Quantitative interactome analysis reveals a chemoresistant edgotype

Abstract

Chemoresistance is a common mode of therapy failure for many cancers. Tumours develop resistance to chemotherapeutics through a variety of mechanisms, with proteins serving pivotal roles. Changes in protein conformations and interactions affect the cellular response to environmental conditions contributing to the development of new phenotypes. The ability to understand how protein interaction networks adapt to yield new function or alter phenotype is limited by the inability to determine structural and protein interaction changes on a proteomic scale. Here, chemical crosslinking and mass spectrometry were employed to quantify changes in protein structures and interactions in multidrug-resistant human carcinoma cells. Quantitative analysis of the largest crosslinking-derived, protein interaction network comprising 1,391 crosslinked peptides allows for 'edgotype' analysis in a cell model of chemoresistance. We detect consistent changes to protein interactions and structures, including those involving cytokeratins, topoisomerase-2-alpha, and post-translationally modified histones, which correlate with a chemoresistant phenotype.

In Vivo Conformational Dynamics of Hsp90 and Its Interactors

“…Using a qXL-MS approach (Chavez et al, 2015) we developed a workflow to study conformational changes induced in Hsp90 due to inhibitor treatment in live cells (Fig. 1).…”

“…S3C). Cross-linked peptides were quantified using SILAC as previously demonstrated (Chavez et al, 2015) and confidence intervals (α = 0.05) were calculated for cross-linked peptides with measured ratios across biological replicates (Table S1). Perturbations to the network at both the edge and node levels were observed due to 17-AAG treatment and are indicated by node and edge colors (Fig.…”

“…Previously we developed a qXL-MS approach, combining Protein Interaction Reporter (PIR) technology (Tang and Bruce, 2010) with SILAC, to compare a multi-drug resistant cancer cell phenotype with the drug sensitive parental cell line (Chavez et al, 2015). Here, we extend these methods and apply qXL-MS to monitor large scale protein structural and interaction changes exhibited by Hsp90 in cells upon inhibitor treatment.…”

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

“…Using the protein interaction reporter concept based on gas phase cleavable, affinity tagged crosslinking reagents, Bruce and coworkers have shown applications to a diverse range of organisms such as Shewanella oneidensis [111], Escherichia coli [112 114], Pseudomonas aeruginosa [115], and even human cell lines [116]; other groups have reported similar concepts [117 119]. Recently, the group of Bruce also demonstrated that a quantitative dimension can be added to proteome wide crosslinking data with the help of metabolic stable isotope labeling [120]. These potentially exciting approaches still have difficulty in identifying larger numbers of interactions between proteins or protein complexes that are not highly abundant like the ribosome, certain chaperones, or histones, but improvements in experimental protocols and instrumentation will increase the coverage in the future, as exemplified by recent work from Heck and coworkers [121].…”

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

“…The resulting spectra are mined for protein sequence information using traditional proteomic search engines such as Mascot (21) and SE-QUEST (22). PIR-derived cross-linked sites identified between or within proteins are then used as distance constraints to develop structural models that enable visualization of proteins and protein complexes, as they exist in cells during cross-linker application (19,23,24). A recent study using in vivo PIR application confirmed OmpA homodimer existence and interfacial regions that were originally identified in bacterial cells by traditional MS methods (25), demonstrating the ability to derive meaningful structural information from this experimental approach.…”