In-Cell Labeling and Mass Spectrometry for Systems-Level Structural Biology.

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.

Differential Proteome and Interactome Analysis Reveal the Basis of Pleiotropy Associated With the Histidine Methyltransferase Hpm1p.

The methylation of histidine is a post-translational modification whose function is poorly understood. Methyltransferase histidine protein methyltransferase 1 (Hpm1p) monomethylates H243 in the ribosomal protein Rpl3p and represents the only known histidine methyltransferase in Saccharomyces cerevisiae. Interestingly, the hpm1 deletion strain is highly pleiotropic, with many extraribosomal phenotypes including improved growth rates in alternative carbon sources. Here, we investigate how the loss of histidine methyltransferase Hpm1p results in diverse phenotypes, through use of targeted mass spectrometry (MS), growth assays, quantitative proteomics, and differential crosslinking MS. We confirmed the localization and stoichiometry of the H243 methylation site, found unreported sensitivities of Δhpm1 yeast to nonribosomal stressors, and identified differentially abundant proteins upon hpm1 knockout with clear links to the coordination of sugar metabolism. We adapted the emerging technique of quantitative large-scale stable isotope labeling of amino acids in cell culture crosslinking MS for yeast, which resulted in the identification of 1267 unique in vivo lysine-lysine crosslinks. By reproducibly monitoring over 350 of these in WT and Δhpm1, we detected changes to protein structure or protein-protein interactions in the ribosome, membrane proteins, chromatin, and mitochondria. Importantly, these occurred independently of changes in protein abundance and could explain a number of phenotypes of Δhpm1, not addressed by expression analysis. Further to this, some phenotypes were predicted solely from changes in protein structure or interactions and could be validated by orthogonal techniques. Taken together, these studies reveal a broad role for Hpm1p in yeast and illustrate how crosslinking MS will be an essential tool for understanding complex phenotypes.

Applications and advancements of FT-ICR-MS for interactome studies.

The set of all intra- and intermolecular interactions, collectively known as the interactome, is currently an unmet challenge for any analytical method, but if measured, could provide unparalleled insight on molecular function in living systems. Developments and applications of chemical cross-linking and high-performance mass spectrometry technologies are beginning to reveal details on how proteins interact in cells and how protein conformations and interactions inside cells change with phenotype or during drug treatment or other perturbations. A major contributor to these advances is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) technology and its implementation with accurate mass measurements on cross-linked peptide-pair precursor and fragment ions to enable improved identification methods. However, these applications place increased demands on mass spectrometer performance in terms of high-resolution spectral acquisition rates for on-line MSn experiments. Moreover, FT-ICR-MS also offers unique opportunities to develop and implement parallel ICR cells for multiplexed signal acquisition and the potential to greatly advance accurate mass acquisition rates for interactome studies. This review highlights our efforts to exploit accurate mass FT-ICR-MS technologies with chemical cross-linking and developments being pursued to realize parallel MS array capabilities that will further advance visualization of the interactome.

Mitochondrial protein interaction landscape of SS-31.

Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.

Improved Interpretation of Protein Conformational Differences and Ligand Occupancy in Large-Scale Cross-Link Data.

Chemical cross-linking of proteins in complex samples, cells, or even tissues is emerging to provide unique structural information on proteins and complexes that exist within native or nativelike environments. The public database XLinkDB automatically maps cross-links to available structures based on sequence homology. Structures most likely to reflect protein conformations in the cross-linked sample are routinely identified by having cross-linked residues separated by Euclidean distances within the maximum span of the applied cross-linker. Solvent accessible surface distance (SASD), which considers the accessibility of the cross-linked residues and the path connecting them, is a better predictor of consistency than the Euclidean distance. However, SASDs of structures are not publicly available, and their calculation is computationally intensive. Here, we describe in XLinkDB version 4.0 the automatic calculation of SASDs using Jwalk for all cross-links mapped to structures, both with and without regard to ligands, and derive empirical maximum SASD spans for BDP-NHP and DSSO cross-linkers of 51 and 43 Å, respectively. We document ligands proximal to cross-links in structures and demonstrate how SASDs can be used to help infer sample protein conformations and ligand occupancy, highlighting cross-links sensitive to ADP binding in mitochondria isolated from HEK293 cells.

