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Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation


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Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation

Bottom-up proteomics is widely accepted as a primary method to characterize proteins. To ensure efficient protein analysis researchers must optimize key steps in the workflow to avoid potential pitfalls such as poor protein sample preparation and inconsistent LC-MS instrument performance. In this presentation, we will:
• Investigate the cause of incomplete trypsin digestion and solution to this problem.
• Discuss the advantage of alternative proteases for mass spec protein analysis.
• Review the impact of mass spec compatible surfactants on protein digestion in gel and protein extraction from animal tissues.
• Detail new reference mass spec protein and peptide materials designed to optimize protein sample preparation steps and monitor key instrument performance parameters.
The presentation should prove valuable to any researcher using bottom-up proteomics, and who is concerned with improving protein mass spec sample preparation and mass spec instrument performance.

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Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation

  1. 1. Overcoming key challenges of Protein Mass Spectrometry Sample preparation Mourad Ferhat, Ph.D
  2. 2. Promega Corporation Manufacturer of reagents, kits and integrated systems for life science market  Promega Headquarters Madison, WI  Founded in 1978  1,300 employees in 15 countries  Over 3,500 products  ISO 13485 certified  ~ 750 patents Operations in:  San Luis Obispo, CA  Sunnyvale, CA  Seoul and Shanghai The Feynman center cGMP facility (260,000 square feet = (24,155 m2), Madison for IVD manufacturing
  3. 3. Drug Discovery Solutions by Promega Bioassays Cell Health In vivo imaging Cell signaling Ab characterization Ab purification ADMECell metabolism Target engagement Alternative Proteases MS compatible Surfactants Reference MS Protein Material Trypsin digestion Mass Spectrometry Reagents
  4. 4. Protein fractionation Mass spec analysis Protein mass spec sample preparation peptides Incomplete digestion Trypsin is not suitable foranalysis Poor peptide recovery Long and laborious sample prep procedure Poor protein extraction fromtissues Protein extraction Inadequate instrument performancemonitoring Protein digestion ❶ Extraction ❷ Fractionation ❸ Digestion❹ Analysis
  5. 5. Enhanced Proteolysis Trypsin, Rapid Trypsin, Trypsin/LysC Mix
  6. 6. Trypsin, Sequencing Grade  The highest digestion efficiency  Tolerance to protease inhibiting agents Digestion efficiency  Higher digestion efficiency  Higher purity (TPCK treatment + affinity purification)  Resistant to autolysis (Lysine residues are modified by reductive methylation) Overall good performance Enhancing of trypsin performance Trypsin Gold, Mass Spec Grade Trypsin/Lys-C mix
  7. 7. Nature of incomplete proteolysis in trypsin digests Overnight trypsin digest of yeast protein extract 22.2% missed cleavages MissedR 3.6% MissedK 18.6% 2.6 4 Majority of missed cleavages occurs at lysine sites. Missed cleavages Trypsin cleavage sites NNNNR NNNNKNNNN K R K K K K K K R : Arginine K : Lysine
  8. 8. Solution: supplementing trypsin with Lys-C Lys-C is an ideal means to compensate for trypsin lysine cleavage inefficiency. Trypsin NNNN(R/K) NNNN Lysines are cleaved less efficiently. NNNN K NNNN Lysines are cleaved with high efficiency. Lys-C
  9. 9. Enhanced proteolysis with Trypsin/Lys-C Missed R 3.6% MissedK 18.6% Trypsin digest Trypsin/Lys-C digest 3.6% 4% Trypsin/Lys-C eliminates majority of missed cleavage sites. Overnight digestion at 37oC Digestion of yeast protein extract containing trypsin inhibiting agents
  10. 10. Study #1: Analysis of FFPE skin tissue 21.5% 8.5% Trypsin/Lys-C Missed Cleavages, % Trypsin Trypsin Identified Peptides Trypsin/Lys-C Identified Proteins Trypsin Trypsin/Lys-C 24% increase 10% increase2.5 fold drop 705 887 165 182 Sample prep is difficult due to extensive protein crosslinking in FFPE tissue. Courtesy of Chris Adams, Stanford U Trypsin/Lys-C increased number of identified peptides and proteins in FFPE tissue.
