REAL-TIME ANALYSIS FOR CROSS-LINKED PEPTIDES

摘要

Disclosed herein are methods for large-scale, high-throughput identification of protein-protein interactions and the topologies thereof under physiologically relevant conditions. In one aspect, the disclosure provides methods for identifying one or a plurality of interacting peptides within a biological system comprising obtaining a population of proteins cross-linked with a cleavable protein interaction reporter (PIR) cross-linker, cleaving the PIR crosslinker to produce released peptides and cleaved reporter ions, and analyzing the population of released peptides to identify interacting peptides. Also disclosed are methods for identifying candidate drug compounds, as well as methods of data processing and visualization of protein-protein interactions.

主权项

1. A method for identifying one or a plurality of interacting peptides within a biological system, comprising:
(a) obtaining a population of cross-linked precursor peptides produced by digestion of a population of proteins cross-linked with a cleavable protein interaction reporter (PIR) cross-linker; (b) subjecting the population of cross-linked precursor peptides to mass spectrometry (MS) to produce precursor ions; (c) subjecting precursor ions with a charge state equal to or greater than a cutoff charge state to conditions under which the cleavable PIR cross-linker is cleaved, thereby producing a population of released peptides and cleaved reporter ions; and (d) analyzing the population of released peptides to identify interacting peptides, wherein identifying interacting peptides comprises identifying released peptides that, when added to the mass of the reporter ion, have a combined mass equal to the mass of the corresponding precursor ion.