ࡱ>  Zbjbj =xx^JGt t 8$|wN,,"NNN)? KLLLLLLL$#PR~LqS))SSLNN1NSNNLSLBXF`kW"D(LGN0wN*DWSyWSPFFSSSLLSSSwNSSSSWSSSSSSSSSSt * :    Preparing Whole Cell Protein Extracts for LC/MS Analysis in the Proteomics and Metabolomics Core (PMC) David Kakhniashvili, Ph.D., Director Before you start your project!!! All users must contact Dr. Kakhniashvili and discuss specific project details before submitting samples to the PMC. Read the Sample Preparation Basics SOP for the PMC. Important Information for Sample Preparation LC/MS analys is used for identification of proteins in analyzed samples and mapping of post-translational modifications (PTM) in identified proteins. Protein sample is digested with a proteolytic enzyme (usually trypsin) and generated peptide mix is subjected to LC/MS analysys Cell Lysis and Protein Extraction The main objective of this procedure is: a. to efficiently lyse cells and extract proteins b. to preserve proteins from degradation and other uncontrolled modifications There is no absolute single best way to lyse cells and extract proteins. A variety of homemade (published) and commercial buffers have been optimized for different cell (or sample) types. Conditions optimal for a specific sample should be selected. Quality and Amount of Protein Extract Required Processing/preparation of protein extracts for LC/MS analysis include trypsin digestion. Before trypsin digestion, protein extracts must be essentially free of a) protease inhibitors, denaturing agents, detergents, etc. that inhibit trypsin digestion, and b) protein stabilizers glycerol, PEG, which severely interfere with MS analysis. Protein extracts can be separated from these low MW components by filtration using centrifugal filter devices of a low MWCO (e.g. FASP columns) or by acetone precipitation. Purified protein extracts are then dissolved and trypsin digested in an appropriate buffer. The required amount of digested protein in submitted samples is at least 0.2g of cell lysate per LC/MS analysis; however, sample processing/preparation including trypsin digestion may require 5-100g per sample (per replicate) depending on application for best result. Protein Extracts Containing Extremely Abundant Proteins Analysis of medium and low abundant proteins is extremely difficult (or impossible) in the presence of highly abundant proteins (e.g. hemoglobin in red blood cells, albumin in blood plasma). Selective depletion of abundant proteins from protein extracts (to at least average abundance level) is required to facilitate analysis of less abundant proteins of interest. Preparing Whole Cell Protein Extracts Basic Protocol Introduction The Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells enables reproducible processing of cultured mammalian cells for proteomic mass spectrometry (MS) analysis. The kit contains all of the necessary buffers, reagents, MS-grade enzymes; an optimized protocol generates MS-compatible peptide samples from whole-cell lysates. Sample preparation can be performed in 2 alternative ways using Acetone precipitation (refer to appendix A) or FASP processing (refer to appendix B) Appendix A Preparing Whole Cell Protein Extracts via Acetone Precipitation Materials Required Pierce"! Mass Spec Sample Prep Kit for Cultured Cells, P/N 84840 Kit Contents (sufficient for processing 20 samples of 100g of cell lysate protein): " Cell Lysis Buffer, 5ml " Digestion Buffer, 5ml " No-Weigh"! DTT, 24 microtubes, each containing 7.7mg of dithiothreitol (DTT) " Iodoacetamide, Single-Use, 24 microtubes, each containing 9.3mg of iodoacetamide (IAA) " Trypsin Storage Solution, 250l " Pierce Digestion Indicator, 10g " Lys-C Protease, MS Grade, 20g " Pierce"! Trypsin Protease, MS Grade, 2 20g Storage: Upon receipt, remove Insert A (containing Pierce Digestion Indicator, Lys-C Protease and Pierce Trypsin Protease, MS Grade) and store at -20C. Store the remaining components at 4C. Product is shipped on dry ice. Additional Materials Required Microcentrifuge polypropylene tubes Microtip probe sonicator or nuclease (e.g., Thermo Scientific Pierce Universal Nuclease for Cell Lysis, P/N. 88700) Protein assay kit (e.g., Thermo Scientific BCA Protein Assay Kit, P/N 23227) (Optional) Pierce Quantitative Colorimetric peptide Assay (P/N 23275) Heating block Chilled (-20C) 100% acetone and 90% acetone Trifluoroacetic acid (TFA) Phosphate-buffered saline (PBS) Vacuum concentrator (e.g., Thermo Scientific SpeedVac Vacuum Concentrator) Procedure for Preparation of Peptides from Cultured Cells for MS Analysis A. Material Preparation Pre-chilled 90% acetone: Prepare 90% acetone in ultrapure water (e.g mix 45mL of 100% acetone with 5mL of ultrapure water) and store at -20C Pre-chilled 100% acetone: Store 100% acetone at -20C Warm the Cell Lysis Buffer and Digestion Buffers to room temperature before use. Store buffers at 4C. B. Cell Lysis 1. Culture cells to harvest at least 100g of protein. For best results, culture a minimum of 2 106 cells. Note: Rinse cell pellets 3 times with 1X PBS to remove cell culture media. Pellet cells using low-speed centrifugation (i.e., < 1000 g) to prevent premature cell lysis. 2. Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 100l of Cell Lysis Buffer for a 20l cell pellet). Pipette sample up and down to break up the cell clumps and gently vortex sample to mix. 3. Incubate the lysate at 95C for 5 minutes. 4. Cool the lysate on ice for 5 minutes, spin down.. 5. Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity by shearing DNA. Alternatively, use Pierce Universal Nuclease for Cell Lysis (P/N 88700) to enzymatically digest DNA and RNA. If using nuclease, add 25 units of nuclease per 1ml of cell lysate and incubate at room temperature for 15 minutes. 6. Centrifuge lysate at 16,000 g for 10 minutes at 4C. 7. Carefully separate the supernatant and transfer into a new tube. 8. Determine the protein concentration of the supernatant using established methods such as the BCA Protein Assay Kit (e.g., Thermo Scientific BCA Protein Assay Kit, P/N 23227) C. Reduction, Alkylation and Acetone Precipitation Note: This procedure is optimized for 100g of cell lysate protein at 1mg/mL concentration; however, the procedure may be used for 10-200g of cell lysate protein with an appropriate amount of reagents (DTT, IAA, Lys-C and trypsin). When using 10g of cell lysate, a protein concentration of 0.2-1mg/ml may be used. 1. Warm and equilibrate the Pierce Digestion Indicator to room temperature. 2. Add 100g of lysate protein to a polypropylene microcentrifuge tube and adjust the sample volume to 100L using Cell Lysis Buffer to a final concentration of 1mg/ml. 3. Add 0.5g (0.5% w/w) of Pierce Digestion Indicator to the sample. Note: The actual concentration is printed on the bottle label. Refer to the label to determine the required volume. 4. Immediately before use, puncture the foil covering of the Thermo Scientific"! No-Weigh"! DTT tube with an empty pipette tip. Add 100l of ultrapure water to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of DTT is ~500mM. Note: To preserve DTT stability between uses, return unused micro-tubes to the pouch containing the desiccant pack. 5. Add 2.1l of DTT solution to the sample (final DTT concentration is ~10mM). Mix and incubate at 50C for 45 minutes. Discard any unused DTT solution. 6. Cool the sample to room temperature for 10 minutes, spin down. 7. Immediately before use, puncture the foil covering of the Single-Use Iodoacetamide tube with an empty pipette tip. Add 100l of Cell Lysis Buffer to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of IAA is ~500mM. Protect solution from light. 8. Add 11.5l of IAA solution to the sample (final IAA concentration is ~50mM). Mix and incubate at room temperature for 20 minutes protected from light. Discard any unused IAA solution. 9. After alkylation with IAA, immediately add 690l (6 volumes) of pre-chilled (-20C) 100% acetone to sample. Vortex tube and incubate at -20C for four hour to overnight to precipitate proteins. 10. Centrifuge at 16,000 g for 10 minutes at 4C. Carefully remove acetone without dislodging the protein pellet. 11. Add 50l of pre-chilled (-20C) 90% acetone, vortex to mix and centrifuge at 16,000 g for 5 minutes at 4C. 12. Carefully remove acetone without dislodging the protein pellet. Allow the pellet to dry for 2-3 minutes and immediately proceed to Section D. Enzymatic Protein Digestion. Note: Do not dry the acetone-precipitated protein pellet for more than 2-3 minutes; excess drying will make the pellet difficult to re-suspend in the Digestion Buffer. D. Enzymatic Protein Digestion 1. Add 100l of Digestion Buffer to the acetone-precipitated protein pellet and resuspend by gentle pipetting up and down to break the pellet. Note: An acetone-precipitated protein pellet may not completely dissolve; however, after proteolysis at 37C, all the protein will be solubilized. 