Abstract
Proteins provide the verbs to biology, and proteomics provides the nouns for their analytical and discovery-driven studies. The term proteomics was coined in the 1990s and deals with the protein complement of the genome—the proteome. Following the classical proteomics era, the development of new mass spectrometric methods for peptide analysis permitted the identification of proteins in peptide mixtures obtained by proteolytic digestion of complex samples, e.g., shotgun proteomics. Since its introduction, shotgun proteomics became the standard technique for the analysis of protein hydrolyzates in a high-throughput way. In this chapter, we provide a survey in shotgun proteomics highlighting instruments and techniques used in modern second and third proteomics generation.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Hancock W, LaBaer J, Marko-Varga GA (2011) Journal of proteome research—10th anniversary. J Proteome Res 10:1–2
Wasinger VC, Cordwell SJ, Cerpa-Poljak A et al (1995) Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis 16:1090–1094
Yates JR (2013) The revolution and evolution of shotgun proteomics for large-scale proteome analysis. J Am Chem Soc 135:1629–1640
O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021
Karas M, Hillenkamp F (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 60:2299–2301
Tanaka K, Waki H, Ido Y et al (1988) Protein and polymer analyses up to m/z 100 000 by laser ionization time?: of? flight mass spectrometry. Rapid Commun Mass Spectrom 2: 151–153
Fenn JB, Mann M, Meng CK et al (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246:64–71
Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038
Mann M, Højrup P, Roepstorff P (1993) Use of mass spectrometric molecular weight information to identify proteins in sequence databases. Biol Mass Spectrom 22:338–345
Henzel WJ, Billeci TM, Stults JT et al (1993) Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci 90:5011–5015
Yates JR, Speicher S, Griffin PR et al (1993) Peptide mass maps: a highly informative approach to protein identification. Anal Biochem 214:397–408
James P, Quadroni M, Carafoli E et al (1993) Protein identification by mass profile fingerprinting. Biochem Biophys Res Commun 195:58–64
Eng JK, McCormack AL, Yates Iii JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5:976–989
Patterson SD, Aebersold RH (2003) Proteomics: the first decade and beyond. Nat Gen 33:311–323
Link AJ, Eng J, Schieltz DM et al (1999) Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol 17:676–682
Wolters DA, Washburn MP, Yates JR (2001) An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem 73:5683–5690
Larsen MR, Roepstorff P (2000) Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis. Fresenius J Anal Chem 366:677–690
Harvey DJ (2001) Identification of protein-bound carbohydrates by mass spectrometry. Proteomics 1:311–328
MacCoss MJ, McDonald WH, Saraf A et al (2002) Shotgun identification of protein modifications from protein complexes and lens tissue. Proc Natl Acad Sci U S A 99: 7900–7905
Mann M, Jensen ON (2003) Proteomic analysis of post-translational modifications. Nat Biotechnol 21:255–261
Cournoyer JJ, Pittman JL, Ivleva VB et al (2005) Deamidation: differentiation of aspartyl from isoaspartyl products in peptides by electron capture dissociation. Protein Sci 14: 452–463
Edelson-Averbukh M, Pipkorn R, Lehmann WD (2007) Analysis of protein phosphorylation in the regions of consecutive serine/threonine residues by negative ion electrospray collision-induced dissociation. Approach to pinpointing of phosphorylation sites. Anal Chem 79:3476–3486
Harvey DJ (2009) Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: an update for 2003–2004. Mass Spectrom Rev 28:273–361
Gygi SP, Rist B, Gerber SA et al (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotech 17:994–999
Ong SE, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics MCP 1:376–386
Zhu H, Pan S, Gu S et al (2002) Amino acid residue specific stable isotope labeling for quantitative proteomics. Rapid Commun Mass Spectrom RCM 16:2115–2123
Ross PL, Huang YN, Marchese JN et al (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics MCP 3:1154–1169
Ishihama Y, Oda Y, Tabata T et al (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics MCP 4:1265–1272
Pratt JM, Simpson DM, Doherty MK et al (2006) Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes. Nat Protoc 1:1029–1043
Wang G, Wu WW, Zeng W et al (2006) Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: reproducibility, linearity, and application with complex proteomes. J Proteome Res 5:1214–1223
Zhang Y, Fonslow BR, Shan B et al (2013) Protein analysis by shotgun/bottom-up proteomics. Chem Rev 113:2343–2394
Glish GL, Vachet RW (2003) The basics of mass spectrometry in the twenty-first century. Nat Rev Drug Discov 2:140–150
Gelpi E (2008) From large analogical instruments to small digital black boxes: 40 years of progress in mass spectrometry and its role in proteomics. Part I 1965-1984. J Mass Spectrom JMS 43:419–435
