Shotgun proteome analysis utilising mixed mode (reversed phase-anion exchange chromatography) in conjunction with reversed phase liquid chromatography mass spectrometry analysis.

Abstract:

:The 2-D peptide separations employing mixed mode reversed phase anion exchange (MM (RP-AX)) HPLC in the first dimension in conjunction with RP chromatography in the second dimension were developed and utilised for shotgun proteome analysis. Compared with strong cation exchange (SCX) typically employed for shotgun proteomic analysis, peptide separations using MM (RP-AX) revealed improved separation efficiency and increased peptide distribution across the elution gradient. In addition, improved sample handling, with no significant reduction in the orthogonality of the peptide separations was observed. The shotgun proteomic analysis of a mammalian nuclear cell lysate revealed additional proteome coverage (2818 versus 1125 unique peptides and 602 versus 238 proteins) using the MM (RP-AX) compared with the traditional SCX hyphenated to RP-LC-MS/MS. The MM analysis resulted in approximately 90% of the unique peptides identified present in only one fraction, with a heterogeneous peptide distribution across all fractions. No clustering of the predominant peptide charge states was observed during the gradient elution. The application of MM (RP-AX) for 2-D LC proteomic studies was also extended in the analysis of iTRAQ-labelled HeLa and cyanobacterial proteomes using nano-flow chromatography interfaced to the MS/MS. We demonstrate MM (RP-AX) HPLC as an alternative approach for shotgun proteomic studies that offers significant advantages over traditional SCX peptide separations.

journal_name

Proteomics

journal_title

Proteomics

authors

Phillips HL,Williamson JC,van Elburg KA,Snijders AP,Wright PC,Dickman MJ

doi

10.1002/pmic.200900669

subject

Has Abstract

pub_date

2010-08-01 00:00:00

pages

2950-60

issue

16

eissn

1615-9853

issn

1615-9861

journal_volume

10

pub_type

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