Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling.
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.
Back
Jaap Willem
JW
jwback@science.uva.nl
Notenboom
Valerie
V
de Koning
Leo J
LJ
Muijsers
Anton O
AO
Sixma
Titia K
TK
de Koster
Chris G
CG
de Jong
Luitzen
L
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D008025:Ligases / Q000032:analysis / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards*; D010103:Oxygen Isotopes; D010449:Peptide Mapping / Q000379:methods / Q000592:standards*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D010103:Oxygen Isotopes; D011506:Proteins; C063607:RAD18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases