sample: article# Agilent Bond Elut C18 Omix tips, 1 tip/sample, article# A Micro centrifuge suitable for 1.5 ml reaction vials Incubator for digestion at 37 C Adjustable pipettes Speedvac Recommended Reagents Name Supplier Article number 2-chloroaetamide (CAA) Sigma G-F Urea GE Healthcare Dithiotreitol (DTT) Sigma D9163-5G Trishydroxymethylaminomethane (Tris) GE Healthcare Lysyl endopeptidase C (LysC) WAKO Chemicals Sequencing grade modified trypsin C=0.5μg/μl Promega V Water, Milli-Q - - Ammonium bicarbonate (ABC) Sigma A-6141 Trifluoroacetic acid (TFA) Pierce 9470 Acetonitrile HPLC-S grade (ACN) Biosolve Formic Acid (FA) Merck Solutions Adjust volumes if necessary mm Ammonium bicarbonate (ABC) Dissolve 200 mg in 50 ml Milli-Q. Prepare fresh, discard remaining solution mm dithiothreitol (reduction buffer) Dissolve 7.7 mg in 5 ml Milli-Q. Prepare fresh, discard remaining solution mm 2-chloroacetamide in 50 mm ABC (alkylation buffer) Dissolve mg in 5 ml solution 1. CAA is light sensitive so store the solution in the dark. Prepare fresh, discard remaining solution mm TRIS-HCl ph 8.0 Dissolve mg Tris in 45 ml Milli-Q. Adjust to ph 8.0 with HCl. Adjust volume to 50 ml with Milli-Q. Store 10 ml aliquots in -20 C up to 1 year. 8 V1.0 8/10/2016

9 5. 8 M Urea in 10 mm TRIS ph 8.0 Dissolve g urea in 50 ml solution 4. Prepare fresh, discard remaining solution. 6. 2% Trifluoroacetic acid Dilute 1 ml TFA with 49 ml Milli-Q. Store in glass, shelf life 12 months µg/µl Lysyl endopeptidase C (LysC) If no aliquots are available, make aliquots: Dissolve the stock-vial of Lys-C to a final concentration of 0.5μg/μl in buffer A. The amount of Lys-C in the vial can be found in the certificate of analysis. If not available, this can be ordered via mail from Wako (see Wako website for contact details). After diluting, prepare aliquots of 50 or 100 μl and store these in -80 C. Using an aliquot: after first use, store in -20 C for further usage (maximally: 1 year). 8. Buffer A: 0.1% FA in H 2 O (Milli-Q) 50 μl FA filled up till 50 ml with water. 9. Buffer B: 0.1% FA in ACN 50 μl FA filled up till 50 ml with ACN. Procedure Digestion 1. Dilute samples 1:1 with 8 M urea/10 mm Tris ph 8.0 (solution 5). For optimal digestion of the sample, the final urea concentration should be 4 M and the ph should be near ph Add 1 μl reduction buffer (solution 2) for every 50 μg sample protein and incubate 30 min at room temperature. (In this procedure all steps prior to digestion are done at room temperature to reduce unwanted derivatization of amino acid side-chains by the denaturants) 3. Add 1 μl alkylation buffer (solution 3) for every 50 μg sample protein and incubate 20 min at room temperature in the dark. 4. Add 1 μg LysC/50 μg total protein and incubate for at least 3 hours at room temperature. 5. Dilute sample 4x with 50mM ABC (solution 1). 6. Add 1 μg trypsin/50 μg total protein and incubate overnight at 37 C. 7. After incubation, spin down the water droplets condensed inside the lid of the test tube. 8. Proceed to Sample cleanup. Sample cleanup For more detailed information see the Pierce Detergent Removal Column manual 1. Remove bottom closure from Pierce Detergent Removal Column and loosen cap (do not remove cap). 2. Place column into a collection tube. 3. Centrifuge at 1500 x g for 1 minute to remove storage solution, do not exceed the indicated speed! Note: When using fixed-angle rotors, place a mark on the side of the column where the compacted resin is slanted upward. Place column in centrifuge with the mark facing outward in all subsequent centrifugation steps. Improper orientation will result in reduced detergent removal efficiency. 4. Add 400 μl 50 mm ABC (solution 1) and centrifuge for 1 minute at 1500 x g. Discard the buffer and repeat this step two additional times. 9 V1.0 8/10/2016

