Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes

被引：131

作者：

Faca, Vitor ^{[1
]}

Pitteri, Sharon J. ^{[1
]}

Newcomb, Lisa ^{[1
]}

Glukhova, Veronika ^{[1
]}

Phanstiel, Doug ^{[1
]}

Krasnoselsky, Alexei ^{[1
]}

Zhang, Qing ^{[1
]}

Struthers, Jason ^{[1
]}

Wang, Hong ^{[1
]}

Eng, Jimmy ^{[1
]}

Fitzgibbon, Matt ^{[1
]}

McIntosh, Martin ^{[1
]}

Hanash, Samir ^{[1
]}

机构：

[1] Fred Hutchinson Canc Res Ctr, Seattle, WA 98109 USA

来源：

JOURNAL OF PROTEOME RESEARCH | 2007年 / 6卷 / 09期

关键词：

human plasma proteins; human serum proteins; intact protein fractionation; liquid chromatography; LC-MS/MS;

D O I：

10.1021/pr070233q

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：3558 / 3565

页数：8

共 28 条

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