Identification of cross-linked peptides by high-resolution precursor ion scan.
Chemical cross-linking coupled to mass spectrometry analysis has become a realistic alternative to the study of proteins structure and interactions, especially when these systems are not amenable to high-resolution techniques such as protein crystallography or nuclear magnetic resonance. One of the main bottlenecks of this approach relies on the detection of cross-linked peptides, as they are usually present in substoichiometric amounts in complex samples. It was shown that one of the main fragmentation pathways of disuccinimidyl suberate (DSS) cross-linked peptides yields diagnostic ions, whose structure is composed of a rearranged lysine side chain and the spacer arm of the linker. In this report, we demonstrate the feasibility of detecting these modified peptides based on a precursor ion scan in a quadrupole time-of-flight (Q-TOF) instrument. It was shown that the fragmentation of nonmodified tryptic peptides hardly generates ions with the same nominal mass of the diagnostic ions, making the precursor ion scan very specific to N-hydroxysuccinimide (NHS)-based cross-linkers. Moreover, the experimental setup is the same as in the case of a regular cross-linking experiment, not demanding any additional experimental steps that would increase sample handling. The results obtained with protein samples allowed us to propose an algorithm that could be implemented in a software to process data from cross-linking experiments in an automated and high-throughput way.
Iglesias
Amadeu H
AH
Institute of Chemistry, University of Campinas, CP 6154, 13083-970 Campinas, São Paulo, Brazil.
Santos
Luiz Fernando A
LF
Gozzo
Fábio C
FC
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013388:Succinimides / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
D003432:Cross-Linking Reagents; D010455:Peptides; D013388:Succinimides; C001426:N-hydroxysuccinimide; C019358:disuccinimidyl suberate