10 5. Place column in a new 1.5 ml collection tube. Slowly apply sample (max 100 μl) to the top of the compact resin bed and incubate for 2 minutes at room temperature. (If sample volume exceeds 1500 μl use a speedvac to concentrate or apply sample to more columns and pool afterwards) 6. Centrifuge at 1500 x g for 2 minutes to collect the detergent-free sample. Discard the used column. 7. Proceed to Sample desalting and concentration. Sample desalting and concentration For more detailed information see the OMIX tips manual. Maximum loading capacity is 10 μg! Divide sample over more tips if necessary. 1. Dilute sample 1:1 with 2% TFA (solution 6). 2. Prepare Omix tip (1 tip/sample): Aspirate 100 μl buffer B and discard solvent, repeat 1x Aspirate 100 μl buffer A and discard solvent, repeat 1x 3. Aspirate up to 100 μl sample. Dispense and aspirate 5 times then discard liquid. If sample volume is more than 100 μl: repeat until all sample has been passed through the tip. 4. Aspirate 100 μl buffer A and discard solvent, repeat 1x. 5. Aspirate 100 μl buffer B and dispense in a new collection tube. Discard the OMIX tip. 6. Speedvac sample to a volume of 2 μl. 7. Add buffer A to obtain a total volume of 20 μl. 8. Store sample in -20 C until shipment. Literature Kinter, M., and Sherman, N. E. 2000, Protein sequencing and identification using tandem mass spectrometry. JohnWiley & Sons, Inc. pp Nielsen ML, Vermeulen M, Bonaldi T, Cox J, Moroder L & Mann M, Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nature Methods 2008, 5 Pierce Detergent Removal Column manual, available from the Pierce website or in the package containing the columns Agilent Bond Elut Omix C18 manual, available from the Agilent website or in the package containing the tips 10 V1.0 8/10/2016

11 Protocol 4 - In-gel digestion for immunopurifications/affinity purifications/purified proteins Sample requirements and precautions Maximum amount of sample is 20μg total protein for 1 gel lane. If total protein content is limited (f.e. in affinity or immunopurifications) concentrate and load the whole sample on gel Samples are not radioactive Special care must be taken to avoid contamination with keratins from skin or hair (wear gloves, lab coat at all times and clean equipment vigorously) Do NOT use silverstaining! Only coomassie! Only cut gels on a clean glass plate Use a new scalpel blade Equipment Recommended: Mini-Protean 12% TGX pre-cast gel ( , Bio-Rad) Recommended: Colloidal Coomassie Blue stain ( , Severn Biotech) Recommended: BioRad Laemmli Sample Buffer 2x or 4x ( , , BioRad) Agilent Bond Elut C18 Omix tips, 1 tip/sample, article# A Micro centrifuge suitable for 1.5 ml reaction vials. Incubator for digestion at 37 C Adjustable pipets. Speedvac Recommended: 2D-Quant kit for protein concentration determination (# GE Healthcare) Recommended: Sample Grinding kit ( , GE Healthcare) Recommended: BioRad Laemmli Sample Buffer 2x or 4x ( , , BioRad) Recommended: Mini-Protean 12% TGX pre-cast gel ( , Bio-Rad) Recommended: Colloidal Coomassie Blue stain ( , Severn Biotech) Agilent Bond Elut C18 Omix tips, 1 tip/sample, (article# A ) Micro centrifuge suitable for 1.5 ml reaction vials Incubator for digestion at 37 C Adjustable pipettes Speedvac Reagents Name Supplier Article number 2-chloroaetamide (CAA) Sigma G-F Dithiotreitol (DTT) Sigma D9163-5G Sequencing grade modified trypsin C=0.5μg/μl Promega V Water, Milli-Q - - Ammonium bicarbonate (ABC) Sigma A-6141 Trifluoroacetic acid (TFA) Pierce 9470 Acetonitrile HPLC-S grade (ACN) Biosolve Formic Acid (FA) Merck V1.0 8/10/2016

