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Reference: Back JW, et al. (2002) Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling. Anal Chem 74(17):4417-22

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Abstract

A new method is presented to screen proteolytic mass maps of cross-linked protein complexes for the presence of cross-linked peptides and for the verification of proposed structures. On the basis of the incorporation of 18O from isotopically enriched water into the C-termini of proteolytic peptides, cross-linked peptides are readily distinguished in mass spectra by a characteristic 8 amu shift. This is due to the incorporation of two 18O atoms in each C-terminus, so that normal and surface-labeled peptides shift 4 amu and cross-linked peptides containing two C-termini will shift 8 amu compared with their unlabeled counterparts. The method is fast, sensitive, and reliable and can be combined with any available cross-linking reagent and a wide range of proteolytic agents. As proof of principle, we successfully applied the method to a complex of two DNA repair proteins (Rad18-Rad6) and identified the interaction domain.

Reference Type
Journal Article
Authors
Back JW, Notenboom V, de Koning LJ, Muijsers AO, Sixma TK, de Koster CG, de Jong L
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