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Analytical strategies for the global quantification of intact proteins

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Abstract

The quantification of intact proteins is a relatively recent development in proteomics. In eukaryotic organisms, proteins are present as multiple isoforms as the result of variations in genetic code, alternative splicing, post-translational modification and other processing events. Understanding the identities and biological functions of these isoforms and how their concentrations vary across different states is the central goal of proteomics. To date, the bulk of proteomics research utilizes a “bottom-up” approach, digesting proteins into their more manageable constitutive peptides, but sacrificing information about the specific isoform and combinations of post-translational modifications present on the protein. Very specific strategies for protein quantification such as the enzyme-linked immunosorbent assay and Western blot are commonplace in laboratories and clinics, but impractical for the study of global biological changes. Herein, we describe strategies for the quantification of intact proteins, their distinct advantages, and challenges to their employment. Techniques contained in this review include the more traditional and widely employed methodology of differential gel electrophoresis and more recently developed mass spectrometry-based techniques including metabolic labeling, chemical labeling, and label-free methodologies.

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References

  • Ardelt P, Dorka P, Jaquet K, Meilmeyer LMG, Kortke H, Korfer R, Notohamiprodjo G (1998) Microanalysis and distribution of cardiac troponin I phospho species in heart areas. Biol Chem 379(3):341–347

    Article  PubMed  CAS  Google Scholar 

  • Berger SJ, Lee S, Anderson GA, Pasa-Tolic L, Tolic N, Shen Y, Zhao R, Smith DL (2002) High-throughput global peptide proteomic analysis by combining stable isotope amino acid labeling and data-dependent multiplexed-MS/MS. Anal Chem 74(19):4994–5000

    Article  PubMed  CAS  Google Scholar 

  • Bogdanov B, Smith RD (2005) Proteomics by FTICR mass spectrometry: top down and bottom up. Mass Spectrom Rev 24(2):168–200

    Article  PubMed  CAS  Google Scholar 

  • Collier TS, Hawkridge AM, Georgianna DR, Payne GA, Muddiman DC (2008) Top-down identification and quantification of stable isotope labeled proteins from Aspergillus flavus using online nano-flow reversed-phase liquid chromatography coupled to a LTQ-FTICR mass spectrometer. Anal Chem 80(13):4994–5001

    Article  PubMed  CAS  Google Scholar 

  • Collier TS, Sarkar P, Rao B, Muddiman DC (2010) Quantitative top-down proteomics of SILAC labeled human embryonic stem cells. J Am Soc Mass Spectrom 21(6):879–889

    Article  PubMed  CAS  Google Scholar 

  • Das T, Sen A, Kempf T, Pramanik SR, Mandal C, Mandal C (2003) Induction of glycosylation in human C-reactive protein under different pathological conditions. Biochem J 373:345–355

    Article  PubMed  CAS  Google Scholar 

  • Das T, Mandal C, Mandal C (2004) Variations in binding characteristics of glycosylated human C-reactive proteins in different pathological conditions. Glycoconjugate J 20:537–543

    Article  CAS  Google Scholar 

  • de Godoy LMF, Olsen JV, de Souza GA, Li G, Mortensen P, Mann M (2006) Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system. Genome Biol 7(6):R50-R50.15

    Google Scholar 

  • Dillon TM, Bondarenko PV (2004) Development of an analytical reversed-phase high-performance liquid chromatography electrospray ionization mass spectrometry method for characterization of recombinant antibodies. J Chromatogr A 1053:299–305

    PubMed  CAS  Google Scholar 

  • Du Y, Meng F, Patrie SM, Miller LM, Kelleher NL (2004) Improved molecular weight-based processing of intact proteins for interrogation by quadrupole-enhanced FT MS/MS. J Proteome Res 3:801–806

    Article  PubMed  CAS  Google Scholar 

  • Du Y, Parks BA, Sohn S, Kwast KE, Kelleher NL (2006) Top-down approaches for measuring expression ratios of intact yeast proteins using Fourier transform mass spectrometry. Anal Chem 78(3):686–694

    Article  PubMed  CAS  Google Scholar 

  • Ge Y, Lawhorn BG, El Naggar M, Strauss E, Park JH, Begley TP, McLafferty FW (2002) Top down characterization of larger proteins (45 kda) by electron capture dissociation mass spectrometry. J Am Chem Soc 124(4):672–678

