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

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?? 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?? of one another in solution. These technical improvements allow this method to be used for investigating protein-protein interactions in complex biological samples.