Gelpi E (2009) From large analogical instruments to small digital black boxes: 40 years of progress in mass spectrometry and its role in proteomics. Part II 1985–2000. J Mass Spectrom JMS 44:1137–1161
Thelen JJ, Miernyk JA (2012) The proteomic future: where mass spectrometry should be taking us. Biochem J 444:169–181
Yates JR 3rd, Washburn MP (2013) Quantitative proteomics. Anal Chem 85:8881–8881
Domon B, Aebersold R (2006) Mass spectrometry and protein analysis. Science 312: 212–217
Spengler B (1997) Post-source decay analysis in matrix-assisted laser desorption/ionization mass spectrometry of biomolecules. J Mass Spectrom 32:1019–1036
Spengler B, Kirsch D, Kaufmann R et al (1992) Peptide sequencing by matrix-assisted laser-desorption mass spectrometry. Rapid Commun Mass Spectrom 6:105–108
Brown RS, Lennon JJ (1995) Sequence-specific fragmentation of matrix-assisted laser-desorbed protein/peptide ions. Anal Chem 67:3990–3999
Medzihradszky KF, Campbell JM, Baldwin MA et al (2000) The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer. Anal Chem 72:552–558
Yost RA, Enke CG (1979) Triple quadrupole mass spectrometry for direct mixture analysis and structure elucidation. Anal Chem 51: 1251–1264
Kanu AB, Dwivedi P, Tam M et al (2008) Ion mobility-mass spectrometry. J Mass Spectrom JMS 43:1–22
Cooks RG, Glish GL, Mc Luckey SA et al (1991) Ion trap mass spectrometry. Chem Eng News 69:12, Medium:–X
March RE (1992) Ion trap mass spectrometry. Int J Mass Spectrom Ion Process 118–119:71–135
Marshall AG, Hendrickson CL, Jackson GS (1998) Fourier transform ion cyclotron resonance mass spectrometry: a primer. Mass Spectrom Rev 17:1–35
Schaub TM, Hendrickson CL, Horning S et al (2008) High-performance mass spectrometry: Fourier transform ion cyclotron resonance at 14.5 Tesla. Anal Chem 80: 3985–3990
Schwartz JC, Senko MW, Syka JEP (2002) A two-dimensional quadrupole ion trap mass spectrometer. J Am Soc Mass Spectrom 13: 659–669
Yates JR, Ruse CI, Nakorchevsky A (2009) Proteomics by mass spectrometry: approaches, advances, and applications. Annu Rev Biomed Eng 11:49–79
Hager JW (2002) A new linear ion trap mass spectrometer. Rapid Commun Mass Spectrom 16:512–526
Makarov A (2000) Electrostatic axially harmonic orbital trapping: a high-performance technique of mass analysis. Anal Chem 72: 1156–1162
Makarov A, Denisov E, Kholomeev A et al (2006) Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer. Anal Chem 78:2113–2120
Hu Q, Noll RJ, Li H et al (2005) The Orbitrap: a new mass spectrometer. J Mass Spectrom JMS 40:430–443
Hebert AS, Richards AL, Bailey DJ et al (2013) The one hour yeast proteome. Mol Cell Proteomics MCP 13(1):339–47
Roepstorff P, Fohlman J (1984) Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom 11(11):601
Biemann K (1988) Contributions of mass spectrometry to peptide and protein structure. Biomed Environ Mass spectrom 16: 99–111
Jones AW, Cooper HJ (2011) Dissociation techniques in mass spectrometry-based proteomics. Analyst 136:3419–3429
Paizs B, Suhai S (2005) Fragmentation pathways of protonated peptides. Mass Spectrom Rev 24:508–548
Ting L, Cowley MJ, Hoon SL et al (2009) Normalization and statistical analysis of quantitative proteomics data generated by metabolic labeling. Mol Cell Proteomics 8: 2227–2242
McAlister GC, Phanstiel D, Wenger CD et al (2010) Analysis of tandem mass spectra by FTMS for improved large-scale proteomics with superior protein quantification. Anal Chem 82:316–322
Pichler P, Kocher T, Holzmann J et al (2011) Improved precision of iTRAQ and TMT quantification by an axial extraction field in an Orbitrap HCD cell. Anal Chem 83: 1469–1474
Olsen JV, Macek B, Lange O et al (2007) Higher-energy C-trap dissociation for peptide modification analysis. Nat Methods 4: 709–712
Zubarev RA, Horn DM, Fridriksson EK et al (2000) Electron capture dissociation for structural characterization of multiply charged protein cations. Anal Chem 72:563–573
Syka JE, Coon JJ, Schroeder MJ et al (2004) Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc Natl Acad Sci U S A 101:9528–9533
Coon JJ, Ueberheide B, Syka JE et al (2005) Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proc Natl Acad Sci U S A 102:9463–9468
Mikesh LM, Ueberheide B, Chi A et al (2006) The utility of ETD mass spectrometry in proteomic analysis. Biochim Biophys Acta 1764:1811–1822
Martin AJ, Synge RL (1941) Separation of the higher monoamino-acids by counter-current liquid-liquid extraction: the amino-acid composition of wool. Biochem J 35:91–121
Washburn MP, Wolters D, Yates JR 3rd (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19:242–247
Thakur SS, Geiger T, Chatterjee B et al (2011) Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation. Mol Cell Proteom MCP 10:M110–M003699
Nagaraj N, Kulak NA, Cox J et al (2012) System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap. Mol Cell Proteom MCP 11: M111–M013722