12 Solutions Adjust volumes if necessary mm Ammonium bicarbonate (ABC) Dissolve 200 mg in 50 ml Milli-Q. Prepare fresh, discard remaining solution mm dithiothreitol (reduction buffer) Dissolve 7.7 mg in 5 ml Milli-Q. Prepare fresh, discard remaining solution mm 2-chloroacetamide in 50 mm ABC (alkylation buffer) Dissolve mg in 5 ml solution 1. CAA is light sensitive so store the solution in the dark. Prepare fresh, discard remaining solution. 4. 2% Trifluoroacetic acid Dilute 1 ml TFA with 49 ml Milli-Q. Store in glass, shelf life 12 months ,5 ng/μl trypsin in 50 mm ABC (Digestion buffer) Dilute 1 μl trypsin with 39 μl 50 mm ABC. Alternatively, for concentrated samples in a small total gel volume, 100 ng trypsin/sample in 50 mm ABC can be used. Use immediately. 6. Buffer A: 0.1% FA in H 2 O (Milli-Q) 50 μl FA filled up till 50 ml with water. 7. Buffer B: 0.1% FA in ACN 50 μl FA filled up till 50 ml with ACN. Procedure Digestion All volumes are for reference purposes only; please adjust the volumes so that all gel particles are covered with liquid. Skip step 1-4 if sample is already in a gel band, otherwise proceed to step 1 1. Add Laemmli buffer to the samples and heat samples for 5 min at 95 C. 2. Run samples on 12% SDS PAGE. (We recommend to use pre-cast gels like the 12% TGX gel from Bio-Rad) To cut out a specific band: run the gel until front reaches the end of the gel. SDS-PAGE as sample clean-up: run sample approx. 1-2 cm into the gel. 3. Stain and destain gel (Only Coomassie stainings!). (We recommend the ready to use Colloidal Coomassie Blue from Severn Biotech) 4. Cut out desired gel band or if SDS-PAGE is used as sample clean-up, cut out the entire sample up to the sample front. 5. Chop the excised fractions in pieces of approximately 1 x 1 mm with use of a clean scalpel. Transfer the gel pieces into clean microcentrifuge tubes. Any excess gel will lead to background noise, so cut precisely. 6. Add 100 μl 50 mm ABC. Incubate for 5 minutes at room temperature. Discard all liquid. 7. Add 100 μl 50% ACN/50% H2O Incubate 5 minutes at room temperature. Discard all liquid. 8. Add 100 μl acetonitrile. Incubate for 5 minutes at room temperature. Discard all liquid. 9. Repeat steps 6 to 8 twice. 10. Add 100 μl reduction buffer (solution 2) and incubate for 20 minutes at 56 C, discard all liquid. 11. Add 100 μl acetonitrile. Incubate for 5 minutes at room temperature. Discard all liquid. 12. Add 100 μl alkylation buffer (solution 3) and incubate 20 min at room temperature in the dark. 13. Add 100 μl acetonitrile. Incubate for 5 minutes at room temperature. Discard all liquid. 12 V1.0 8/10/2016

13 14. Add 100 μl 50 mm ABC. Incubate for 5 minutes at room temperature. Discard all liquid. 15. Add 100 μl acetonitrile. Incubate for 5 minutes at room temperature. Discard all liquid. 16. Repeat steps 14 and Add digestion buffer (solution 5) until all gel pieces are covered. Incubate for minutes at room temperature. Add more digestion buffer if the digestion buffer is completely absorbed. Incubate for 30 minutes at room temperature. Add μl 50 mm ABC to cover all gel pieces and keep them wet during digestion. 18. Incubate overnight at 37 C. 19. After incubation, spin down the water droplets condensed inside the lid of the test tube. 20. Add 2% TFA (solution 4) 1:1 and incubate 20 minutes, shaking at room temperature. 21. Transfer supernatant to a new tube. 22. Repeat steps 20 and 21 with buffer B (solution 7) and pool supernatants after incubation. 