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J Biomol Tech. 2007 February; 18(1): 57–58.
PMCID: PMC2291922

P166-S Novel Method for High-Throughput Cross-Linking Sites Analysis


We developed a new strategy and computer algorithm for high-throughput analysis of protein-protein or intra-protein interaction sites from chemically cross-linked protein(s). In this strategy, we directly analyze LC-MS/MS data obtained with LTQ-FTMS without any comparison with control sample or isotopic labeling. We developed a computer algorithm, X!Link, to find cross-links of two peptides, which takes only ~10 seconds to analyze ~5000 MS/MS spectra. It is very sensitive and has low false-positive rate. We applied this method for the cross-linking site analysis of cytochrome c and Dnm1 G385D homodimer chemically cross-linked by BS3. We also adopted X!Tandem and Sequest for intra-peptide cross-linking and monolink modification analysis. Total of twenty one cross-links are identified in a single LC-MS/MS data of cytochrome c, in which thirteen is inter-peptide cross-links in 42 MS/MS spectra. High coverage of BS3 modified Lys (84%, 16/19) is owing to the high sensitivity of the present method. Monomer and dimer SDS-PAGE bands of Dnm1 G385D were studied to investigate inter-protein interaction sites of this homodimer. Total of forty five cross-links including thirty seven inter-peptide cross-links from 243 MS/MS spectra were identified in four LC-MS/MS datasets, which is the most cross-links identified so far in a single protein. Closer look of the data demonstrates importance of careful manual inspection for the correct assignment of cross-linking sites when multiple sites exist in a peptide or other similar sequences exist. Two such cases are shown in Figure 11.

Top:Two internal lys residues in a peptide. Bottom: exactly the same parent mass between cross-linked peptides of two subsequent peptide sequences and a single peptide with monolink modification.

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