title: Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes.
abstract: In-depth analysis of the serum and plasma proteomes by mass spectrometry is challenged by the vast dynamic range of protein abundance and substantial complexity. There is merit in reducing complexity through fractionation to facilitate mass spectrometry analysis of low-abundance proteins. However, fractionation reduces throughput and has the potential of diluting individual proteins or inducing their loss. Here, we have investigated the contribution of extensive fractionation of intact proteins to depth of analysis. Pooled serum depleted of abundant proteins was fractionated by an orthogonal two-dimensional system consisting of anion-exchange and reversed-phase chromatography. The resulting protein fractions were aliquotted; one aliquot was analyzed by shotgun LC-MS/MS, and another was further resolved into protein bands in a third dimension using SDS-PAGE. Individual gel bands were excised and subjected to in situ digestion and mass spectrometry. We demonstrate that increased fractionation results in increased depth of analysis based on total number of proteins identified in serum and based on representation in individual fractions of specific proteins identified in gel bands following a third-dimension SDS gel analysis. An intact protein analysis system (IPAS) based on a two-dimensional plasma fractionation schema was implemented that resulted in identification of 1662 proteins with high confidence with representation of protein isoforms that differed in their chromatographic mobility. Further increase in depth of analysis was accomplished by repeat analysis of aliquots from the same set of two-dimensional fractions resulting in overall identification of 2254 proteins. We conclude that substantial depth of analysis of proteins from milliliter quantities of serum or plasma and detection of isoforms are achieved with depletion of abundant proteins followed by two-dimensional protein fractionation and MS analysis of individual fractions.
authors: Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109, USA. vfaca@fhcrc.org
authors: Vitor
authors: V
authors: Faca
authors: Sharon J
authors: SJ
authors: Pitteri
authors: Lisa
authors: L
authors: Newcomb
authors: Veronika
authors: V
authors: Glukhova
authors: Doug
authors: D
authors: Phanstiel
authors: Alexei
authors: A
authors: Krasnoselsky
authors: Qing
authors: Q
authors: Zhang
authors: Jason
authors: J
authors: Struthers
authors: Hong
authors: H
authors: Wang
authors: Jimmy
authors: J
authors: Eng
authors: Matt
authors: M
authors: Fitzgibbon
authors: Martin
authors: M
authors: McIntosh
authors: Samir
authors: S
authors: Hanash
mesh_terms: D000838:Anions; D001798:Blood Proteins / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D020033:Protein Isoforms; D011506:Proteins / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D044967:Serum / Q000378:metabolism
chemical_list: D000838:Anions; D001798:Blood Proteins; D020033:Protein Isoforms; D011506:Proteins; D020543:Proteome