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Proteomics. 2011 Oct;11(20):4109-15. doi: 10.1002/pmic.201100015. Epub 2011 Sep 6.

Photo-assisted peptide enrichment in protein complex cross-linking analysis of a model homodimeric protein using mass spectrometry.

Author information

1
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA. funing.yan@einstein.yu.edu

Abstract

MS analysis of cross-linked peptides can be used to probe protein contact sites in macromolecular complexes. We have developed a photo-cleavable cross-linker that enhances peptide enrichment, improving the signal-to-noise ratio of the cross-linked peptides in mass spectrometry analysis. This cross-linker utilizes nitro-benzyl alcohol group that can be cleaved by UV irradiation and is stable during the multiple washing steps used for peptide enrichment. The enrichment method utilizes a cross-linker that aids in eliminating contamination resulting from protein-based retrieval systems, and thus, facilitates the identification of cross-linked peptides. Homodimeric pilM protein from Pseudomonas aeruginosa 2192 (pilM) was investigated to test the specificity and experimental conditions. As predicted, the known pair of lysine side chains within 14 Å was cross-linked. An unexpected cross-link involving the protein's amino terminus was also detected. This is consistent with the predicted mobility of the amino terminus that may bring the amino groups within 19 Å of one another in solution. These technical improvements allow this method to be used for investigating protein-protein interactions in complex biological samples.

PMID:
21834138
PMCID:
PMC3465073
DOI:
10.1002/pmic.201100015
[Indexed for MEDLINE]
Free PMC Article

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