Expanding the Scope of Cross-Link Identifications by Incorporating Collisional Activated Dissociation and Ultraviolet Photodissociation Methods

With
the advent of new cross-linking chemistries, analytical technologies,
and search algorithms, cross-linking has become an increasingly popular
strategy for evaluating tertiary and quaternary structures of proteins.
Collisional activated dissociation remains the primary MS/MS method
for identifications of peptide cross-links in high throughput workflows.
Ultraviolet photodissociation (UVPD) at 193 nm has emerged as an alternative
ion activation method well-suited for characterization of peptides
and has been found in some cases to identify different peptides or
provide distinctive sequence information than obtained by collisional
activation methods. Complementary high energy collision dissociation
(HCD) and UVPD were used in the present study to characterize protein
cross-linking for bovine serum albumin, hemoglobin, and E.
coli ribosome. Cross-links identified by HCD and UVPD using
bis­(sulfosuccinimidyl)­suberate (BS3), a homobifunctional amine-to-amine
cross-linker, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride (DMTMM), a heterofunctional amine-to-carboxylic acid cross-linker,
were evaluated in the present study. While more unique BS3 cross-links
were identified by HCD, UVPD, and HCD provided a complementary panel
of DMTMM cross-links which extended the degree of structural insight
obtained for the proteins.