  11. 11. Study #2: Developing biomarker quantitative assay for human plasma Courtesy of Matt Szapacs, GSK 674099 Trypsin/Lys-C digest 9139 Trypsin/Lys-C digest Trypsin digest 3743 Trypsin/Lys-C digest 1180 Trypsin/Lys-C digest Trypsin digestTrypsin digest Peptide peak area Trypsin digest SAP protein 145305 8976 2207 555 Trypsin/Lys-C provided conditions for more accurate quantitation of the targeted protein in plasma.
  12. 12. Study #3: Increased tolerance to trypsin inhibiting agents Inhibitor:GuClInhibitor:protease inhibitorcocktail 1252 1495 Trypsin/Lys-C mix assures efficient proteolysis even if a protein sample is contaminated with trypsin inhibiting agents. Inhibitor Protease Missed cleavages Protease inhibitor cocktail, 1X Trypsin 44.4% Trypsin/Lys-C 21.5% GuCl, 0.5 M Trypsin 55.9% Trypsin/Lys-C 24.6% Digestion of yeast protein extract containing trypsin inhibiting agents Missed (undigested) cleavage sites Identified proteins Trypsin Trypsin/ Lys-C Trypsin 13-20% increase 1364 1204 Trypsin/ Lys-C
  13. 13. Rapid Trypsin : Trypsin digestion in as little as 60 min 2. Heat increases enzymatic activity 1. Heat induces protein unfolding providing easy protease accessto cleavage sites. Heat advantages for proteolysis Rapid Trypsin is a thermostable formulation of trypsin. It rapidly digests proteins at high temperature.  30 min digestion with no need for reduction and alkylation.  Rapid digestion with Rapid Trypsin  30 min at70oC
  14. 14. Alternatives Proteases Protease Cleavage site Property and application Lys-C NNNNK NNN  Active under denaturing conditions  Digest proteolytically resistant proteins Glu-C NNNNE NNN  Used as trypsin alternative if trypsin cleavage sites have disadvantageous distribution Asp-N NNNN DNNN Arg-C NNNNR NNN (also cleave at K at a lesser degree)  Analysis of histone posttranslational modifications Chymotrypsin NNNN(F/Y/W) NNN  Digests hydrophobic proteins (i.e. membrane proteins) Pepsin Nonspecific protease  Works at low pH  Used in HDX studies Thermolysin Nonspecific protease  Works at elevated temperature  Digest proteolytically difficult proteins; structural studies Elastase Nonspecific protease  Used to increase protein coverage
  15. 15. Pepsin and thermolysin are a better alternative for membrane proteins than trypsin  Fully digest membrane proteins  Low pH and high temperature used by these proteases help unfolding these proteins. Case study: digestion of membrane proteins Toofew tryptic cleavage sites Tight folding prevents trypsin access to cleavage sites
  16. 16. 20 0 Bacteriorhodopsin coverage was dramatically increased when digested with thermolysin and pepsin. 40 60 100 80 Pepsin Thermolysin Trypsin Sequencecoverage,% Bacteriorhodopsin Coverage with trypsin Coverage with pepsin Bacteriorhodopsin sequence coverage Increased protein sequence coverage with pepsin and thermolysin
  17. 17. Biotherapeutics Characterization AccuMap™ Low pH digest, IdeS/IdeZ proteases, Glycosidases
  18. 18. IdeS and IdeZ proteases Antibody specific proteases
  19. 19. IdeS – Immunoglobulin Degrading Enzyme from Streptococcus pyogenes IdeS is an IgG-specific protease. It cleaves IgG at a unique site below the hinge. ’ Fc/2 (+Glycans) LC Mass spec 30 min digestion Fd’ IdeS IdeS advantage for IgG characterization  Ready separation of IgG Fragments  Rapid analysis of major protein modifications
  20. 20. IdeZ and IdeS protease cleavage specificity We have recently added IdeZ protease to our mass spec reagent portfolio. IdeZ offers further improvement for IgG analysis. In contrast to IdeS, which preferentially cleaves human antibodies, IdeZ also efficiently cleaves mouse antibodies.