Multiplexed Isobaric Quantitative Cross-Linking Reveals Drug-Induced Interactome Changes in Breast Cancer Cells.

The study of protein structures and interactions is critical to understand their function. Chemical cross-linking of proteins with mass spectrometry (XL-MS) is a rapidly developing structural biology technique able to provide valuable insight into protein conformations and interactions, even as they exist within their native cellular environment. Quantitative analysis of cross-links can reveal protein conformational and interaction changes that occur as a result of altered biological states, environmental conditions, or pharmacological perturbations. Our laboratory recently developed an isobaric quantitative protein interaction reporter (iqPIR) cross-linking strategy for comparative interactome studies. This strategy relies on isotope encoded chemical cross-linkers that have the same molecular mass yet produce unique and specific isotope signatures upon fragmentation in the mass spectrometer which can be used for quantitative analysis of cross-linked peptides. The initial set of iqPIR molecules allowed for binary comparisons. Here, we describe the in vivo application of an extended set of six iqPIR reagents (6-plex iqPIR), allowing multiplexed quantitative interactome analysis of up to six biological samples in a single LC-MS acquisition. Multiplexed iqPIR is demonstrated on MCF-7 breast cancer cells treated with five different Hsp90 inhibitors revealing large scale protein conformational and interaction changes specific to the molecular class of the inhibitors.

Self-Assembled Monolayer of Monomercaptoundecahydro-closo-dodecaborate on a Polycrystalline Gold Surface.

In this work, we present an electrochemical study of the boron cage monomercaptoundecahydro-closo-dodecaborate [B12H11SH]2- in solution and in a self-assembled monolayer over a polycrystalline gold electrode. Cyclic voltammetry of the anion [B12H11SH]2- in solution showed a shift in the peak potentials related to the redox processes of gold hydroxides, which evidences the interaction between the boron cage and the gold surface. For an Au electrode modified with the anion [B12H11SH]2-, cyclic voltammetry response of the probe Fe(CN)63-/Fe(CN)64- showed a ΔEp value typical for a surface modification. Electrochemical impedance spectroscopy presented Rtc and Cdl values related to the formation of a self-assembled monolayer (SAM). A comparison of electrochemical responses of a modified electrode with thioglycolic acid (TGA) reveals that the boron cage [B12H11SH]2- diminishes the actives sites over the Au surface due to the steric effects. Differential capacitance measurements for bare gold electrode and those modified with [B12H11SH]2- and (TGA), indicate that bulky thiols enhance charge accumulation at the electrode-solution interface. In addition to electrochemical experiments, DFT calculations and surface plasmon resonance measurements (SPR) were carried out to obtain quantum chemical descriptors and to evaluate the molecular length and the dielectric constant of the Boron cage. From SPR experiments, the adsorption kinetics of [B12H11SH]2- were studied. The data fit for a Langmuir kinetic equation, typical for the formation of a monolayer.

[Consensus on the use of iSGLT2 in the treatment of patients with type 2 diabetes mellitus]. / Documento de consenso sobre el uso de los iSGLT2 en el tratamiento de pacientes con diabetes mellitus tipo 2.

Although in recent years in Mexico the quality of diabetes mellitus (DM) care has improved and access to health services and medications has increased, there is a lack of adherence to the recommendations of the clinical guidelines, which could explain the poor glycemic control in many of the patients with DM. Sodium-glucose cotransporter type 2 (iSGLT2) inhibitors have been the last class of antidiabetic agents to receive approval from the Food and Drug Administration (FDA) and COFEPRIS (Mexico). In order to improve the use of SGLT2i in clinical practice in Mexico, this paper presents the recommendations issued by a panel of eleven Mexican experts based on the new published evidence for the treatment of patients with DM2.

Aunque en los últimos años en México ha mejorado la calidad de la atención de la diabetes mellitus (DM) y ha aumentado el acceso a servicios de salud y medicamentos, existe una falta de apego a las recomendaciones de las guías de práctica clínica, que podría explicar la falta de un control glucémico adecuado en muchos de los pacientes con DM. Los inhibidores del cotransportador de sodio-glucosa tipo 2 (iSGLT2) han sido la última clase de agentes antidiabéticos en recibir la aprobación de la Food and Drug Administration (FDA) y de la Comisión Federal para la Protección contra Riesgos Sanitarios de México (COFEPRIS). Con el fin de mejorar el uso de los iSGLT2 en la práctica clínica en México, en este documento se presentan las recomendaciones emitidas por un panel de 11 expertos mexicanos con base en las nuevas evidencias publicadas para el tratamiento de los pacientes con DM2.

