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

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 Proteins form stable and dynamic multisubunit complexes under different physiological conditions to maintain cell viability and normal cell homeostasis. Detailed knowledge of protein interactions and protein complex structures is fundamental to understanding how individual proteins function within a complex and how the complex functions as a whole. However, structural elucidation of large multisubunit protein complexes has been difficult because of a lack of technologies that can effectively handle their dynamic and heterogeneous nature. Traditional methods such as nuclear magnetic resonance (NMR) analysis and x-ray crystallography can yield detailed information on protein structures; however, NMR spectroscopy requires large quantities of pure protein in a specific solvent, whereas x-ray crystallography is often limited by the crystallization process.In recent years, chemical cross-linking coupled with mass spectrometry (MS) has become a powerful method for studying protein interactions (1-3). Chemical cross-linking stabilizes protein interactions through the formation of covalent bonds and allows the detection of stable, weak, and/or transient protein-protein interactions in native cells or tissues (4 -9). 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, 10) or protein complex (11-13). The application of chemical cross-linking, enzymatic digestion, and subsequent mass spectrometric and computational analyses for the elucidation of three-dimensional protein structures offers distinct advantages over traditional methods because of its speed, sensitivity, and versatility. Identification of cross-linked peptides provides distance constraints that aid in constructing...

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

“…Over the past few years the field has seen significant progress and several methods to enrich crosslinks [13][14][15], various crosslinking chemistries [9,10,[15][16][17][18][19][20][21][22], and the introduction of multiple detection and identification strategies [9][10][11][23][24][25][26][27][28]. Statistical models that differentiate true from false identifications have also been developed [10].…”

“…However, due to multiple major technical obstacles it had been impossible until recently to directly and reliably identify crosslinked peptides from protein complexes by MS (see [1][2][3][4] for recent reviews). After early work on individual proteins [5][6][7] and protein complexes [8,9], notably by the Sinz and Rappsilber groups, Aebersold and coworkers introduced the first robust general workflow by optimizing wet-lab protocols and the development of the publicly available xQuest/xProphet open-source software suite for the analysis and validation of crosslinks from large protein complexes by MS [10][11][12].Over the past few years the field has seen significant progress and several methods to enrich crosslinks [13][14][15], various crosslinking chemistries [9,10,[15][16][17][18][19][20][21][22], and the introduction of multiple detection and identification strategies [9][10][11][23][24][25][26][27][28]. Statistical models that differentiate true from false identifications have also been developed [10].…”

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

“…Because of the similarity between DSBSO and DSSO, the general data analysis workflow for the identification of DSBSO interlinked peptides via LC-MS n is the same as the analysis of DSSO crosslinked peptides (20,36 Homo sapiens was set as the species (20,501 entries) for analyzing data from human cells. The mass tolerances for parent ions and fragment ions were set as Ϯ20 ppm and 0.6 Da, respectively.…”

“…The in-house program Link-Hunter is a revised version of the previously written Link-Finder program, designed to automatically validate and summarize cross-linked peptide sequences based on MS n data and database searching results as previously described (20,36). In addition to checking MS 2 spectra for predicted patterns, Link-Hunter automatically correlates sequence data from MS 3 to MS 2 and MS 1 parent masses and reports identified interlinked peptides with two associated sequences.…”

“…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.…”