28. Speedvac samples to ensure all ACN has been evaporated. (tip: set a mark on the new tube after transferring the first supernatant. When during speedvac the level of sample reaches the mark, as the second supernatant consists of ACN, almost all ACN has been evaporated) 29. Dilute sample 1:1 with 2% TFA (solution 4). 30. Proceed to Sample desalting and concentration. Sample desalting and concentration For more detailed information see the OMIX tips manual. Maximum loading capacity is 10 μg! Divide sample over more tips if necessary. 1. Prepare Omix tip (1 tip/sample): Aspirate 100 μl buffer B and discard solvent, repeat 1x Aspirate 100 μl buffer A and discard solvent, repeat 1x 2. Aspirate up to 100 μl sample. Dispense and aspirate 5 times then discard liquid. If sample volume is more than 100 μl: repeat until all sample has been passed through the tip. 3. Aspirate 100 μl buffer A and discard solvent, repeat 1x. 4. Aspirate 100 μl buffer B and dispense in a new collection tube. Discard the OMIX tip. 5. Speedvac sample to a volume of 2 μl. 6. Add buffer A to obtain a total volume of 20 μl. 7. Store sample in -20 C until shipment. Literature Nielsen ML, Vermeulen M, Bonaldi T, Cox J, Moroder L & Mann M, Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nature Methods 2008, 5 Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S. et al., Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 1996, 379, Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes Nature Protocols (2006), 1(6), Agilent Bond Elut Omix C18 manual, available from the Agilent website or in the package containing the tips 13 V1.0 8/10/2016

14 Protocol 5 - In-solution digestion protocol for human body fluids (plasma, serum, CSF) This protocol is only intended for samples of human origin. For samples of other species follow the in-gel digestion protocol (protocol 2) or use a depletion column designed for the organism. Sample requirements and precautions Preferred amount of total protein in a sample before depletion is 100μg Samples are not radioactive Samples are detergent-free (e.g. Triton X-100, PEG-44) Salt concentrations should be less than 500 μm No organic solvents present Special care must be taken to avoid contamination with keratins from skin or hair (wear gloves, lab coat at all times and clean equipment vigorously) Equipment Recommended: 2D-Quant kit for protein concentration determination (# GE Healthcare) MARS14 depletion spin column (Multiple Affinity Removal Spin cartridge, Hu-14, # , Agilent) Agilent Bond Elut C18 Omix tips, 1 tip/sample, article# A kDa MWCO cut off filter (Amicon Ultra, UFC500396, Merck Millipore) Micro centrifuge suitable for 1.5 ml reaction vials Incubator for digestion at 37 C Adjustable pipettes Speedvac Recommended Reagents Name Supplier Article number 2-chloroaetamide (CAA) Sigma G-F Urea GE Healthcare Dithiotreitol (DTT) Sigma D9163-5G Trishydroxymethylaminomethane (Tris) GE Healthcare Lysyl endopeptidase C (LysC) WAKO Chemicals Sequencing grade modified trypsin C=0.5μg/μl Promega V Water, Milli-Q - - Ammonium bicarbonate (ABC) Sigma A-6141 Trifluoroacetic acid (TFA) Pierce 9470 Acetonitrile HPLC-S grade (ACN) Biosolve Formic Acid (FA) Merck Solutions Adjust volumes if necessary mm Ammonium bicarbonate (ABC) Dissolve 200 mg in 50 ml Milli-Q. Prepare fresh, discard remaining solution mm dithiothreitol (reduction buffer) 14 V1.0 8/10/2016

