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J Proteome Res. 2017 Feb 3;16(2):720-727. doi: 10.1021/acs.jproteome.6b00752. Epub 2016 Nov 30.

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

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Department of Genome Sciences, University of Washington , Seattle, Washington 98109, United States.


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.


PIR; in vivo protein interactions; isotope labeling; proteome-scale; quantitative CL−MS

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