Post-digestion 18O Exchange/Labeling for Quantitative Shotgun Proteomics of Membrane Proteins

  • Xiaoying Ye
  • Brian T. Luke
  • Donald J. JohannJr
  • King C. Chan
  • DaRue A. Prieto
  • Akira Ono
  • Timothy D. Veenstra
  • Josip BlonderEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 893)


The role of membrane proteins is critical for regulation of physiologic and pathologic cellular processes. Hence it is not surpassing that membrane proteins make ∼70% of contemporary drug targets. Quantitative profiling of membrane proteins using mass spectrometry (MS)-based proteomics is critical in a quest for disease biomarkers and novel cancer drugs. Post-digestion 18O exchange is a simple and efficient method for differential 18O/16O stable isotope labeling of two biologically distinct specimens, allowing relative quantitation of proteins in complex mixtures when coupled with shotgun MS-based proteomics. Due to minimal sample consumption and unrestricted peptide tagging, 18O/16O stable isotope labeling is particularly suitable for amount-limited protein specimens typically encountered in membrane and clinical proteomics. This chapter describes a protocol that relies on shotgun proteomics for quantitative profiling of the detergent-insoluble membrane proteins isolated from HeLa cells, differentially transfected with plasmids expressing HIV Gag protein and its myristylation-defective N-terminal mutant. Whilst this protocol depicts solubilization of detergent-insoluble membrane proteins coupled with post-digestion 18O labeling, it is amenable to any complex membrane protein mixture. Described approach relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (v/v) methanol followed by differential 18O/16O labeling of protein digests in 20% (v/v) methanol buffer. After mixing, the differentially labeled peptides are fractionated using off-line strong cation exchange (SCX) followed by on-line reversed phase nanoflow reversed-phase liquid chromatography (nanoRPLC)-MS identification/quantiation of peptides/proteins. The use of methanol-based buffers in the context of the post-digestion 18O exchange/labeling eliminates the need for detergents or chaotropes that interfere with LC separations and peptide ionization. Sample losses are minimized because solubilization, digestion, and stable isotope labeling are carried out in a single tube, avoiding any sample transfer or buffer exchange between these steps.

Key words

O/16O stable isotope labeling Membrane protein quantiation Quantitative shotgun proteomics Mass spectrometry 



This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contracts HHSN261200800001E and NO1-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the United States Government.


