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Mol Cell Proteomics. 2012 Mar;11(3):M111.014126. doi: 10.1074/mcp.M111.014126. Epub 2012 Jan 27.

Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography.

Author information

  • 1Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule Zurich, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland.

Abstract

Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe.

PMID:
22286754
[PubMed - indexed for MEDLINE]
PMCID:
PMC3316732
Free PMC Article

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