2. Immediately before use, add 40L of ultrapure water to the bottom of the vial containing 20g Lys-C and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining Lys-C solution in single-use volumes at -80C. 3. Add 2l of Lys-C (1g, enzyme-to-substrate ratio = 1:100) to the sample. Mix and incubate at 37C for 2 hours. 4. Immediately before use, add 40l of Trypsin Storage Solution to the bottom of the vial Containing 20g trypsin and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining trypsin solution in single-use volumes at -80C for long-term storage. 5. Add 4l of trypsin (2g, enzyme-to-substrate ratio = 1:50) to the sample. Mix and incubate overnight at 37C. 6. Acidify the sample with TFA (to 0.1%) to stop digestion, spin down. 7. Speed vac the sample (106l) for at least 2 hr. to remove the (volatile) Digestion Buffer. Record the protein amount per sample. 8. The samples are ready to be submitted to the facility for LC/MS analysis. Appendix B Preparing Whole Cell Protein Extracts via FASP Processing Materials Required Pierce Mass Spec Sample Prep Kit for Cultured Cells, P/N 84840 Kit Contents (sufficient for processing 20 samples of 100g of cell lysate protein): " Cell Lysis Buffer, 5ml " Digestion Buffer, 5ml " No-Weigh"! DTT, 24 micro-tubes, each containing 7.7mg of dithiothreitol (DTT) " Iodoacetamide, Single-Use, 24 microtubes, each containing 9.3mg of iodoacetamide (IAA) " Trypsin Storage Solution, 250l " Pierce Digestion Indicator, 10g " Lys-C Protease, MS Grade, 20g " Pierce"! Trypsin Protease, MS Grade, 2 20g Storage: Upon receipt, remove Insert A (containing Pierce Digestion Indicator, Lys-C Protease and Pierce Trypsin Protease, MS Grade) and store at -20C. Store the remaining components at 4C. Product is shipped on dry ice. FASP Protein Digestion Kit, Expedeon P/N 44250, Thermo Fisher P/N EX44250 Kit Contents (sufficient for processing 8 samples): Spin Columns, 8 Collection Tubes, 16 Tris-HCl solution,100mM, pH 8.5, 20ml Ammonium Bicarbonate Solution, 50mM, 20ml Do not use for this protocol Urea, single-use, 8 micro-tubes, each containing 0.75g of urea NaCl solution, 500mM, 20ml Iodoacetamide (IAA), single-use, 8 micro-tubes - Do not use for this protocol Additional Materials Required Microtip probe sonicator or nuclease (e.g., Thermo Scientific Pierce Universal Nuclease for Cell Lysis, P/N. 88700) Protein assay kit (e.g., Thermo Scientific BCA Protein Assay Kit, P/N 23227) Pierce Quantitative Colorimetric peptide Assay (P/N 23275) Micro-centrifuge polypropylene tubes Heating block Vortex Trifluoroacetic acid (TFA) Phosphate-buffered saline (PBS) Vacuum concentrator (e.g., Thermo Scientific SpeedVac Vacuum Concentrator) Procedure for Preparation of Peptides from Cultured Cells A. Material Preparation 1. Warm the Cell Lysis Buffer and Digestion Buffer provided with Pierce kit to room temperature before use. Store buffers at 4C. 2. Urea Sample Solution: Add 1 mL Tris Hydrochloride Solution provided with the FASP Kit to one tube of Urea, also provided with the FASP Kit. Vortex the tube until all the powder dissolves. 3. TEAB Solution, 50mM: e.g. add 1ml of 1M TEAB to 19ml of ultrapure water, mix. B. Cell Lysis 1. Culture cells to harvest at least 100g of protein. For best results, culture a minimum of 2 106 cells. Note: Rinse cell pellets 3 times with 1X PBS to remove cell culture media. Pellet cells using low-speed centrifugation (i.e., < 1000 g) to prevent premature cell lysis. 2. 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Pipette sample up and down to break up the cell clumps and gently vortex sample to mix. 3. Incubate the lysate at 95C for 5 minutes. 4. Cool the lysate on ice for 5 minutes, spin down.. 5. Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity by shearing DNA. Alternatively, use Pierce Universal Nuclease for Cell Lysis (P/N 88700) to enzymatically digest DNA and RNA. If using nuclease, add 25 units of nuclease per 1ml of cell lysate and incubate at room temperature for 15 minutes. 6. Centrifuge lysate at 16,000 g for 10 minutes at 4C. 7. Carefully separate the supernatant and transfer into a new tube. 8. Determine the protein concentration of the supernatant using established methods such as the BCA Protein Assay Kit (e.g., Thermo Scientific"! BCA Protein Assay Kit, P/N 23227) C. Reduction and Alkylation Note: This procedure is optimized for 100g of cell lysate protein at 1mg/mL concentration; however, the procedure may be used for 50-100g of cell lysate protein with an appropriate (proportional) amount of reagents (DTT, IAA, Pierce Digestion Indicator, Lys-C and trypsin). 