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

Eilon Barnea, Raya Sorkin, Tamar Ziv, Ilan Beer, Arie Admon*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Prefractionations of proteins prior to their proteolysis, chromatography, and MS/MS analyses help reduce complexity and increase the yield of protein identifications. A number of methods were evaluated here for prefractionating serum samples distributed to the participating laboratories as part of the human Plasma Proteome Project. These methods include strong cation exchange (SCX) chromatography, slicing of SDS-PAGE gel bands, and liquid-phase IEF of the proteins. The fractionated proteins were trypsinized and the resulting peptides were resolved and analyzed by multidimensional protein identification technology coupled to IT MS/MS. The MS/MS spectra were clustered, combined, and searched against the IPI protein databank using PepMiner. The identification results were evaluated for the efficacy of the different prefractionation methodologies to identify larger numbers of proteins at higher confidence and to achieve the best coverage of the proteins with the identified peptides. Prefractionation based on SCX resulted in the largest number of identified proteins, followed by gel slices and then the liquid-phase IEF. An important observation was that each of the methods revealed a set of unique proteins, some identified with high confidence. Therefore, for comprehensive identification of the serum proteins, several different prefractionation approaches should be used in parallel.

Original languageEnglish
Pages (from-to)3367-3375
Number of pages9
Issue number13
StatePublished - Aug 2005
Externally publishedYes


  • Plasma Proteome Project
  • Prefractionation
  • Serum


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