Xu T, Venable JD, Park SK et al. (2006) ProLuCID: a fast and sensitive tandem mass spectra-based protein identification program. Mol Cell Pro 5(10 suppl):S174
Perkins DN, Pappin DJ, Creasy DM et al (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20: 3551–3567
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372
Colinge J, Bennett KL (2007) Introduction to computational proteomics. PLoS Comput Biol 3
Deutsch EW, Lam H, Aebersold R (2008) Data analysis and bioinformatics tools for tandem mass spectrometry in proteomics. Phys Genomic 33:18–25
Eng JK, Searle BC, Clauser KR et al (2011) A face in the crowd: recognizing peptides through database search. Mol Cell proteom MCP 10:R111–R009522
Zhou H, Ranish JA, Watts JD et al (2002) Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nat Biotechnol 20:512–515
Hansen KC, Schmitt-Ulms G, Chalkley RJ et al (2003) Mass spectrometric analysis of protein mixtures at low levels using cleavable 13C-isotope-coded affinity tag and multidimensional chromatography. Mol Cell Proteom MCP 2:299–314
Schmidt A, Kellermann J, Lottspeich F (2005) A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics 5:4–15
Hsu JL, Huang SY, Chow NH et al (2003) Stable-isotope dimethyl labeling for quantitative proteomics. Anal Chem 75:6843–6852
Boersema PJ, Aye TT, van Veen TA et al (2008) Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates. Proteomics 8:4624–4632
Thompson A, Schafer J, Kuhn K et al (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75: 1895–1904
Thingholm TE, Palmisano G, Kjeldsen F et al (2010) Undesirable charge-enhancement of isobaric tagged phosphopeptides leads to reduced identification efficiency. J Proteome Res 9:4045–4052
Pichler P, Kocher T, Holzmann J et al (2010) Peptide labeling with isobaric tags yields higher identification rates using iTRAQ 4-plex compared to TMT 6-plex and iTRAQ 8-plex on LTQ Orbitrap. Anal Chem 82: 6549–6558
Ting L, Rad R, Gygi SP et al (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 8:937–940
Wenger CD, Lee MV, Hebert AS et al (2011) Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging. Nat Methods 8:933–935
Miyagi M, Rao KC (2007) Proteolytic 18O-labeling strategies for quantitative proteomics. Mass Spectrom Rev 26:121–136
Oda Y, Huang K, Cross FR et al (1999) Accurate quantitation of protein expression and site-specific phosphorylation. Proc Natl Acad Sci U S A 96:6591–6596
Liao L, McClatchy DB, Park SK et al (2008) Quantitative analysis of brain nuclear phosphoproteins identifies developmentally regulated phosphorylation events. J Proteome Res 7:4743–4755
McClatchy DB, Liao L, Park SK et al (2007) Quantification of the synaptosomal proteome of the rat cerebellum during post-natal development. Genome Res 17:1378–1388
Ong SE, Kratchmarova I, Mann M (2003) Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC). J Proteome Res 2:173–181
Blagoev B, Ong SE, Kratchmarova I et al (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat Biotechnol 22:1139–1145
Olsen JV, Blagoev B, Gnad F et al (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127:635–648
Schwanhausser B, Gossen M, Dittmar G et al (2009) Global analysis of cellular protein translation by pulsed SILAC. Proteomics 9: 205–209
Cambridge SB, Gnad F, Nguyen C et al (2011) Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover. J Proteome Res 10: 5275–5284
Geiger T, Cox J, Ostasiewicz P et al (2010) Super-SILAC mix for quantitative proteomics of human tumor tissue. Nat Methods 7: 383–385
Ong SE (2012) The expanding field of SILAC. Anal Bioanalytical Chem 404:967–976
Bondarenko PV, Chelius D, Shaler TA (2002) Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry. Anal Chem 74:4741–4749
Chelius D, Bondarenko PV (2002) Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry. J Proteome Res 1:317–323
Bantscheff M, Lemeer S, Savitski MM et al (2012) Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present. Anal Bioanal Chem 404: 939–965
Gallien S, Duriez E, Domon B (2011) Selected reaction monitoring applied to proteomics. J Mass Spectrom JMS 46:298–312
Gerber SA, Rush J, Stemman O et al (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A 100:6940–6945
Kirkpatrick DS, Gerber SA, Gygi SP (2005) The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications. Methods 35:265–273
Acknowledgements
Conselho Nacional de Pesquisas (CNPq), Brazil, grant # 308819/ 2011-0, and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Brazil, grant # E-26/110.138/2013. We thank Dr Magno Junqueira, Proteomics Unit, UFRJ, for critically reading the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Nogueira, F.C.S., Domont, G.B. (2014). Survey of Shotgun Proteomics. In: Martins-de-Souza, D. (eds) Shotgun Proteomics. Methods in Molecular Biology, vol 1156. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0685-7_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0685-7_1
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0684-0
Online ISBN: 978-1-4939-0685-7
eBook Packages: Springer Protocols