    Article  PubMed  CAS  Google Scholar 

  • Gharbi S, Gaffney P, Yang A, Zvelebil MJ, Cramer R, Waterfield MD, Timms JF (2002) Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system. Mol Cell Proteomics 1(2):91–98

    Article  PubMed  CAS  Google Scholar 

  • Gordon EF, Mansoori BA, Carroll CF, Muddiman DC (1999) Hydropathic influences on the quantification of equine heart cytochrome c using relative ion abundance measurements by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. J Mass Spectrom 34(10):1055–1062

    Article  PubMed  CAS  Google Scholar 

  • Greengauz-Roberts O, Stoppler H, Nomura S, Yamaguchi H, Goldenring JR, Podolsky RH, Lee JR, Dynan WS (2005) Saturation labeling with cysteine-reactive cyanine fluorescent dyes provides increased sensitivity for protein expression profiling of laser-microdissected clinical specimens. Proteomics 5(7):1746–1757

    Article  PubMed  CAS  Google Scholar 

  • Hanke S, Besir H, Oesterhelt D, Mann M (2008) Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level. J Proteome Res 7:1118–1130

    Article  PubMed  CAS  Google Scholar 

  • Hawkridge AM, Heublein DM, Bergen RH III, Cataliotti A, Burnett JC Jr, Muddiman DC (2005) Quantitative mass spectral evidence for the absence of circulating brain natriuretic peptide (BNP-32) in severe human heart failure. Proc Nat Acad Sci USA 102(48):17442–17447

    Article  PubMed  CAS  Google Scholar 

  • Horn DM, Zubarev RA, McLafferty FW (2000) Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules. J Am Soc Mass Spectrom 11(4):320–332

    Article  PubMed  CAS  Google Scholar 

  • Hu QZ, Noll RJ, Li HY, Makarov A, Hardman M, Cooks RG (2005) The Orbitrap: a new mass spectrometer. J Mass Spectrom 40(4):430–433

    Article  PubMed  CAS  Google Scholar 

  • Jennings KR (2000) The changing impact of the collision-induced decomposition of ions on mass spectrometry. Int J Mass Spectrom 200(1–3):479–493

    CAS  Google Scholar 

  • Johnson KL, Mason CJ, Muddiman DC, Eckel JE (2004) Analysis of the low molecular weight fraction of serum by LC–dual ESI-FT-ICR mass spectrometry: precision of retention time, mass, and ion abundance. Anal Chem 76(17):5097–5103

    Article  PubMed  CAS  Google Scholar 

  • Julka S, Regnier F (2004) Quantification in proteomics through stable isotope coding: a review. J Proteome Res 3(3):350–363

    Article  PubMed  CAS  Google Scholar 

  • Karp NA, Lilley KS (2005) Maximising sensitivity for detecting changes in protein expression: experimental design using minimal CyDyes. Proteomics 5(12):3105–3115

    Article  PubMed  CAS  Google Scholar 

  • Karp NA, Kreil DP, Lilley KS (2004) Determining a significant change in protein expression with decyder (TM) during a pair-wise comparison using two-dimensional difference gel electrophoresis. Proteomics 4(5):1421–1432

    Article  PubMed  CAS  Google Scholar 

  • Katrukha AG (1998) Degradation of cardiac troponin I: Implication for reliable immunodetection. Clin Chem 44(12):2433

    PubMed  CAS  Google Scholar 

  • Kelleher NL, Taylor SV, Grannis D, Kinsland C, Chiu HJ, Begley TP, McLafferty FW (1998) Efficient sequence analysis of the six gene products (7–74 kda) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry. Protein Sci 7(8):1796–1801

    Article  PubMed  CAS  Google Scholar 

  • Kelleher NL, Lin HY, Valaskovic GA, Aaserud DJ, Fridriksson EK, McLafferty FW (1999) Top down versus bottom up protein characterization by tandem high-resolution mass spectrometry. J Am Chem Soc 121(4):806–812

    Article  CAS  Google Scholar 

  • Kenyon GL, De Marini DM, Fuchs E, Galas DJ, Kirsch JF, Leyh TS, Moos WH, Petsko GA, Ringe D, Rubin GM, Sheahan LC (2002) Defining the mandate of proteomics in the post-genomics era: workshop report. Mol Cell Proteomics 1(10):763–780

    PubMed  CAS  Google Scholar 

  • Kreil DP, Karp NA, Lilley KS (2004) DNA microarray normalization methods can remove bias from differential protein expression analysis of 2D difference gel electrophoresis results. Bioinformatics 20(13):2026–2034

