Identification of protein ubiquitylation by electrospray ionization tandem mass spectrometric analysis of sulfonated tryptic peptides

Dongxia Wang, Dario Kalume, Cecile Pickart, Akhilesh Pandey, Robert J. Cotter

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

We report here the application of electrospray ionization tandem mass spectrometry for the characterization of protein ubiquitylation, an important posttranslational modification of cellular proteins. Trypsin digestion of ubiquitin-conjugated proteins produces diglycine branched peptides containing the modification sites. Chemical derivatization by N-terminal sulfonation was carried out on several model peptides for the formation of a characteristic fragmentation pattern in their MS/MS analysis. The fragmentation of derivatized singly charged peptides results in a product ion distribution similar to that already observed by MALDI-TOF MS/MS. Signature fragments distinguished the diglycine branched peptides from other modified and unmodified peptides, while the sequencing product ions reveal the amino acid sequence and the location of the ubiquitylation site. Doubly charged peptide derivatives fragment in a somewhat different manner, but several fragments characteristic to diglycine branched peptides were observed under low collision energy conditions. These signature peaks can also be used to identify peptides containing ubiquitylation sites. In addition, a marker ion corresponding to a glycine-modified lysine residue produced by high-energy fragmentation provides useful information for identity verification. The method is demonstrated by the analysis of three ubiquitin-conjugated proteins using LC/MS/MS.

Original languageEnglish (US)
Pages (from-to)3681-3687
Number of pages7
JournalAnalytical Chemistry
Volume78
Issue number11
DOIs
StatePublished - Jun 1 2006
Externally publishedYes

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Electrospray ionization
Peptides
Glycylglycine
Proteins
Ions
Ubiquitin
Sulfonation
Glycine
Trypsin
Lysine
Mass spectrometry
Derivatives
Amino Acids

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Identification of protein ubiquitylation by electrospray ionization tandem mass spectrometric analysis of sulfonated tryptic peptides. / Wang, Dongxia; Kalume, Dario; Pickart, Cecile; Pandey, Akhilesh; Cotter, Robert J.

In: Analytical Chemistry, Vol. 78, No. 11, 01.06.2006, p. 3681-3687.

Research output: Contribution to journalArticle

Wang, Dongxia ; Kalume, Dario ; Pickart, Cecile ; Pandey, Akhilesh ; Cotter, Robert J. / Identification of protein ubiquitylation by electrospray ionization tandem mass spectrometric analysis of sulfonated tryptic peptides. In: Analytical Chemistry. 2006 ; Vol. 78, No. 11. pp. 3681-3687.
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