Leveraging the Entirety of the Protein Data Bank to Enable Improved Structure Prediction Based on Cross-Link Data.

XLinkDB is a fast-expanding public database now storing more than 100 000 distinct identified cross-linked protein residue pairs acquired by chemical cross-linking with mass spectrometry from samples of 12 species (J. Proteome Res.2019, 18 (2), 753-758). Mapping identified cross-links to protein structures, when available, provides valuable guidance on protein conformations detected in the cross-linked samples. As more and more structures become available in the Protein Data Bank (Nucleic Acids Res.2000, 28 (1), 235-242), we sought to leverage their utility for cross-link studies by automatically mapping identified cross-links to structures based on sequence homology of the cross-linked proteins with those within structures. This enables use of structures derived from organisms different from those of samples, including large multiprotein complexes and complexes in alternative states. We demonstrate utility of mapping to orthologous structures, highlighting a cross-link between two subunits of mouse mitochondrial Complex I that was mapped to 15 structures derived from five mammals, its distances there of 16.2 ± 0.4 Å indicating strong conservation of the protein interaction. We also show how multimeric structures enable reassessment of cross-links presumed to be intraprotein as potentially homodimeric interprotein in origin.

Multiplexed Cross-Linking with Isobaric Quantitative Protein Interaction Reporter Technology.

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 MS1-based 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 13C and 15N 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.

Characterizing environmental stress responses of aposymbiotic Astrangia poculata to divergent thermal challenges.

Anthropogenic climate change threatens corals globally and both high and low temperatures are known to induce coral bleaching. However, coral stress responses across wide thermal breadths remain understudied. Disentangling the role of symbiosis on the stress response in obligately symbiotic corals is challenging because this response is inherently coupled with nutritional stress. Here, we leverage aposymbiotic colonies of the facultatively symbiotic coral, Astrangia poculata, which lives naturally with and without its algal symbionts, to examine how broad thermal challenges influence coral hosts in the absence of symbiosis. A. poculata were collected from their northern range limit and thermally challenged in two independent 16-day common garden experiments (heat and cold challenge) and behavioural responses to food stimuli and genome-wide gene expression profiling (TagSeq) were performed. Both thermal challenges elicited significant reductions in polyp extension. However, there were five times as many differentially expressed genes (DEGs) under cold challenge compared to heat challenge. Despite an overall stronger response to cold challenge, there was significant overlap in DEGs between thermal challenges. We contrasted these responses to a previously identified module of genes associated with the environmental stress response (ESR) in tropical reef-building corals. Cold challenged corals exhibited a pattern consistent with more severe stressors while the heat challenge response was consistent with lower intensity stressors. Given that these responses were observed in aposymbiotic colonies, many genes previously implicated in ESRs in tropical symbiotic species may represent the coral host's stress response in or out of symbiosis.

Acute Demands and Recovery From Common Interval Training Protocols.

ABSTRACT: Elmer, DJ, Barron, EN, and Chavez, JL. Acute demands and recovery from common interval training protocols. J Strength Cond Res 35(11): 3041-3049, 2021-The definition of interval training is quite broad, with no accepted procedure for classifying protocols with different workloads and work and recovery interval times. In addition, little is known about the differences in training load and recovery from common interval protocols. The purpose of this study was to evaluate the differences in acute physiological demands and perceived difficulty between 3 common interval training protocols and a moderate, continuous exercise session. Eight subjects completed the training sessions on a cycle ergometer in a randomized order, with at least 1 week between sessions: 30-second/4-minute, 1-minute/1-minute, 4-minute/3-minute, and a 45-minute continuous session. Metabolic variables were measured throughout exercise and 30 minutes of recovery. Training impulse (TRIMP), session-RPE, and RPE-training load were also measured. There were significant differences between protocols, including between interval training protocols, for average V̇o2 (p < 0.001) and heart rate (HR) (p = 0.02), total O2 consumption (p < 0.001), peak lactate (p < 0.001), TRIMP (p = 0.02), session-RPE (p = 0.01), and RPE-training load (p < 0.001). There were no significant differences between interval training protocols for peak V̇o2 or peak HR attained during exercise. There were also no differences in V̇o2 or HR after 5 or 30 minutes of recovery. Blood lactate was only significantly higher after 30 minutes of recovery from the 30-second/4-minute compared with the 4-minute/3-minute protocol (p = 0.001) and the 45-minute session (p < 0.001). These findings show a range of differences in acute physiological demands and perceptions from interval training protocols, which should be accounted for when planning training sessions or research studies or when interpreting past research.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analysis of Citrus limon Response to Graft Inoculation by Candidatus Liberibacter asiaticus.

