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Reducing Complexity? Cysteine Reduction and S-Alkylation in Proteomic Workflows: Practical Considerations

  • Caroline A. EvansEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1977)

Abstract

Reduction and alkylation are common processing steps in sample preparation for qualitative and quantitative proteomic analyses. In principle, these steps mitigate the limitations resulting from the presence of disulfide bridges. There has been recurring debate in the proteomics community around their use, with concern over negative impacts that result from overalkylation (off-target, non-thiol sites) or incomplete reduction and/or S-alkylation of cysteine. This chapter integrates findings from a number of studies on different reduction and alkylation strategies, to guide users in experimental design for their optimal use in proteomic workflows.

Key words

N-terminal alkylation Alkylation Reduction Cysteine Artifact Proteomic 

References

  1. 1.
    Wedemeyer WJ, Welker E, Narayan M et al (2000) Disulfide bonds and protein folding. Biochemistry 39:4207–4216CrossRefGoogle Scholar
  2. 2.
    Lindahl M, Mata-Cabana A, Kieselbach T (2011) The disulfide proteome and other reactive cysteine proteomes: analysis and functional significance. Antioxid Redox Signal 14:2581–2642CrossRefGoogle Scholar
  3. 3.
    Lundell N, Schreitmüller T (1999) Sample preparation for peptide mapping — a pharmaceutical quality-control perspective. Anal Biochem 266:31–47CrossRefGoogle Scholar
  4. 4.
    Herbert B, Galvani M, Hamdan M et al (2001) Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how? Electrophoresis 22:2046–2057CrossRefGoogle Scholar
  5. 5.
    Padula MP, Berry IJ, Raymond B et al (2017) A comprehensive guide for performing sample preparation and top-down protein analysis. Proteome 5:11CrossRefGoogle Scholar
  6. 6.
    Sechi S, Chait BT (1998) Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification. Anal Chem 70:5150–5158CrossRefGoogle Scholar
  7. 7.
    Müller T, Winter D (2017) Systematic evaluation of protein reduction and alkylation reveals massive unspecific side effects by iodine-containing reagents. Mol Cell Proteomics 6:1173–1187CrossRefGoogle Scholar
  8. 8.
    Wojdyla K, Rogowska-Wrzesinska A (2015) Differential alkylation-based redox proteomics–lessons learnt. Redox Biol 6:240–252CrossRefGoogle Scholar
  9. 9.
    Dzieciatkowska M, Hill R, Hansen KC (2014) GeLC-MS/MS analysis of complex protein mixtures. In: Shotgun proteomics. Humana Press, New York, NY, pp 53–66CrossRefGoogle Scholar
  10. 10.
    Shevchenko A, Tomas H, Havlis J et al (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860CrossRefGoogle Scholar
  11. 11.
    Giansanti P, Tsiatsiani L, Low TY et al (2016) Six alternative proteases for mass spectrometry-based proteomics beyond trypsin. Nat Protoc 11:993CrossRefGoogle Scholar
  12. 12.
    Bittremieux W, Tabb DL, Impens F et al (2018) Quality control in mass spectrometry-based proteomics. Mass Spectrom Rev 37:697–711CrossRefGoogle Scholar
  13. 13.
    Herbert B, Hopwood F, Oxley D et al (2003) β-elimination: an unexpected artefact in proteome analysis. Proteomics 3:826–831CrossRefGoogle Scholar
  14. 14.
    Galvani M, Rovatti L, Hamdan M et al (2001) Protein alkylation in the presence/absence of thiourea in proteome analysis: a matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation. Electrophoresis 22:2066–2074CrossRefGoogle Scholar
  15. 15.
    Liang X, Wang JR, Wong KWV et al (2014) Optimization of 2-dimensional gel electrophoresis for proteomic studies of solid tumor tissue samples. Mol Med Rep 9:626–632CrossRefGoogle Scholar
