US 9,612,246 B2Granted

Real-time analysis for cross-linked peptides

AssigneeUniversity of Washington Though Its Center For Commercialization

InventorsJames E. Bruce, Juan Chavez, Chad Weisbrod

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Abstract

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.

Claims22

Dependent claims

A method for identifying at least two different interacting peptide pairs within a biological system comprising:

(a) obtaining a sample comprising 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 sample to mass spectrometry (MS) to:
(i) produce a population of precursor ions, and
(ii) generate a 1° mass spectrum used to determine the charge states and masses of precursor ions within the population of precursor ions;
(c) selecting, from within the 1° mass spectrum, a precursor ion with a charge state equal to or greater than a cutoff charge state;
subjecting the selected precursor ion-to conditions under which the cleavable PIR cross-linker, if present in the selected precursor ion, is cleaved, thereby producing a population of released peptides and, if present in the selected precursor ion, cleaved reporter ions;
and determining the masses of the released peptides and, if present in the selected precursor ion, cleaved reporter ions by tandem mass spectrometry (MS2);
(d) analyzing the population of released peptides to identify one or more pairs of interacting peptides, where a pair of released peptides is identified as an interacting peptide pair if the combined mass of the pair of released peptides and cleaved reporter ion, as determined by MS2 in step (c), is equal to the mass of the selected precursor ion, as determined by MS in step (b)(ii);
and
(e) repeating steps (b)-(d) at least once, and until at least two different interacting peptide pairs are identified within the biological system, wherein the same cutoff charge state is used for each repetition.

7 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/283,902Filed 21 May 2014 · us provisional application
Priority: 21 May 2013
Published: US 2014/0349871 A127 Nov 2014
Granted: 4 Apr 2017
Term ends: Not estimatedThe source records don’t support an estimate.

Classification13 codes

G01N 33/6848
General methods of protein analysis not limited to specific proteins or families of proteins: methods of protein analysis involving mass spectrometry
C07K 7/06
Linear peptides containing only normal peptide links: having 5 to 11 amino acids
C07K 7/08
Linear peptides containing only normal peptide links: having 12 to 20 amino acids
G01N 33/56911
For microorganisms, e.g. protozoa, bacteria, viruses: bacteria