    Article  PubMed  CAS  Google Scholar 

  • Labugger R, Organ L, Collier C, Atar D, Van Eyk JE (2000) Extensive troponin I and t modification detected in serum from patients with acute myocardial infarction. Circulation 102(11):1221–1226

    Article  PubMed  CAS  Google Scholar 

  • Lilley KS, Razzaq A, Dupree P (2002) Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation. Curr Opin Chem Biol 6(1):46–50

    Article  PubMed  CAS  Google Scholar 

  • Little DP, Speir JP, Senko MW, O’Connor PB, McLafferty FW (1994) Infrared multiphoton dissociation of large multiply charged ions for biomolecule sequencing. Anal Chem 66(18):2809–2815

    Article  PubMed  CAS  Google Scholar 

  • Martinovic S, Veenstra TD, Anderson GA, Pasa-Tolic L, Smith RD (2002) Selective incorporation of isotopically labeled amino acids for identification of intact proteins on a proteome-wide level. J Mass Spectrom 37(1):99–107

    Article  PubMed  Google Scholar 

  • Mazur MT, Cardasis HL, Spellman DS, Liaw A, Yates NA, Hendrickson RC (2010) Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry. Proc Nat Acad Sci USA 107(17):7728–7733

    Article  PubMed  CAS  Google Scholar 

  • Meng FY, Cargile BJ, Patrie SM, Johnson JR, McLoughlin SM, Kelleher NL (2002) Processing complex mixtures of intact proteins for direct analysis by mass spectrometry. Anal Chem 74(13):2923–2929

    Article  PubMed  CAS  Google Scholar 

  • Meng F, Wiener MC, Sachs JR, Burns C, Verma P, Paweletz CP, Mazur MT, Deyanova EG, Yates NA, Hendrickson CL (2007) Quantitative analysis of complex peptide mixtures using FTMS and differential mass spectrometry. J Am Soc Mass Spectrom 18:226–233

    Article  PubMed  CAS  Google Scholar 

  • Oda Y, Huang K, Cross FR, Cowburn D, Chait BT (1999) Accurate quantitation of protein expression and site-specific phosphorylation. Proc Nat Acad Sci USA 96(12):6591–6596

    Article  PubMed  CAS  Google Scholar 

  • Ong S, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386

    Article  PubMed  CAS  Google Scholar 

  • Ong S, 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

    Article  PubMed  CAS  Google Scholar 

  • Parks BA, Jiang L, Thomas PM, Wenger CD, Roth MJ, Boyne Ii MT, Burke PV, Kwast KE, Kelleher NL (2007) Top-down proteomics on a chromatographic time scale using linear ion trap Fourier transform hybrid mass spectrometers. Anal Chem 79(21):7984–7991

    Article  PubMed  CAS  Google Scholar 

  • Patton WF (2000) A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis 21(6):1123–1144

    Article  PubMed  CAS  Google Scholar 

  • Pesavento JJ, Mizzen CA, Kelleher NL (2006) Quantitative analysis of modified proteins and their positional isomers by tandem mass spectrometry: human histone h4. Anal Chem 78(13):4271–4280

    Article  PubMed  CAS  Google Scholar 

  • Quadroni M, James P (1999) Proteomics and automation. Electrophoresis 20(4–5):664–677

    Article  PubMed  CAS  Google Scholar 

  • Shaw J, Rowlinson R, Nickson J, Stone T, Sweet A, Williams K, Tonge R (2003) Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes. Proteomics 3(7):1181–1195

    Article  PubMed  CAS  Google Scholar 

  • Siuti N, Kelleher NL (2007) Decoding protein modifications using top-down mass spectrometry. Nat Methods 4(10):817–821

    Article  PubMed  CAS  Google Scholar 

  • Syka JEP, Marto JA, Bai DL, Horning S, Senko MW, Schwartz JC, Ueberheide B, Gacia B, Busby S, Muratore T, Shabanowitz J, Hunt DF (2004) Novel linear quadrupole ion trap/FT mass spectrometer: Performance characterization and use in the comparative analysis of histone h3 post-translational modifications. J Proteome Res 3(3):621–626

    Article  PubMed  CAS  Google Scholar 

  • Tonge R, Shaw J, Middleton B, Rowlinson R, Rayner S, Young J, Pognan F, Hawkins E, Currie I, Davison M (2001) Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1(3):377–396

