Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome.

Abstract:

:Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14 416 confidently identified peptides covering 4294 different proteins with an estimated 10% gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390 proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein M(r) , pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.

journal_name

Proteomics

journal_title

Proteomics

authors

Liu T,Qian WJ,Chen WN,Jacobs JM,Moore RJ,Anderson DJ,Gritsenko MA,Monroe ME,Thrall BD,Camp DG 2nd,Smith RD

doi

10.1002/pmic.200401055

subject

Has Abstract

pub_date

2005-04-01 00:00:00

pages

1263-73

issue

5

eissn

1615-9853

issn

1615-9861

journal_volume

5

pub_type

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