Skip to main content

Advertisement

SpringerLink
Log in

A proteomic approach for plasma biomarker discovery with 8-plex iTRAQ labeling and SCX-LC-MS/MS

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Plasma is recognized as a promising source of disease-related biomarkers, and proteomic approaches for identifying novel plasma biomarkers are in great demand. However, the complexity and dynamic protein concentration range of plasma remain the main obstacles for current research in this field. In this study, plasma proteins were prefractioned by immunodepletion and Protein Equalizer Technology to remove high abundant proteins, then labeled with an 8-plex isobaric tags for relative and absolute quantitation (iTRAQ) to improve the peptide ionization, and analyzed by strong-cation-exchange(SCX) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our results showed that both prefraction methods were complementary, with regard to the number of identified proteins. Good chromatographic technique is important to further fractionate the iTRAQ labeling peptides, which allowed 320 and 248 different proteins to be characterized from two prefraction methods, respectively, encompassing a wide array of biological functions and a broad dynamic range of 107. Furthermore, the accuracy of iTRAQ relative quantitation for differentially expressed proteins is associated with the number of peptides hits per protein.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Omenn GS, States DJ, Adamski M et al (2005) Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database. Proteomics 5:3226–3245

    Article  CAS  PubMed  Google Scholar 

  2. Lochnit G, Geyer R (2004) An optimized protocol for nano-LC-MALDI-TOF-MS coupling for the analysis of proteolytic digests of glycoproteins. Biomed Chromatogr 18:841–848

    Article  CAS  PubMed  Google Scholar 

  3. Zolotarjova N, Martosella J, Nicol G et al (2005) Differences among techniques for high-abundant protein depletion. Proteomics 5:3304–3313

    Article  CAS  PubMed  Google Scholar 

  4. Righetti PG, Boschetti E, Lomas L et al (2006) Protein Equalizer Technology: the quest for a “democratic proteome”. Proteomics 6:3980–3992

    Article  CAS  PubMed  Google Scholar 

  5. Tonack S, Aspinall-O’Dea M, Jenkins RE et al (2009) A technically detailed and pragmatic protocol for quantitative serum proteomics using iTRAQ. J Proteomics 73:352–356

    Article  CAS  PubMed  Google Scholar 

  6. Bouchal P, Roumeliotis T, Hrstka R et al (2009) Biomarker discovery in low-grade breast cancer using isobaric stable isotope tags and two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) based quantitative proteomic analysis. J Proteome Res 8:362–373

    Article  CAS  PubMed  Google Scholar 

  7. Choe L, D’Ascenzo M, Relkin NR et al (2007) 8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer’s disease. Proteomics 7:3651–3660

    Article  CAS  PubMed  Google Scholar 

  8. Ow SY, Cardona T, Taton A et al (2008) Quantitative shotgun proteomics of enriched heterocysts from Nostoc sp. PCC 7120 using 8-plex isobaric peptide tags. J Proteome Res 7:1615–1628

    Article  CAS  PubMed  Google Scholar 

  9. Ernoult E, Bourreau A, Gamelin E et al (2010) A proteomic approach for plasma biomarker discovery with iTRAQ labelling and OFFGEL fractionation. J Biomed Biotechnol 2010:917–927

    Google Scholar 

  10. Song X, Bandow J, Sherman J et al (2008) iTRAQ experimental design for plasma biomarker discovery. J Proteome Res 7:2952–2958

    Article  CAS  PubMed  Google Scholar 

  11. Hergenroeder G, Redell JB, Moore AN et al (2008) Identification of serum biomarkers in brain-injured adults: potential for predicting elevated intracranial pressure. J Neurotrauma 25:79–93

    Article  PubMed  Google Scholar 

  12. Rai AJ, Gelfand CA, Haywood BC et al (2005) HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples. Proteomics 5:3262–3277

    Article  CAS  PubMed  Google Scholar 

  13. Shilov IV, Seymour SL, Patel AA et al (2007) The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra. Mol Cell Proteomics 6:1638–1655

    Article  CAS  PubMed  Google Scholar 

  14. Huang L, Fang X (2008) Immunoaffinity fractionation of plasma proteins by chicken IgY antibodies. Methods Mol Biol 425:41–51

    Article  CAS  PubMed  Google Scholar 

  15. Sihlbom C, Kanmert I, Bahr H et al (2008) Evaluation of the combination of bead technology with SELDI-TOF-MS and 2-D DIGE for detection of plasma proteins. J Proteome Res 7:4191–4198

    Article  CAS  PubMed  Google Scholar 

  16. Beausoleil SA, Jedrychowski M, Schwartz D et al (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci USA 101:12130–12135

