Chapter 8. Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins

  1. Prof. Dr. Gilbert S. Omenn
  1. Eilon Barnea,
  2. Raya Sorkin,
  3. Tamar Ziv,
  4. Ilan Beer and
  5. Professor Arie Admon

Published Online: 2 NOV 2006

DOI: 10.1002/9783527609482.ch8

Exploring the Human Plasma Proteome

Exploring the Human Plasma Proteome

How to Cite

Barnea, E., Sorkin, R., Ziv, T., Beer, I. and Admon, A. (2006) Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins, in Exploring the Human Plasma Proteome (ed G. S. Omenn), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. doi: 10.1002/9783527609482.ch8

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. Department of Biology, Technion, Haifa 3200, Israel

  1. Originally published in Proteomics 2005, 13, 3367-3375

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;
  • evaluation of prefractionation methods;
  • multidimensional based chromatography of serum proteins;
  • HUPO plasma proteome project (PPP);
  • serum as a complex sample;
  • depletion from serum albumin;
  • depletion from antibodies;
  • comparisons prefractionation methods;
  • identification of different protein subsets;
  • clustering and comparing raw data

Summary

This chapter contains sections titled:

  • Introduction

    • The HUPO Plasma Proteome Project (PPP) goals and the serum as a complex sample

    • The scope of this manuscript

  • Materials and methods

    • Depletion from serum albumin and antibodies

    • MudPIT and mass segmentation

    • Protein separation by SDS-PAGE

    • SCX separation of intact proteins followed by MudPIT

    • Liquid-phase IEF followed by MudPIT

    • Capillary RP-LC-MS/MS

    • MS data processing and peptide/protein identifications

  • Results

    • Comparisons between the prefractionation methods

    • Identification of different protein subsets

    • Proteins identified by only one prefractionation method

    • Different methods resulted in diverse peptide coverage

  • Discussion

    • Giving every peptide a chance

    • How to identify more of the marginal proteins

    • Clustering and comparing raw data

    • High throughput and ruggedness versus high sensitivity

    • The cost effectiveness of the different methods

  • Concluding remarks

  • References