Protein structure underpins functional roles in all biological processes; therefore, improved understanding of protein structures is of fundamental importance in nearly all biological and biomedical research areas. Traditional techniques such as X‐ray crystallography and more recently, cryo‐EM, can reveal structural features on isolated proteins/protein complexes at atomic resolution level and have become indispensable tools for structural biology. Crosslinking mass spectrometry (XL‐MS), on the other hand, is an emerging technique capable of capturing transient and dynamic information on protein interactions and assemblies in their native environment. The combination of XL‐MS with traditional techniques holds potential for bridging the gap between structural biology and systems biology approaches. Such a combination will enable visualization of protein structures and interactions within the crowded macromolecular environment in living systems that can dramatically increase understanding of biological functions. In this review, we first discuss general strategies of XL‐MS and then survey recent examples to show how qualitative and quantitative XL‐MS studies can be integrated with available protein structural data to better understand biological function at systems level.

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Biological systems have evolved to utilize proteins to accomplish nearly all functional roles needed to sustain life. A majority of biological functions occur within the crowded environment inside cells and subcellular compartments where proteins exist in a densely packed complex network of protein–protein interactions. The structural biology field has experienced a renaissance with recent advances in crystallography, NMR, and CryoEM that now produce stunning models of large and complex structures previously unimaginable. Nevertheless, measurements of such structural detail within cellular environments remain elusive. This review will highlight how advances in mass spectrometry, chemical labeling, and informatics capabilities are merging to provide structural insights on proteins, complexes, and networks that exist inside cells. Because of the molecular detection specificity provided by mass spectrometry and proteomics, these approaches provide systems-level information that not only benefits from conventional structural analysis, but also is highly complementary. Although far from comprehensive in their current form, these approaches are currently providing systems structural biology information that can uniquely reveal how conformations and interactions involving many proteins change inside cells with perturbations such as disease, drug treatment, or phenotypic differences. With continued advancements and more widespread adaptation, systems structural biology based on in-cell labeling and mass spectrometry will provide an even greater wealth of structural knowledge.

Chemical cross-linking with mass spectrometry (XL-MS) has emerged as a useful technique for interrogating protein structures and interactions. When combined with quantitative proteomics strategies, protein conformational and interaction dynamics can be probed. Quantitative XL-MS has been demonstrated with the use of stable isotopes incorporated metabolically or into the cross-linker molecules. Isotope-labeled cross-linkers have primarily utilized deuterium and rely on MS1based quantitation of precursor ion extracted ion chromatograms. Recently the development and application of isobaric quantitative protein interaction reporter (iqPIR) cross-linkers were reported, which utilize 13 C and 15 N isotope labels. Quantitation is accomplished using relative fragment ion isotope abundances in tandem mass spectra. Here we describe the synthesis and initial evaluation of a multiplexed set of iqPIR molecules, allowing for up to six cross-linked samples to be quantified simultaneously. To analyze data for such cross-linkers, the two-channel mode of iqPIR quantitative analysis was adapted to accommodate any number of channels with defined ion isotope peak mass offsets. The summed ion peak intensities in the overlapping channel isotope envelopes are apportioned among the channels to minimize the difference with respect to the predicted ion isotope envelopes. The result is accurate and reproducible relative quantitation enabling direct comparison among six differentially labeled cross-linked samples. The approach described here is generally extensible for the iqPIR strategy, accommodating future iqPIR reagent design, and enables large-scale in vivo quantitative XL-MS investigation of the interactome.

Background RNA-binding proteins (RBPs) are well known as key factors in gene expression regulation in eukaryotes. These proteins associate with mRNAs and other proteins to form mRNP complexes that ultimately determine the fate of target transcripts in the cell. This association is usually mediated by an RNA-recognition motif (RRM). In the case of trypanosomatids, these proteins play a paramount role, as gene expression regulation is mostly posttranscriptional. Despite their relevance in the life cycle of Trypanosoma cruzi , the causative agent of Chagas’ disease, to date, few RBPs have been characterized in this parasite. Results We investigated the role of DRBD2 in T. cruzi , an RBP with two RRM domains that is associated with cytoplasmic translational complexes. We show that DRBD2 is an ortholog of the Gbp2 in yeast, an SR-rich protein involved in mRNA quality control and export. We used an immunoprecipitation assay followed by shotgun proteomics and RNA-seq to assess the interaction partners of the DRBD2-mRNP complex in epimastigotes. The analysis identified mostly proteins involved in RNA metabolism and regulation, such as ALBA1, ALBA3, ALBA4, UBP1, UBP2, DRBD3, and PABP2. The RNA-seq results showed that most of the transcripts regulated by the DRBD2 complex mapped to hypothetical proteins related to multiple processes, such as to biosynthetic process, DNA metabolic process, protein modification, and response to stress. Conclusions The identification of regulatory proteins in the DRBD2-mRNP complex corroborates the important role of DRBD2 in gene expression regulation in T. cruzi . We consider these results an important contribution to future studies regarding gene expression regulation in T. cruzi , especially in the field of RNA-binding proteins. Electronic supplementary material The online version of this article (10.1186/s12866-019-1505-8) contains supplementary material, which is available to authorized users.