15 Dissolve 7.7 mg in 5 ml Milli-Q. Prepare fresh, discard remaining solution mm 2-chloroacetamide in 50 mm ABC (alkylation buffer) Dissolve mg in 5 ml solution 1. CAA is light sensitive so store the solution in the dark. Prepare fresh, discard remaining solution mm TRIS-HCl ph 8.0 Dissolve mg Tris in 45 ml Milli-Q. Adjust to ph 8.0 with HCl. Adjust volume to 50 ml with Milli-Q. Store 10 ml aliquots in -20 C up to 1 year M Urea in 10 mm TRIS ph 8.0 Dissolve g urea in 50 ml solution 4. Prepare fresh, discard remaining solution. 6. 2% Trifluoroacetic acid Dilute 1 ml TFA with 49 ml Milli-Q. Store in glass, shelf life 12 months µg/µl Lysyl endopeptidase C (LysC) If no aliquots are available, make aliquots: Dissolve the stock-vial of Lys-C to a final concentration of 0.5μg/μl in buffer A. The amount of Lys-C in the vial can be found in the certificate of analysis. If not available, this can be ordered via mail from Wako (see Wako website for contact details). After diluting, prepare aliquots of 50 or 100 μl and store these in -80 C. Using an aliquot: after first use, store in -20 C for further usage (maximally: 1 year). 8. Buffer A: 0.1% FA in H 2 O (Milli-Q) 50 μl FA filled up till 50 ml with water. 9. Buffer B: 0.1% FA in ACN 50 μl FA filled up till 50 ml with ACN. Procedure Depletion and digestion 1. Determine protein concentration. (In our experience, the 2D-Quant kit gives the best results) 2. We recommend using 100 μg of total protein/sample as starting material. Be sure to use the same protein amount for all samples. 3. Apply sample to MARS14 spin column, following the maunfacturer s instructions. 4. Concentrate sample on 3kDa MWCO cutoff filter and wash with 500 µl MQ for extra desalting. 5. Optional: Check depletion by running samples before and after depletion on SDS-PAGE and stain with Coomassie staining. 6. Dilute samples 1:1 with 8M urea/10 mm Tris ph 8.0 (solution 5) after depletion. For optimal digestion of the sample, the final urea concentration should be 4 M and the ph should be near ph Add 1 μl reduction buffer (solution 2) for every 50 μg sample protein and incubate 30 min at room temperature. (In this procedure all steps prior to digestion are done at room temperature to reduce unwanted derivatization of amino acid side-chains by the denaturants) 8. Add 1 μl alkylation buffer (solution 3) for every 50 μg sample protein and incubate 20 min at room temperature in the dark. 9. Add 1 μg LysC/50 μg total protein and incubate for at least 3 hours, room temperature. 10. Dilute sample 4x with 50 mm ABC (solution 1). 11. Add 1 μg trypsin/50 μg total protein and incubate overnight at 37 C. 12. After incubation, spin down the water droplets condensed inside the lid of the test tube. 15 V1.0 8/10/2016

16 13. Proceed to Sample desalting and concentration. Sample desalting and concentration For more detailed information see the OMIX tips manual. Maximum loading capacity is 10 μg! Divide sample over more tips if necessary. 1. Prepare Omix tip (1 tip/sample): Aspirate 100 μl buffer B and discard solvent, repeat 1x Aspirate 100 μl buffer A and discard solvent, repeat 1x 2. Aspirate up to 100 μl sample. Dispense and aspirate 5 times then discard liquid. If sample volume is more than 100 μl: repeat until all sample has been passed through the tip. 3. Aspirate 100 μl buffer A and discard solvent, repeat 1x. 4. Aspirate 100 μl buffer B and dispense in a new collection tube. Discard the OMIX tip. 5. Speedvac sample to a volume of 2 μl. 6. Add buffer A to obtain a total volume of 20 μl. 7. Store sample in -20 C until shipment. Literature Kinter, M., and Sherman, N. E. 2000, Protein sequencing and identification using tandem mass spectrometry. JohnWiley & Sons, Inc. pp Nielsen ML, Vermeulen M, Bonaldi T, Cox J, Moroder L & Mann M, Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nature Methods 2008, 5 Pierce Detergent Removal Column manual, available from the Pierce website or in the package containing the columns Agilent Bond Elut Omix C18 manual, available from the Agilent website or in the package containing the tips 16 V1.0 8/10/2016