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Protein-protein interaction is one of the key regulatory mechanisms for controlling protein function in various cellular processes. Chemical cross-linking coupled with mass spectrometry has proven to be a powerful method not only for mapping protein-protein interactions of all natures, including weak and transient ones, but also for determining their interaction interfaces. One critical challenge remaining in this approach is how to effectively isolate and identify cross-linked products from a complex peptide mixture. In this work, we have developed a novel strategy using conjugation chemistry for selective enrichment of cross-linked products. An azide-tagged cross-linker along with two biotinylated conjugation reagents were designed and synthesized. Cross-linking of model peptides and cytochrome c as well as enrichment of the resulting cross-linked peptides has been assessed. Selective conjugation of azide-tagged cross-linked peptides has been demonstrated using two strategies: copper catalyzed cycloaddition and Staudinger ligation. While both methods are effective, Staudinger ligation is better suited for enriching the cross-linked peptides since there are fewer issues with sample handling. LC MS(n) analysis coupled with database searching using the Protein Prospector software package allowed identification of 58 cytochrome c cross-linked peptides after enrichment and affinity purification. The new enrichment strategy developed in this work provides useful tools for facilitating identification of cross-linked peptides in a peptide mixture by MS, thus presenting a step forward in future studies of protein-protein interactions of protein complexes by cross-linking and mass spectrometry.

Selective enrichment and identification of azide-tagged cross-linked peptides using chemical ligation and mass spectrometry