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

Abstract

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. (J Am Soc Mass Spectrom 2010, 21, 1432-1445) © 2010 American Society for Mass Spectrometry P roteins form stable and/or dynamic multisubunit protein complexes under different physiologic conditions to maintain cell viability and normal cell homeostasis. A thorough understanding of protein interactions and structures of protein complexes is fundamental to the understanding of protein function and regulation. Chemical cross-linking coupled with mass spectrometry (MS) is a powerful method that can be used to study protein-protein interactions [1][2][3][4][5][6]. The unique capability of chemical cross-linking to stabilize protein interactions through covalent bond formation allows not only the structural elucidation [7][8][9][10][11][12][13][14][15][16][17], but also the detection of stable, weak, and/or transient protein-protein interactions in native cells or tissues [18 -25].In addition to capturing protein interacting partners, many studies have shown that chemical cross-linking can yield low-resolution structural information about the constraints within a molecule [2][3][4]. Traditional methods such as NMR analysis and X-ray crystallography can yield detailed information on protein structure, however NMR spectroscopy requires large quantities of pure protein in a specific solvent, and X-ray crystallography is often limited by th...

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

“…In Vitro Cross-linking, Biotin Conjugation, and Enrichment of Azide-A-DSBSO Cross-linked Cytochrome C-Azide-A-DSBSO cross-linking of bovine cytochrome C was similar to that described elsewhere (35). The reaction was quenched with 500 mM NH 4 HCO 3 , and samples were ultracentrifuged on 10-kDa NMWL Amicon Ultra centrifugal filters to remove excess cross-linker.…”

“…Azide-A-DSBSO is membrane permeable and has a spacer length of ϳ14 Å. In addition, it carries a bio-orthogonal azide tag that functions as an enrichment handle permitting selective isolation of cross-linked proteins and peptides through azide-based conjugation chemistry and subsequent affinity purification (35). The incorporation of an acid-cleavable site adjacent to the azide tag facilitates the purification and recovery of cross-linked peptides.…”

“…biotin tag) to Azide-A-DSBSO cross-linked products through azide-based conjugation chemistry. Although both coppercatalyzed click chemistry and Staudinger ligation were effective, they have proven to be technically challenging (35). In order to develop a more robust methodology, we adopted a copper-free click chemistry reaction using a biotin-tagged strained alkyne BARAC (47).…”

“…Various efforts have been made to address the limitations of XL-MS studies, resulting in new developments in bioinformatics tools for improved data interpretation (28 -32) and new designs of cross-linking reagents for enhanced MS analysis of cross-linked peptides (24,(33)(34)(35)(36)(37)(38)(39). Among these approaches, the development of new cross-linking reagents holds great promise for mapping PPIs on the systems level.…”

Topographic Studies of the GroEL-GroES Chaperonin Complex by Chemical Cross-linking Using Diformyl Ethynylbenzene

“…Hence, isotopic labeling schemes only serve as an aid to correct identification rather than a means to increase the yield of cross-linked peptides that may be selected for MS/MS. Other strategies incorporate a chemical tag into the cross-linking reagent that allows enrichment of labeled peptides (19,(22)(23)(24)(25).…”