Methods for analyzing peptides and proteins on a chromatographic timescale by electron-transfer dissociation mass spectrometry

Namrata D. Udeshi, Philip D. Compton, Jeffrey Shabanowitz, Donald F. Hunt, Kristie L. Rose*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

71 Scopus citations

Abstract

Advancement in proteomics research relies on the development of new, innovative tools for identifying and characterizing proteins. Here, we describe a protocol for analyzing peptides and proteins on a chromatographic timescale by coupling nanoflow reversephase (RP) liquid chromatography (LC) to electron-transfer dissociation (ETD) mass spectrometry. For this protocol, proteins can be proteolytically digested before ETD analysis, although digestion is not necessary for all applications. Proteins <30 kDa can be analyzed intact, particularly if the objective is protein identification. Peptides or proteins are loaded onto a RP column and are gradient-eluted into an ETD-enabled mass spectrometer. ETD tandem mass spectrometry (MS/MS) provides extensive sequence information required for the unambiguous identification of peptides and proteins and for characterization of posttranslational modifications. ETD is a powerful MS/MS technique and does not compromise the sensitivity and speed necessary for online chromatographic separations. The described procedure for sample preparation, column packing, sample loading and ETD analysis can be implemented in 5-15 h.

Original languageEnglish (US)
Pages (from-to)1709-1717
Number of pages9
JournalNature Protocols
Volume3
Issue number11
DOIs
StatePublished - Oct 16 2008

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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