Web Release Date: May 5,
Identification of Protein Ubiquitylation by Electrospray Ionization Tandem Mass Spectrometric Analysis of Sulfonated Tryptic Peptides
and
Department of Pharmacology and Molecular Sciences, Department of Biological Chemistry, Johns Hopkins University School of Medicine, and Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205
Received for review October 25, 2005. Accepted March 8, 2006.
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.
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