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Modification of citrulline residues with 2,3‐butanedione facilitates their detection by liquid chromatography/mass spectrometry
Author(s) -
De Ceuleneer Marlies,
De Wit Vanessa,
Van Steendam Katleen,
Van Nieuwerburgh Filip,
Tilleman Kelly,
Deforce Dieter
Publication year - 2011
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.5015
Subject(s) - deamidation , chemistry , citrullination , mass spectrometry , chromatography , citrulline , arginine , peptide , bottom up proteomics , biochemistry , liquid chromatography–mass spectrometry , amino acid , target peptide , racemization , protein mass spectrometry , tandem mass spectrometry , enzyme , organic chemistry
Citrullination is a post‐translational modification (PTM) that results from the deimination of the amino acid arginine into citrulline by Peptidyl Arginine Deiminase enzymes and occurs in a wide range of proteins in health and disease. This modification causes a 1 Da mass shift, which can be used to identify citrullination sites in proteins by the use of mass spectrometry. However, other PTMs, such as deamidation from asparagine to aspartic acid or from glutamine to glutamic acid, can also cause a 1 Da mass shift, making correct interpretation of the data more difficult. We developed a chemical tagging strategy which, combined with an open source search application, allowed us to selectively pinpoint citrullinated peptides in a complex mixture after liquid chromatography/mass spectrometry (LC/MS) analysis. After incubation of a peptide mixture with 2,3 butanedione, citrulline residues were covalently modified which resulted in a 50 Da shift in singly charged mass. By comparison of the peptide mass fingerprint from a modified and an unmodified version of the same sample, our in‐house search application was able to identify the citrullinated peptides in the mixture. This strategy was optimized on synthetic peptides and validated on a digest of in vitro citrullinated fibrinogen, where different proteolytic enzymes were used to augment the protein coverage. This new method results in easy detection of citrullinated residues, without the need for complex mass spectrometry equipment. Copyright © 2011 John Wiley & Sons, Ltd.