  21. 21. Analysis of Glycoproteins with LC/MS and PNGase F Asn-linked type glycans can be cleaved enzymatically by PNGase F :  The cleavage separate intact oligosaccharides from slightly modified proteins (Asn residues at the site of N-glycosylation are converted to Asp)  The deglycosylated peptides can be analyzed by mass spectrometry
  22. 22. Using EndoH and PNGase F to monitor protein trafficking
  23. 23. AccuMAP™ Low pH digestion
  24. 24. Nonenzymatic PTMs induced during protein sample preparation LC-MS or UV-HPLC Denaturation Digestion Reduction Alkylation Biotherapeutic protein Digested peptides  Alkaline pH causes artificial PTMs  Urea can cause protein carbamylation  Deamidation  Alkaline pH reagents causes artificial PTMs  Excipients/impurities induce protein oxidation  Incomplete digestion  Baseline noise  Long overnight incubation  Poor reproducibility  Disulfide bond scrambling  Oxidation AccuMAP™ Low pH digest
  25. 25. Suppression of Artificial, Nonenzymatic PTMs with AccuMAP™ Unmodifiedpeptide Deamidated form Scrambling issuppressed 46.13 47.44 46.08 Conventional digestion (pH 8) Low pH digestion HC319-LC134 scrambledbond Mass= 497.7491;Z= 4 SGTASVVCLLNNFYPR CK 39 40 41 42 43 44 45 46 47 Retentiontime(minutes) 48 49 50 51 52 0 50 100 0 50 100 68.5 69.5 71.5 72.5 70.93 70.83 70.78 70.68 70.45 70.62 70.16 70.36 71.06 71.31 71.46 71.58 71.77 72.15 69.0 70.0 70.5 71.0 Retentiontime(minutes) 72.0 73.0 B. 100 80 Conventional digestion (pH 8) 40 20 0 60 40 20 0 100 80 60 Low pH digestion LC134 HC319 Deamidationissuppressed A. GLEWIGAIYPGNGDTSYNQK  Deamidation suppression  Disulfide bond scrambling suppression Rituximab (Rituxan™, Roche) Panitumumab (Vectibix®, Amgen) + AccuMAP™ + AccuMAP™
  26. 26. Protein digestion in Gel with MS Compatible surfactant Protease Max™
  27. 27. In-gel protein digestion : Advantages and challenges Advantages of SDS-PAGE protein fractionation  Rapid removal of mass spec interfering impurities  Efficient reduction of sample complexity
  28. 28. Shortcomings of in-gel protein digestion  Inefficient peptide recovery from gel  Extensive peptide loss due to adsorption to a plastic ware  Lengthy and laborious procedure In-gel protein digestion : Advantages and challenges
  29. 29. ProteaseMAX™ Surfactant Cleavable bonds Degradation by temperature or acid LC/MS compatible compounds ZwitterionicheadHydrophobic tail + DegradationProducts ProteaseMAX™ mass spec compatible surfactant Mass spec compatible anionic surfactant Cleavable bonds Self-degradable mass spec compatible surfactant ProteaseMAX™ is designed to self-degrade over the course of mass spec protein sample preparation onto mass spec innocuous compounds.
  30. 30. Peptide Increase in peptide recovery with ProteaseMAX™,fold AGGALCANGAVR 1.45 QGDDGAALEVIEVHR 2.06 EHLPLPSEAGPTPCAPASFER 1.80 Improved peptide recovery ProteaseMAX™ increases peptide recovery from gel. MALDI-TOF spectrum of HTR1A protein digested in gel Peptides recovered with ProteaseMAX™ Peptides recovered in conventional digestion Saveliev et al. Analytical Chemistry 2013, 85 (2), pp 907–914.
