Use of a combination of lsotopically coded cross-linkers and lsotopically coded N-terminal modification reagents for selective identification of inter-peptide crosslinks

Evgeniy V. Petrotchenko, Jason J. Serpa, Christoph H. Borchers

Research output: Contribution to journalArticlepeer-review

28 Citations (Scopus)

Abstract

Cross-linking combined with mass spectrometry has great potential for determining three-dimensional structures of proteins and protein assemblies. One of the main analytical challenges of this method is the specific detection and identification of the inter-peptide crosslinks in the peptide mixture after enzymatic digestion of the cross-linked protein complex. These inter-peptide crosslinks are important because they provide the critical distance information needed for structural proteomics studies. In this paper, we demonstrate the use of isotopically coded N-terminal modification (ICNTM) in combination with isotopically coded cross-linkers (ICCL) for specific detection of inter-peptide crosslinks. Inter-peptide crosslinks contain two amino termini, compared to one in the case of free peptides, dead-end crosslinks, or intra-peptide crosslinks. Therefore, N-terminal modification with a 1:1 mixture of heavy and light isotopically coded reagents produces inter-peptide crosslinks with a distinct isotopic signature (a 1:2:1 ratio). Modification also occurs at the ε-amino groups of non-cross-linked lysine residues, resulting in two modifications per free lysine-containing peptide. However, if ICCL and ICNTM are used together, inter-peptide crosslinks can be distinguished from free lysine-containing peptides. Specialized software has also been developed for the analysis of ICCL + ICNTM experimental data. This procedure, combined with software for data analysis, provides a simple and rapid method for specific detection of inter-peptide crosslinks.

Original languageEnglish
Pages (from-to)817-823
Number of pages7
JournalAnalytical Chemistry
Volume82
Issue number3
DOIs
Publication statusPublished - 1 Feb 2010
Externally publishedYes

Fingerprint

Dive into the research topics of 'Use of a combination of lsotopically coded cross-linkers and lsotopically coded N-terminal modification reagents for selective identification of inter-peptide crosslinks'. Together they form a unique fingerprint.

Cite this