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