  31. 31. Minimized peptide adsorption to plastic ware Peptide Increase in soluble peptide with ProteaseMAX (fold) PLSRTLSVAAK 16.6 TTYADFIASGRTGRRNAIHD 9.2 AAKIQASFRGHMARKK 4.6 EPPLSQEAFADLWKK 2.05 Saveliev et al. Analytical Chemistry 2013, 85 (2), pp 907–914. ProteaseMAX™ minimizes peptide adsorption to a plastic ware.
  32. 32. Enhanced analysis of a complex protein mixture with ProteaseMAX™-assisted in-gel digestion Gel-LC Analysis of Mouse Protein Extract Courtesy of Dr. Chris Adams, Stanford U ProteaseMAX™ increases number of peptide and protein identifications in a cell extract digested in gel.
  33. 33. Conventional In-gel Protein Digestion Peptide extraction (1.5 – 2 h) Mass spec analysis Mass spec analysis In-gel Protein Digestion with ProteaseMAX™ Digestion/extraction step (1 h) Protein digestion and peptide extraction are complete in a single 1 h step. 12-18 h Streamlined and rapid in-gel digestion with ProteaseMAX™
  34. 34. Protein Extraction from Tissues with MS Compatible surfactant Surfactant 3273
  35. 35. Surfactant 3273 – MS compatible SDS analog for tissue proteomics Surfactant3273 Degradationby a strong acid Cleavable bonds Mass spec compatible anionic surfactant DegradationProducts Hydrophobic tail Zwitterionichead + LC/MS compatible compounds 3273 is designed for efficient protein extraction from tissues and other biological samples and solubilization of protein pellets.  Enhanced protein extracting and solubilizing capability  Tolerates harsh treatment, including boiling
  36. 36. Improved protein extraction from pig heart with surfactant 3273 Protein IDs in pig liver extractsTotal extracted protein SDS-PAGE Chang et al. J. Proteome Res. 2015, 14 (3), pp 1587–1599. RapiGest Control 3273 Control Total membrane protein IDs in tissue extracts 3273 3273 enhances protein extraction from animal tissues.  Protein extraction efficiency is comparable to SDS.  Number of recovered membrane proteins is significantly increased.
  37. 37. Reference MS protein Materials Yeast and Human Protein extracts 6 X 5 Peptide Mix
  38. 38. Highly complex reference protein material for:  Mass spec instrument performance monitoring  Sample preparation method development Features  Compatible with LC/MS  Pre-processed for immediate use  Lot to lot consistency in protein composition and abundance Provided in intact and pre-digested formats. MS-compatible whole cell protein extracts Model proteomic material K562 human cells Yeast Reference Protein Materials address the critical question: Do my mass spec instrument, reagents and method work properly?
  39. 39. Mass spec instrument performance monitoring RT: 0.00 - 106.14 SM: 5G 0 30 40 50 60 70 80 90 100 Time (min) 40 20 0 80 60 100 0 40 20 80 60 100 40 20 0 80 60 100 87.6345.71 38.74 23.45 82.0432.20 35.72 79.9543.26 52.11 58.3726.04 61.02 77.63 90.0665.57 98.66 21.09 15.17 19.385.10 44.31 87.37 30.83 37.51 41.9722.59 81.30 81.96 49.48 50.78 57.2021.37 89.7775.56 76.69 97.6218.5711.088.74 38.22 45.10 87.9122.92 31.60 82.66 35.2025.48 58.4548.88 82.4565.43 72.43 96.42 98.61 20.61 14.88 18.94 10 20 8.10 st1 week nd2 week 3rd week Detecting deterioration of an instrument performance in a timely Manner (1 µg of human predigested protein extract) Relativeabundance
  40. 40. Example of compromised instrument performance 10 20 30 40 50 60 70 80 40 30 20 10 0 42.18 58.19 90.22 86.15 46.10 56.29 59.8135.74 83.5729.61 63.33 63.41 70.5054.44 75.9225.88 92.56 97.95 1 90 100 23.91 11.10 20.39 Courtesy by MS BioWorks, Ann Arbor,MI Good quality chromatogram (an instrument properly works) Time(min) Poor quality chromatogram (an instrument needs maintenance) Peptide ionization and retention times are compromised Detecting deterioration of an instrument performance in a timely manner. RelativeabundanceRelativeabundance 40 30 20 10 0 40.09 51.15 60.3328.07 32.39 92.54 92.7866.58 74.10 82.28 12.00 25.9823.17
  41. 41. 6x5 peptide mix Isomer # Sequence MW M 1 LLSLGAGEFK 1072.67318 0.00 2 LLSLGAGEFK 1062.64598 10.03 3 LLSLGAGEFK 1055.62878 7.02 4 LLSLGAGEFK 1048.61158 7.02 5 LLSLGAGEFK 1041.59448 7.02 most hydrophilic peptideMSPeakIntensity LC Chromatogram C18 LC Gradient (increasing hydrophobicity) most hydrophobic peptide Intensity Peptide Retention Time Linear Dynamic range m/z  Six peptides.  Each peptide is represented by five isotopologues mixed within linear concentration dynamic range.