1. Warm and equilibrate the Pierce Digestion Indicator to room temperature. 2. Add 100g of lysate protein to a polypropylene microcentrifuge tube and adjust the sample volume to 100L using Cell Lysis Buffer to a final concentration of 1mg/ml. 3. Add 0.5g (0.5% w/w) of Pierce Digestion Indicator to the sample (i.e. 0.005g of Pierce Digestion Indicator per g of sample protein). Note: The actual concentration is printed on the bottle label. Refer to the label to determine the required volume. 4. Immediately before use, puncture the foil covering of the Thermo Scientific"! No-Weigh"! DTT tube with an empty pipette tip. Add 100l of ultrapure water to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of DTT is ~500mM. Note: To preserve DTT stability between uses, return unused micro-tubes to the pouch containing the desiccant pack. 5. Add 2.1l of 500mM DTT solution to the sample (final DTT concentration is ~10mM). Mix and incubate at 50C for 45 minutes. Discard any unused DTT solution. 6. Cool the sample to room temperature for 10 minutes, spin down. 7. Immediately before use, puncture the foil covering of the Single-Use Iodoacetamide tube with an empty pipette tip. Add 100l of Cell Lysis Buffer to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of IAA is ~500mM. Protect solution from light. 8. Add 11.5l of 500mM IAA solution to the sample (final IAA concentration is ~50mM). Mix and incubate at room temperature for 20 minutes protected from light. Discard any unused IAA solution. 9. After alkylation with IAA, immediately add 100l of Urea Sample Solution and proceed to Section D, FASP Protein digestion D. FASP Protein Digestion 1. Add 200l of Urea Sample Solution to a Spin Filter and centrifuge at 14,000 x g for 5 min. Note: The centrifugation times may need adjustment  keep it short but long enough to let all solvent flow through the filter to the collection tube. 2. Transfer the alkylated protein sample (step C9) into the Spin Filter. Centrifuge at 14,000 x g for 15 min. Discard the flow-through from the collection tube 3. Add 200L of Urea Sample Solution to the Spin Filter, cap the filter, vortex and centrifuge at 14,000 x g for 12 min. Discard the flow-through from the collection tube. Repeat this step once. 4. Add 100l of 50 mM TEAB Solution to the Spin Filter, cap the filter, vortex and centrifuge at 14,000 x g for 10 min. Discard the flow-through from the collection tube. Repeat this step twice. 5. Add 100l of Digestion Buffer provided with Pierce kit 6. Immediately before use, add 40L of ultrapure water to the bottom of the vial containing 20g Lys-C and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining Lys-C solution in single-use volumes at -80C. 7. Add 2l of Lys-C (1g, enzyme-to-substrate ratio = 1:100) to the sample, cap the filter, vortex 1 min, and incubate at 37C for 2 hours. 8. Immediately before use, add 40l of Trypsin Storage Solution to the bottom of the vial Containing 20g trypsin and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining trypsin solution in single-use volumes at -80C. 9. Add 4l of trypsin (2g, enzyme-to-substrate ratio = 1:50) to the sample, cap the filter, vortex, and Incubate overnight at 37C. Wrap the tops of the tubes with Parafilm to minimize the effects from evaporation. 10. Transfer the Spin Filter to a new collection tube and centrifuge at 14,000 x g for 10 min. Do not discard the filtrate. 11. Add 50l of 50 mM TEAB Solution to the Spin Filter and centrifuge at 14,000 x g for 10 min. Do not discard the combined filtrate. 12. Add 50l 0.5 M Sodium Chloride Solution provided with the FASP Kit and centrifuge the Spin Filter at 14,000 x g for 10 min. Save the combined (206l) filtrate. 11. Speed vac the sample (206l, containing ~ 100g of digested proteins) to ~20-50l and desalt using C18 ZipTips (or equivalent) of appropriate capacity according to the manufacturer s protocol. 11. Speed vac the desalted sample to dryness. 12. Resuspend the sample in 100l of 10% acetonitrile. 13. Determine the peptide concentration in the samples using Pierce Quantitative Colorimetric Peptide Assay (P/N 23275) according to the manufacturer s protocol. 14. Transfer at least 25g of the digested protein sample into a new tube; record the transferred amount. 15. Speed vac the samples to dryness. The samples are ready to be submitted to the facility for LC/MS analysis.     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