Chapter 10. Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome Project

  1. Prof. Dr. Gilbert S. Omenn
  1. Xiaohai Li,
  2. Yan Gong,
  3. Ying Wang,
  4. Songfeng Wu,
  5. Yun Cai,
  6. Ping He,
  7. Zhuang Lu,
  8. Wantao Ying,
  9. Yangjun Zhang,
  10. Liyan Jiao,
  11. Hongzhi He,
  12. Zisen Zhang,
  13. Fuchu He,
  14. Xiaohang Zhao and
  15. Dr. Xiaohong Qian Ph.D.

Published Online: 2 NOV 2006

DOI: 10.1002/9783527609482.ch10

Exploring the Human Plasma Proteome

Exploring the Human Plasma Proteome

How to Cite

Li, X., Gong, Y., Wang, Y., Wu, S., Cai, Y., He, P., Lu, Z., Ying, W., Zhang, Y., Jiao, L., He, H., Zhang, Z., He, F., Zhao, X. and Qian, X. (2006) Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome Project, in Exploring the Human Plasma Proteome (ed G. S. Omenn), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. doi: 10.1002/9783527609482.ch10

Editor Information

  1. Internal Medicine, University of Michigan, A520 MSRB 1 Bldg, 1150 West Medical Center Dr. Ann Arbor, MI 48109–0626, USA

Author Information

  1. Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China

  1. Originally published in Proteomics 2005, 13, 3423–3441

Publication History

  1. Published Online: 2 NOV 2006
  2. Published Print: 6 OCT 2006

ISBN Information

Print ISBN: 9783527317578

Online ISBN: 9783527609482

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Keywords:

  • human plasma proteome;
  • alternative analytical techniques;
  • characterisation of human serum proteome;
  • HUPO plasma proteome project;
  • materials and methods;
  • fractionation of depleted serum samples;
  • anion-exchange HPLC;
  • 2-D HPLC;
  • nonporous RP-HPLC;
  • integrated analysis;
  • protein grouping analysis

Summary

This chapter contains sections titled:

  • Introduction

  • Materials and methods

    • Materials

    • Human serum samples

    • Integrated strategy for characterising analytical approaches

    • Depletion of the highly abundant serum proteins by MARS

    • Desalting and concentrating the flow-through fractions by centrifugal ultrafiltration

    • Fractionation of depleted serum samples by anion-exchange HPLC

    • Protein fractionation by 2-D HPLC with nonporous RP-HPLC

    • The 2-DE strategy for the analysis of serum proteins

      • 2-DE

      • In-gel digestion via automated workstation

      • Protein spot identification by MALDI-TOF-MS/MS

    • Shotgun strategy for the analysis of serum proteins

      • Trypsin digestion of serum proteins

      • Protein identification by micro2-D LC-ESI-MS/MS

      • Data processing

    • Protein fractionation strategy for the analysis of serum proteins

      • 2-D LC fractionation of serum proteins

      • Digestion of the 2-D LC separated fractions

      • 1-D microRP-HPLC-ESI-MS/MS identification of digested serum proteins

    • Offline shotgun strategy for the analysis of serum proteins

      • Offline SCX for first-dimension chromatographic separation of peptides

      • 1-D capillary RP-HPLC/microESI-IT-MS/MS analysis for the SCX-separated peptide fractions

    • Optimised nanoRP-HPLC-nanoESI IT-MS/MS for the reanalysis of offline SCX-separated peptides (offline-nanospray strategy)

  • Integrated analysis of the whole data sets

    • Protein grouping analysis

    • Sequence clustering

  • Results and discussion

    • Depletion of the highly abundant serum proteins

    • The 2-DE strategy for the analysis of serum proteins

    • 2-D HPLC fractionation for the analysis of serum proteins

    • Shotgun strategy for the analysis of serum proteins with online SCX

    • Shotgun strategy for the analysis of serum proteins with offline SCX

    • Offline SCX shotgun-nanospray strategy for the analysis of serum proteins

    • Comparison of the five strategies for the analysis of the human serum proteome

  • Concluding remarks

  • References