An enhanced protein crosslink identification strategy using CID-cleavable chemical crosslinkers and LC/MS(n)  analysis.
We describe a novel two-step LC/MS(n) strategy to effectively and confidently identify numerous crosslinked peptides from complex mixtures. This method incorporates the use of our gas-phase cleavable crosslinking reagent, disuccinimidyl-succinamyl-aspartyl-proline (SuDP), and a new data-processing algorithm CXLinkS (Cleavable Crosslink Selection), which enables unequivocal crosslink peptide selection and identification on the basis of mass measurement accuracy, high resolving power, and the unique fragmentation pattern of each crosslinked peptide. We demonstrate our approach with well-characterized monomeric and multimeric protein systems with and without database searching restrictions where inter-peptide crosslink identification is increased 8-fold over our previously published data-dependent LC/MS³ method and discuss its applicability to other CID-cleavable crosslinkers and more complex protein systems.
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, USA.
Fan
F
Liu
Cong
C
Wu
Jonathan V
JV
Sweedler
Michael B
MB
Goshe
D000465:Algorithms; D002853:Chromatography, Liquid / Q000379:methods; D045424:Complex Mixtures / Q000032:analysis*; D030562:Databases, Protein; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism
D045424:Complex Mixtures; D006454:Hemoglobins; D010455:Peptides; D011506:Proteins