Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Athit Kao,Chi-Li Chiu,Danielle Vellucci,Yingying Yang,Vishal R Patel,Shenheng Guan,Arlo Randall,Pierre Baldi,Scott D Rychnovsky,Lan Huang

doi:10.1074/mcp.m110.002212

Abstract

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS(3) analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS(3)) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.

Highlights

From the Departments of ‡Physiology and Biophysics and Developmental and Cell Biology and §Chemistry and ¶Institute for Genomics and Bioinformatics, University of California, Irvine, California 92697 and ʈDepartment of Pharmaceutical Chemistry, University of California, San Francisco, California 94158
It is well documented that methionine sulfoxide-containing peptides have preferential fragmentation at the C–S bond adjacent to the sulfoxide during collision-induced dissociation (CID) analysis [40], and this fragmentation is dominant and much more labile than peptide bonds
We have presented a novel cross-linking strategy for structural analysis of model proteins and the yeast 20 S proteasome complex by combining a newly designed mass spectrometry (MS)-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO), with an integrated data analysis work flow

Summary

Introduction

From the Departments of ‡Physiology and Biophysics and Developmental and Cell Biology and §Chemistry and ¶Institute for Genomics and Bioinformatics, University of California, Irvine, California 92697 and ʈDepartment of Pharmaceutical Chemistry, University of California, San Francisco, California 94158. The respective MS3 analysis of ␣A and ␣S ions (Fig. 2, D and E) allowed unambiguous identification of the peptide sequence and cross-linked site based on a series of y and b ions.

Results

Conclusion