    Article  PubMed  CAS  Google Scholar 

  • Tran JC, Doucette AA (2008) Gel-eluted liquid fraction entrapment electrophoresis: An electrophoretic method for broad molecular weight range proteome separation. Anal Chem 80(5):1568–1573

    Article  PubMed  CAS  Google Scholar 

  • Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18(11):2071–2077

    Article  PubMed  CAS  Google Scholar 

  • Van Hoof D, Pinkse MW, Oostwaard DW, Mummery CL, Heck AJ, Krijgsveld J (2007) An experimental correction for arginine to proline conversion artifacts in SILAC-based quantitative proteomics. Nat Methods 4:677–678

    Article  PubMed  Google Scholar 

  • Veenstra TD, Martinovic S, Anderson GA, Pasa-Tolic L, Smith RD (2000) Proteome analysis using selective incorporation of isotopically labeled amino acids. J Am Soc Mass Spectrom 11(1):78–82

    Article  PubMed  CAS  Google Scholar 

  • Vellaichamy A, Tran JC, Catherman AD, Lee JE, Kellie JF, Sweet SMM, Zamdborg L, Thomas PM, Ahlf DR, Durbin KR, Valaskovic GA, Kelleher NL (2010) Size-sorting combined with improved nanocapillary liquid chromatography–mass spectrometry for identification of intact proteins up to 80 kda. Anal Chem 82(4):1234–1244

    Article  PubMed  CAS  Google Scholar 

  • Viswanathan S, Unlu M, Minden JS (2006) Two-dimensional difference gel electrophoresis. Nat Protocols 1(3):1351–1358

    Article  CAS  Google Scholar 

  • Waanders LF, Hanke S, Mann M (2007) Top-down quantitation and characterization of SILAC-labeled proteins. J Am Soc Mass Spectrom 18(11):2058–2064

    Article  PubMed  CAS  Google Scholar 

  • Wang Y (2005) Effects of chromatography conditions on intact protein separations for top-down proteomics. J Chromatogr 1073(1–2):35

    CAS  Google Scholar 

  • Washburn MP, Ulaszek RR, Deciu C, Schieltz DM, Yates JR (2002) Analysis of quantitative proteomic data generated via multidimensional protein identification technology. Anal Chem 74(7):1650–1657

    Article  PubMed  CAS  Google Scholar 

  • Yan X, Xu M, Yang LM, Wang QQ (2010) Absolute quantification of intact proteins via 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid-10-maleimidoethylacetamide-europium labeling and HPLC coupled with species-unspecific isotope dilution ICPMS. Anal Chem 82(4):1261–1269

    Article  PubMed  CAS  Google Scholar 

  • Yates JR, Cociorva D, Liao LJ, Zabrouskov V (2006) Performance of a linear ion trap-orbitrap hybrid for peptide analysis. Anal Chem 78(2):493–500

    Article  PubMed  CAS  Google Scholar 

  • Zamdborg L, LeDuc RD, Glowacz KJ, Kim Y, Viswanathan V, Spaulding IT, Early BP, Bluhm EJ, Babai S, Kelleher NL (2007) Prosight ptm 2.0: Improved protein identification and characterization for top down mass spectrometry. Nucleic Acids Res 35:W701–W706

    Article  PubMed  Google Scholar 

  • Zhang RJ, Sioma CS, Wang SH, Regnier FE (2001) Fractionation of isotopically labeled peptides in quantitative proteomics. Anal Chem 73(21):5142–5149

    Article  PubMed  CAS  Google Scholar 

  • Zubarev RA, Kelleher NL, McLafferty FW (1998) Electron capture dissociation of multiply charged protein cations: a nonergodic process. J Am Chem Soc 120(13):3265–3266

    Article  CAS  Google Scholar 

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Acknowledgments

The authors wish to acknowledge funding support from NSF Grant MCB-0918611, the W. M. Keck Foundation, and North Carolina State University.

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  1. W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, NC, 27695, USA

    Timothy S. Collier & David Charles Muddiman

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  1. Timothy S. Collier
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  2. David Charles Muddiman
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Correspondence to David Charles Muddiman.

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Collier, T.S., Muddiman, D.C. Analytical strategies for the global quantification of intact proteins. Amino Acids 43, 1109–1117 (2012). https://doi.org/10.1007/s00726-012-1285-z

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