A negative ion mass spectrometry approach to identify cross-linked peptides utilizing characteristic disulfide fragmentations.
Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS(3) analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.
School of Chemistry and Physics, The University of Adelaide, Adelaide, SA, 5005, Australia.
Antonio N
AN
Calabrese
Nikki J
NJ
Good
Tianfang
T
Wang
Jingjia
J
He
John H
JH
Bowie
Tara L
TL
Pukala
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D011487:Protein Conformation; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry
D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D025801:Ubiquitin