  16. 16.
    Wu X, Xu C, Wang W (2017) Reduction and alkylation of proteins in 2D gel electrophoresis: before or after isoelectric focusing? Front Chem 5:59CrossRefGoogle Scholar
  17. 17.
    Mineki R, Taka H, Fujimura T et al (2002) In situ alkylation with acrylamide for identification of cysteinyl residues in proteins during one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Proteomics 2:1672–1681CrossRefGoogle Scholar
  18. 18.
    Lane LC (1978) A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis. Anal Biochem 86:655–664CrossRefGoogle Scholar
  19. 19.
    Turko IV, Sechi S (2007) Acrylamide—a cysteine alkylating reagent for quantitative proteomics. Methods Mol Biol 359:1–16CrossRefGoogle Scholar
  20. 20.
    Fischer R, Kessler BM (2015) Gel-aided sample preparation (GASP)—a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells. Proteomics 15:1224–1229CrossRefGoogle Scholar
  21. 21.
    D’Silva AM, Hyett JA, Coorssen JR (2017) A routine “top-down” approach to analysis of the human serum proteome. Proteome 5:13CrossRefGoogle Scholar
  22. 22.
    Naryzhny S (2018) Inventory of proteoforms as a current challenge of proteomics: some technical aspects. J Proteome 191:22–28CrossRefGoogle Scholar
  23. 23.
    Wang J, Zhao X, Zhao Y et al (2013) Influence of off-target in enzymatic digestion on the qualitative and quantitative analysis of proteins. Se Pu 31:927–933PubMedGoogle Scholar
  24. 24.
    Hains PG, Robinson PJ (2017) The impact of commonly used alkylating agents on artifactual peptide modification. J Proteome Res 16:3443–3447CrossRefGoogle Scholar
  25. 25.
    Suttapitugsakul S, Xiao H, Smeekens J et al (2017) Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics. Mol BioSyst 13:2574–2582CrossRefGoogle Scholar
  26. 26.
    Boja ES, Fales HM (2001) Off-target of a protein digest with iodoacetamide. Anal Chem 73:3576–3582CrossRefGoogle Scholar
  27. 27.
    Woods AG, Sokolowska I, Darie CC (2012) Identification of consistent alkylation of cysteine-less peptides in a proteomics experiment. Biochem Biophys Res Commun 419:305–308CrossRefGoogle Scholar
  28. 28.
    Rebecchi KR, Go EP, Xu L et al (2011) A general protease digestion procedure for optimal protein sequence coverage and post-translational modifications analysis of recombinant glycoproteins: application to the characterization of human lysyl oxidase-like 2 glycosylation. Anal Chem 83:8484–8491CrossRefGoogle Scholar
  29. 29.
    Yang Z, Attygalle AB (2007) LC/MS characterization of undesired products formed during iodoacetamide derivatization of sulfhydryl groups of peptides. J Mass Spectrom 42:233–243CrossRefGoogle Scholar
  30. 30.
    Lapko VN, Smith DL, Smith JB (2000) Identification of an artifact in the mass spectrometry of proteins derivatized with iodoacetamide. J Mass Spectrom 35:572–575CrossRefGoogle Scholar
  31. 31.
    Kruger R, Hung CW, Edelson-Averbukh M et al (2005) Iodoacetamide-alkylated methionine can mimic neutral loss of phos- phoric acid from phosphopeptides as exemplified by nano-electrospray ionization quadrupole time-of-flight parent ion scanning. Rapid Commun Mass Spectrom 19:1709–1716CrossRefGoogle Scholar
  32. 32.
    Guo M, Weng G, Yin D et al (2015) Identification of the over alkylation sites of a protein by IAM in MALDI-TOF/TOF tandem mass spectrometry. RSC Adv 5:103662–103668CrossRefGoogle Scholar
  33. 33.
    Xu P, Duong DM, Seyfried NT et al (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137:133–145CrossRefGoogle Scholar
  34. 34.
    Mouchahoir T, Schiel JE (2018) Development of an LC-MS/MS peptide mapping protocol for the NISTmAb. Anal Bioanal Chem 410:2111–2126CrossRefGoogle Scholar
  35. 35.
    Creasy DM, Cottrell JS (2004) Unimod: protein modifications for mass spectrometry. Proteomics 4:1534–1536CrossRefGoogle Scholar
  36. 36.
    Paulech J, Solis N, Cordwell SJ (2013) Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry. Biochim Biophys Acta 1834:372–379CrossRefGoogle Scholar
  37. 37.
    Huang J, Wang J, Li Q et al (2017) Enzyme and chemical assisted N-terminal blocked peptides analysis, ENCHANT, as a selective proteomics approach complementary to conventional shotgun approach. J Proteome Res 17:212–221CrossRefGoogle Scholar
  38. 38.
    Nielsen ML, Vermeulen M, Bonaldi T et al (2008) Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat Methods 5:459CrossRefGoogle Scholar
  39. 39.
    Schnatbaum K, Zolg D, Wenschuh H et al (2016) Fast and accurate determination of cysteine reduction and alkylation efficacy in proteomics workflows. JPT Application NoteGoogle Scholar
  40. 40.
  41. 41.
    Wiśniewski JR, Zougman A, Nagaraj N et al (2009) Universal sample preparation method for proteome analysis. Nat Methods 6:59Google Scholar
  42. 42.
    Stepanova E, Gygi SP, Paulo JA (2018) Filter-based protein digestion (FPD): a detergent-free and scaffold-based strategy for TMT workflows. J Proteome Res 17:1227–1234CrossRefGoogle Scholar
  43. 43.
    Mohammed H, Taylor C, Brown GD et al (2016) Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes. Nat Protoc 11:316CrossRefGoogle Scholar
  44. 44.
    Bayer M, König S (2016) Abundant cysteine side reactions in traditional buffers interfere with the analysis of posttranslational modifications and protein quantification—how to compromise. Rapid Commun Mass Spectrom 30:1823–1828CrossRefGoogle Scholar
  45. 45.
    Ackermann D, König S (2018) Comparative two-dimensional fluorescence gel electrophoresis. In: Difference gel electrophoresis. Humana Press, New York, NY, pp 69–78CrossRefGoogle Scholar
  46. 46.
    Herbert BR, Molloy MP, Gooley AA et al (1998) Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent. Electrophoresis 19:845–851CrossRefGoogle Scholar
  47. 47.
    Liu P, O’Mara BW, Warrack BM et al (2010) A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins. J Am Soc Mass Spectrom 21:837–844CrossRefGoogle Scholar
  48. 48.
    Wang Z, Rejtar T, Zhou ZS et al (2010) Desulfurization of cysteine-containing peptides resulting from sample preparation for protein characterization by mass spectrometry. Rapid Commun Mass Spectrom 24:267–275CrossRefGoogle Scholar
  49. 49.
    Zwyssig A, Schneider EM, Zeltner M et al (2017) Protein reduction and dialysis-free work-up through phosphines immobilized on a magnetic support: TCEP-functionalized carbon-coated cobalt nanoparticles. Chemistry 23:8585–8589CrossRefGoogle Scholar
  50. 50.
    Hale JE, Butler JP, Gelfanova V et al (2004) A simplified procedure for the reduction and alkylation of cysteine residues in proteins prior to proteolytic digestion and mass spectral analysis. Anal Biochem 333:174–181CrossRefGoogle Scholar
  51. 51.
    Svozil J, Bärenfaller K (2017) A cautionary tale on the inclusion of variable posttranslational modifications in database-dependent searches of mass spectrometry data. Methods Enzymol 586:433–452CrossRefGoogle Scholar
  52. 52.
    HaileMariam M, Eguez RV, Singh H et al (2018) S-Trap is an ultrafast sample preparation approach for shotgun proteomics. J Proteome Res 17:2917–2924CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemical and Biological EngineeringUniversity of SheffieldSheffieldUK

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