  42. 42. A mixture of 6x5 = 30 peptides for complete monitoring of LC-MS/MS parameters Each peptide has five chromatographically indistinguishable isotopologues, with abundances spanning four orders of magnitude. Bolded amino acids (in red) are uniformly labeled with stable 13C and 15N atoms. Beri et al. Analytical Chemistry 2015, 87, 11635−11640
  43. 43. Mass Spectrometry solutions by Promega Digestion Trypsin Trypsin Gold, Trypsin/Lys-C Mix, Sequencing grade, immobilized Trypsin, Rapid Trypsin, AccuMAP™ Alternative proteases Lys-C, Arg-C, Glu-C, Asp-N, Chymotrypsin, Pepsin, ThermoLysin, Elastase Glycosidases PNGase, EndoH Antibody specific protease IdeZ IdeS protease Extraction Surfactant for in-gel protein digestion ProteaseMAX™ Surfactant for improved protein digestion, extraction and solubilization Protein extraction from Tissues Surfactant 3273. That surfactant is designed for efficient protein extraction from tissues and other biological samples and solubilization of protein pellets. Instrument performance monitoring Protein extracts for LC/MS Instrument MS compatible Human or Yeast protein extracts for instrument performance monitoring Learn more about Promega solutions at:
  44. 44. Promega solutions for Proteomics Expression Expression Vectors Mammalian expression vectors : regulated /constitutive expression Bacterial strains KRX, BL21 Cell-free protein expression Translation : Rabbit reticulocyte, Wheat Germ extract Transcription and Translation : TnT® system (Reticulocyte, Wheat Germ, Insect Cell), E.coli extracts Purification Affinity-based protein purification His-Tagged proteins : HisLink™ (resin, spin column, well- plates) Biotinylated proteins : SoftLink™, PinPoint™ HaloTag® Fusion proteins : from E.coli & Mammalian cells Magnetic beads (manual/automated) MagneGST™, Magne™ HaloTag®, MagneHis™ Antibody purification Magne™ Protein A/Magne™ Protein G Labeling Cell-free protein labeling FluoroTect™ GreenLys, Transcend™ Biotinylated Lysine tRNA HaloTag® Ligands Coumarin, Alexa Fluor® 488/660, Oregon Green®, TMR, R110 Processing/characterization Membrane vesicles Canine pancreatic microsomal membranes for signal peptide cleavage and core glycosylation studies Trypsin Trypsin Gold, Trypsin/Lys-C Mix, Sequencing grade, immobilized Trypsin Alternative proteases Lys-C, Arg-C, Glu-C, Asp-N, Chymotrypsin, Pepsin, ThermoLysin, Elastase Glycosidases PNGase, EndoH, Fetuin Surfactant ProteaseMAX™ Surfactant for improved protein digestion, extraction, solubilization compatible with MS Protein extracts for LC/MS Instrument MS compatible Human or Yeast protein extracts for instrument performance monitoring Antibody specific protease IdeZ , IdeS Interactions Live cell PPIs NanoBiT NanoBRET Drug discovery Target engagement : NanoBRET TE Target identification Dectection/Capture Western Blotting & ELISA Substrates, Conjugated Secondary antibodies Protein Arrays HaloLink™ Protein Array system