    Article  CAS  PubMed  Google Scholar 

  17. Link AJ, Eng J, Schieltz DM et al (1999) Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol 17:676–682

    Article  CAS  PubMed  Google Scholar 

  18. Ernoult E, Gamelin E, Guette C (2008) Improved proteome coverage by using iTRAQ labelling and peptide OFFGEL fractionation. Proteome Sci 6:27

    Article  PubMed  Google Scholar 

  19. Tanaka Y, Akiyama H, Kuroda T et al (2006) A novel approach and protocol for discovering extremely low-abundance proteins in serum. Proteomics 6:4845–4855

    Article  CAS  PubMed  Google Scholar 

  20. Chao J, Schmaier A, Chen LM et al (1996) Kallistatin, a novel human tissue kallikrein inhibitor: levels in body fluids, blood cells, and tissues in health and disease. J Lab Clin Med 127:612–620

    Article  CAS  PubMed  Google Scholar 

  21. Svendsen CB, Hummelshoj T, Munthe-Fog L et al (2008) Ficolins and Mannose-Binding Lectin in Danish patients with sarcoidosis. Respir Med 102:1237–1242

    Article  PubMed  Google Scholar 

  22. Kamper EF, Papaphilis AD, Angelopoulou MK et al (1999) Serum levels of tetranectin, intercellular adhesion molecule-1 and interleukin-10 in B-chronic lymphocytic leukemia. Clin Biochem 32:639–645

    Article  CAS  PubMed  Google Scholar 

  23. Schwaeble WJ, Reid KB (1999) Does properdin crosslink the cellular and the humoral immune response? Immunol Today 20:17–21

    Article  CAS  PubMed  Google Scholar 

  24. Saito Y, Watanabe Y, Saito E et al (2001) Production and application of monoclonal antibodies to human Selenoprotein P. J Health Sci 47:346–352

    Article  CAS  Google Scholar 

  25. Koenig W, Khuseyinova N, Hoffmann MM et al (2002) CD14 C(-260)→T polymorphism, plasma levels of the soluble endotoxin receptor CD14, their association with chronic infections and risk of stable coronary artery disease. J Am Coll Cardiol 40:34–42

    Article  CAS  PubMed  Google Scholar 

  26. Omenn GS (2005) Exploring the human plasma proteome. Proteomics 5:3223–3225

    Article  CAS  PubMed  Google Scholar 

  27. Haab BB, Geierstanger BH, Michailidis G et al (2005) Immunoassay and antibody microarray analysis of the HUPO Plasma Proteome Project reference specimens: systematic variation between sample types and calibration of mass spectrometry data. Proteomics 5:3278–3291

    Article  CAS  PubMed  Google Scholar 

  28. Wader KF, Fagerli UM, Holt RU et al (2008) Elevated serum concentrations of activated hepatocyte growth factor activator in patients with multiple myeloma. Eur J Haematol 81:380–383

    CAS  PubMed  Google Scholar 

  29. Cunningham SK, Loughlin T, Culliton M et al (1984) Plasma sex hormone-binding globulin levels decrease during the second decade of life irrespective of pubertal status. J Clin Endocrinol Metab 58:915–918

    Article  CAS  PubMed  Google Scholar 

  30. Vinck WJ, Fagard RH, Vlietinck R et al (2002) Heritability of plasma renin activity and plasma concentration of angiotensinogen and angiotensin-converting enzyme. J Hum Hypertens 16:417–422

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was supported by a grant from National Outstanding Young Scientist’s Foundation of China (Grant No. 30725038). We sincerely thank School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School for nano-LC MALDI-TOF/TOF technique support, and Dr Y Wang for his valuable suggestion and editorial assistance.

Author information

Authors and Affiliations

  1. Peking University People’s Hospital, Peking University Institute of Hematology, No. 11, Xizhimen South Street, Beijing, 100044, China

    Haige Ye, Xiaojun Huang & Xiaoshu Zhao

  2. Biosteed Gene Expression Tech. Co. Ltd., No. 330, Wengjiao Road, Haicang, Xiamen, Fujian, 361022, China

    Li Sun & Ping Zhang

Authors
  1. Haige Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Li Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaojun Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ping Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaoshu Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiaojun Huang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ye, H., Sun, L., Huang, X. et al. A proteomic approach for plasma biomarker discovery with 8-plex iTRAQ labeling and SCX-LC-MS/MS. Mol Cell Biochem 343, 91–99 (2010). https://doi.org/10.1007/s11010-010-0502-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-010-0502-x

Keywords

Search

Navigation