  1. 1.
    Choudhary C, Mann M (2010) Decoding signalling networks by mass spectrometry-based proteomics. Nat Rev Mol Cell Biol 11:427–439PubMedCrossRefGoogle Scholar
  2. 2.
    Domon B, Aebersold R (2010) Options and considerations when selecting a quantitative proteomics strategy. Nat Biotechnol 28:710–721PubMedCrossRefGoogle Scholar
  3. 3.
    Ong SE, Mann M (2005) Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol 1:252–262PubMedCrossRefGoogle Scholar
  4. 4.
    Desiderio DM, Kai M (1983) Preparation of stable isotope-incorporated peptide internal standards for field desorption mass spectrometry quantification of peptides in biologic tissue. Biomed Mass Spectrom 10:471–479PubMedCrossRefGoogle Scholar
  5. 5.
    Yao X, Freas A, Ramirez J et al (2001) Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus. Anal Chem 73:2836–2842PubMedCrossRefGoogle Scholar
  6. 6.
    Ye X, Luke B, Andresson T, Blonder J (2009) 18O stable isotope labeling in MS-based proteomics. Brief Funct Genomic Proteomic 8:136–144PubMedCrossRefGoogle Scholar
  7. 7.
    Antonov VK, Ginodman LM, Rumsh LD et al (1981) Studies on the mechanisms of action of proteolytic enzymes using heavy oxygen exchange. Eur J Biochem 117:195–200PubMedCrossRefGoogle Scholar
  8. 8.
    Yao X, Afonso C, Fenselau C (2003) Dissection of proteolytic 18O labeling: endoprotease-catalyzed 16O-to-18O exchange of truncated peptide substrates. J Proteome Res 2:147–152PubMedCrossRefGoogle Scholar
  9. 9.
    Qian WJ, Monroe ME, Liu T et al (2005) Quantitative proteome analysis of human plasma following in vivo lipopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach. Mol Cell Proteomics 4:700–709PubMedCrossRefGoogle Scholar
  10. 10.
    Zang L, Palmer Toy D, Hancock WS et al (2004) Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 16O/18O isotopic labeling. J Proteome Res 3:604–612PubMedCrossRefGoogle Scholar
  11. 11.
    Wu CC, Yates JR 3rd (2003) The application of mass spectrometry to membrane proteomics. Nat Biotechnol 21:262–267PubMedCrossRefGoogle Scholar
  12. 12.
    Speers AE, Wu CC (2007) Proteomics of integral membrane proteins – theory and application. Chem Rev 107:3687–3714PubMedCrossRefGoogle Scholar
  13. 13.
    Blonder J, Conrads TP, Yu LR et al (2004) A detergent- and cyanogen bromide-free method for integral membrane proteomics: application to Halobacterium purple membranes and the human epidermal membrane proteome. Proteomics 4:31–45PubMedCrossRefGoogle Scholar
  14. 14.
    Blonder J, Rodriguez-Galan MC, Chan KC et al (2004) Analysis of murine natural killer cell microsomal proteins using two-dimensional liquid chromatography coupled to tandem electrospray ionization mass spectrometry. J Proteome Res 3:862–870PubMedCrossRefGoogle Scholar
  15. 15.
    Blonder J, Terunuma A, Conrads TP et al (2004) A proteomic characterization of the plasma membrane of human epidermis by high-throughput mass spectrometry. J Invest Dermatol 4:691–699CrossRefGoogle Scholar
  16. 16.
    Ye X, Johann DJ Jr, Hakami RM et al (2009) Optimization of protein solubilization for the analysis of the CD14 human monocyte membrane proteome using LC-MS/MS. J Proteomics 73:112–122PubMedCrossRefGoogle Scholar
  17. 17.
    Blonder J, Hale ML, Chan KC et al (2005) Quantitative profiling of the detergent-resistant membrane proteome of iota-b toxin induced Vero cells. J Proteome Res 4:523–531PubMedCrossRefGoogle Scholar
  18. 18.
    Blonder J, Yu LR, Radeva G et al (2006) Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96. J Proteome Res 5:349–360PubMedCrossRefGoogle Scholar
  19. 19.
    Stockwin LH, Blonder J, Bumke MA et al (2006) Proteomic analysis of plasma membrane from hypoxia-adapted malignant melanoma. J Proteome Res 5:2996–3007PubMedCrossRefGoogle Scholar
  20. 20.
    Ye X, Luke BT, Johann DJ et al (2010) Optimized method for computing (18)O/(16)O ratios of differentially stable-isotope labeled peptides in the context of postdigestion (18)O exchange/labeling. Anal Chem 82:5878–5886PubMedCrossRefGoogle Scholar
  21. 21.
    Freed EO (1998) HIV-1 gag proteins: diverse functions in the virus life cycle. Virology 251:1–15PubMedCrossRefGoogle Scholar
  22. 22.
    Ono A (2010) Relationships between plasma membrane microdomains and HIV-1 assembly. Biol Cell 102:335–350PubMedCrossRefGoogle Scholar
  23. 23.
    Ono A, Waheed AA, Joshi A, Freed EO (2005) Association of human immunodeficiency virus type 1 gag with membrane does not require highly basic sequences in the nucleocapsid: use of a novel Gag multimerization assay. J Virol 79:14131–14140PubMedCrossRefGoogle Scholar
  24. 24.
    Lindwasser OW, Resh MD (2001) Multimerization of human immunodeficiency virus type 1 Gag promotes its localization to barges, raft-like membrane microdomains. J Virol 75:7913–7924PubMedCrossRefGoogle Scholar
  25. 25.
    Fernandez J, Andrews L, Mische SM (1994) An improved procedure for enzymatic digestion of polyvinylidene difluoride-bound proteins for internal sequence analysis. Anal Biochem 218:112–117PubMedCrossRefGoogle Scholar
  26. 26.
    Klibanov AM (2001) Improving enzymes by using them in organic solvents. Nature 409:241–246PubMedCrossRefGoogle Scholar
  27. 27.
    Storms HF, van der Heijden R, Tjaden UR, van der Greef J (2006) Considerations for proteolytic labeling-optimization of 18O incorporation and prohibition of back-exchange. Rapid Commun Mass Spectrom 20:3491–3497PubMedCrossRefGoogle Scholar
  28. 28.
    Chan KC, Muschik GM, Issaq HJ (2000) Solid-state UV laser-induced fluorescence detection in capillary electrophoresis. Electrophoresis 21:2062–2066PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Xiaoying Ye
    • 1
  • Brian T. Luke
    • 2
  • Donald J. JohannJr
    • 3
  • King C. Chan
    • 1
  • DaRue A. Prieto
    • 1
  • Akira Ono
    • 4
  • Timothy D. Veenstra
    • 1
  • Josip Blonder
    • 1
    Email author
  1. 1.Laboratory of Proteomics and Analytical Technologies, Advanced Technology ProgramSAIC-Frederick, Inc., National Cancer Institute at FrederickFrederickUSA
  2. 2.Advanced Biomedical Computing Center, Advanced Technology ProgramSAIC-Frederick, Inc., National Cancer Institute at FrederickFrederickUSA
  3. 3.Medical Oncology Branch, Center for Cancer ResearchNational Cancer InstituteBethesdaUSA
  4. 4.Department of Microbiology and ImmunologyUniversity of Michigan Medical SchoolAnn ArborUSA

Personalised recommendations