Large-Scale and Targeted Quantitative Cross-Linking MS Using Isotope-Labeled Protein Interaction Reporter (PIR) Cross-Linkers

Quantitative
measurement of chemically cross-linked proteins is
crucial to reveal dynamic information about protein structures and
protein–protein interactions and how these are differentially
regulated during stress, aging, drug treatment, and most perturbations.
Previously, we demonstrated how quantitative in vivo cross-linking
(CL) with stable isotope labeling of amino acids in cell culture (SILAC)
enables both heritable and dynamic changes in cells to be visualized.
In this work, we demonstrate the technical feasibility of proteome-scale
quantitative in vivo CL–MS using isotope-labeled protein interaction
reporter (PIR) cross-linkers and E. coli as a model
system. This isotope-labeled cross-linkers approach, combined with
Real-time Analysis of Cross-linked peptide Technology (ReACT) previously
developed in our lab, enables the quantification of 941 nonredundant
cross-linked peptide pairs from a total of 1213 fully identified peptide
pairs in two biological replicate samples through comparison of MS1 peak intensity of the light and heavy cross-linked peptide
pairs. For targeted relative quantification of cross-linked peptide
pairs, we further developed a PRM-based assay to accurately probe
specific site interaction changes in a complex background. The methodology
described in this work provides reliable tools for both large-scale
and targeted quantitative CL–MS that is useful for any sample
where SILAC labeling may not be practical.