Format

Send to

Choose Destination
Anal Chem. 2009 Jul 1;81(13):5524-32. doi: 10.1021/ac900853k.

Identification of cross-linked peptides after click-based enrichment using sequential collision-induced dissociation and electron transfer dissociation tandem mass spectrometry.

Author information

1
Biological Sciences Division and Environmental and Molecular Science Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.

Abstract

Chemical cross-linking combined with mass spectrometry can be a powerful approach for the identification of protein-protein interactions and for providing constraints on protein structures. However, enrichment of cross-linked peptides is crucial to reduce sample complexity before mass spectrometric analysis. In addition compact cross-linkers are often preferred to provide short spacer lengths, surface accessibility to the protein complexes, and must have reasonable solubility under conditions where the native complex structure is stable. In this study, we present a novel compact cross-linker that contains two distinct features: (1) an alkyne tag and (2) a small molecule detection tag (NO(2)) to maintain reasonable solubility in water. The alkyne tag enables enrichment of the cross-linked peptides after proteolytic cleavage and coupling of an affinity tag using alkyne-azido click chemistry. Neutral loss of the small NO(2) moiety provides a secondary means of detecting cross-linked peptides in MS/MS analyses, providing additional confidence in peptide identifications. We show the labeling efficiency of this cross-linker, which we termed CLIP (click-enabled linker for interacting proteins) using ubiquitin. The enrichment capability of CLIP is demonstrated for cross-linked ubiquitin in highly complex E. coli cell lysates. Sequential collision-induced dissociation tandem mass spectrometry (CID-MS/MS) and electron transfer dissociation (ETD)-MS/MS of intercross-linked peptides (two peptides connected with a cross-linker) are also demonstrated for improved automated identification of cross-linked peptides.

PMID:
19496583
PMCID:
PMC2907912
DOI:
10.1021/ac900853k
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for American Chemical Society Icon for PubMed Central
Loading ...
Support Center