Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification

Abstract

In recent years, cross-linking mass spectrometry has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, cross-linking mass spectrometry workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probing chromatin-associated protein complexes using small volumes of mammalian whole cell lysates, single affinity purification, and on-bead cross-linking followed by LC-MS/MS analysis. We first benchmarked xIP-MS using the structurally well-characterized phosphoribosyl pyrophosphate synthetase complex. We then applied xIP-MS to the chromatin-associated cohesin (SMC1A/3), XRCC5/6 (Ku70/86), and MCM complexes, and we provide novel structural and biological insights into their architectures and molecular function. Of note, we use xIP-MS to perform topological studies under cell cycle perturbations, showing that the xIP-MS protocol is sufficiently straightforward and efficient to allow comparative cross-linking experiments. This work, therefore, demonstrates that xIP-MS is a robust, flexible, and widely applicable methodology for interrogating chromatin-associated protein complex architectures. Molecular & Cellular Proteomics 15: 10.1074/mcp.M115.053082, 854-865, 2016.The structural basis of specific protein-protein interactions and higher-order protein complex multimerization crucially informs our understanding of many molecular and cellular processes. AP-MS-based strategies have been a major player in the elucidation of specific protein-protein interactions and core protein complexes (1-3). However, although quantitative AP-MS/MS experiments identify specific interactors for any given bait, they cannot differentiate between direct and indirect interactors or provide structural or topological information on protein complex assemblies. Furthermore, traditional structural methodologies often fail with large or dynamic protein complexes including chromatin remodeling or chromatin associated protein complexes. Cross-linking mass spectrometry (XL-MS), 1 therefore, has emerged as a powerful technique for analyzing protein complex architectures through direct observation of subcomplex interfaces at the peptide level (4 -6). Landmark studies used XL-MS to reveal the architectures of the chromatin remodeling complexes Ino80 and SWR1 (7,8), and other chromatin-associated complexes have been interrogated using traditional XL-MS workflows (9 -14).In general, cross-linked peptides represent a small fraction of the total pool of peptides measured in an MS/MS analysis. Therefore, traditional XL-MS workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows to facilitate the detection of low abundance cross-linked peptides from...

Elucidation of host–pathogen protein–protein interactions to uncover mechanisms of host cell rewiring

“…XL-MS thus yields fixed distance restraints between bound residues, suggesting direct physical intra-protein or inter-protein interactions between crosslinked peptides belonging to the same or distinct proteins respectively [56] (see Figure 1). Chemical crosslinking reactions can be performed on purified protein samples [57] using GFP epitope tags [58], on cell lysates [59] or on living cells such as on the pathogen Pseudomonas aeruginosa [60]. Although having gained popularity in recent years to study the topology of protein networks, decipher the architecture of macromolecular complexes, and provide insights into domain-resolution protein interactions, XL-MS has not yet been widely applied to study HP interactions due to its challenging utilization.…”

compleXView: a server for the interpretation of protein abundance and connectivity information to identify protein complexes

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Structure-based validation can drastically under-estimate error rate in proteome-wide cross-linking mass spectrometry studies

“…Furthermore, we acknowledge that this drastic under-estimation of the error rate by structure-based measurements is unlikely to pose a serious issue for XL-MS studies on specific proteins and individual complexes as long as the cross-link search is performed against only proteins that are included in the experiment. Importantly, this issue is highly relevant for the increasingly popular proteome-wide XL-MS experiments 9, 10 and cross-linking immunoprecipitation MS (xIP-MS) studies 23 . In these studies, the conventional structure-based evaluation (which is used in almost all studies) does not provide an adequate assessment of the data quality and will make even lowquality datasets seem good.…”