Presymptomatic detection of citrus trees infected with Candidatus Liberibacter asiaticus (CLas), the bacterial pathogen associated with Huanglongbing (HLB; citrus greening disease), is critical to controlling the spread of the disease. To test whether infected citrus trees produce systemic signals that may be used for indirect disease detection, lemon (Citrus limon) plants were graft-inoculated with either CLas-infected or control (CLas-) budwood, and leaf samples were longitudinally collected over 46 weeks and analyzed for plant changes associated with CLas infection. RNA, protein, and metabolite samples extracted from leaves were analyzed using RNA-Seq, mass spectrometry, and 1H NMR spectroscopy, respectively. Significant differences in specific transcripts, proteins, and metabolites were observed between CLas-infected and control plants as early as 2 weeks post graft (wpg). The most dramatic differences between the transcriptome and proteome of CLas-infected and control plants were observed at 10 wpg, including coordinated increases in transcripts and proteins of citrus orthologs of known plant defense genes. This integrated approach to quantifying plant molecular changes in leaves of CLas-infected plants supports the development of diagnostic technology for presymptomatic or early disease detection as part of efforts to control the spread of HLB into uninfected citrus groves.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analyses of Citrus sinensis (L.) Osbeck Graft-Inoculated with "Candidatus Liberibacter asiaticus".

"Candidatus Liberibacter asiaticus" (CLas) is the bacterium associated with the citrus disease Huanglongbing (HLB). Current CLas detection methods are unreliable during presymptomatic infection, and understanding CLas pathogenicity to help develop new detection techniques is challenging because CLas has yet to be isolated in pure culture. To understand how CLas affects citrus metabolism and whether infected plants produce systemic signals that can be used to develop improved detection techniques, leaves from Washington Navel orange (Citrus sinensis (L.) Osbeck) plants were graft-inoculated with CLas and longitudinally studied using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry), and metabolomics (proton nuclear magnetic resonance). Photosynthesis gene expression and protein levels were lower in infected plants compared to controls during late infection, and lower levels of photosynthesis proteins were identified as early as 8 weeks post-grafting. These changes coordinated with higher sugar concentrations, which have been shown to accumulate during HLB. Cell wall modification and degradation gene expression and proteins were higher in infected plants during late infection. Changes in gene expression and proteins related to plant defense were observed in infected plants as early as 8 weeks post-grafting. These results reveal coordinated changes in greenhouse navel leaves during CLas infection at the transcript, protein, and metabolite levels, which can inform of biomarkers of early infection.

Isobaric Quantitative Protein Interaction Reporter Technology for Comparative Interactome Studies.

Chemical cross-linking with mass spectrometry (XL-MS) has emerged as a useful tool for the large-scale study of protein structures and interactions from complex biological samples including intact cells and tissues. Quantitative XL-MS (qXL-MS) provides unique information on protein conformational and interaction changes resulting from perturbations such as drug treatment and disease state. Previous qXL-MS studies relied on the incorporation of stable isotopes into the cross-linker (primarily deuterium) or metabolic labeling with SILAC. Here, we introduce isobaric quantitative protein interaction reporter (iqPIR) technology which utilizes stable isotopes selectively incorporated into the cross-linker design, allowing for isobaric cross-linked peptide pairs originating from different samples to display distinct quantitative isotope signatures in tandem mass spectra. This enables improved quantitation of cross-linked peptide levels from proteome-wide samples because of the reduced complexity of tandem mass spectra relative to MS1 spectra. In addition, because of the isotope incorporation in the reporter and the residual components of the cross-linker that remain on released peptides, each fragmentation spectrum can offer multiple independent opportunities and, therefore, improved confidence for quantitative assessment of the cross-linker pair level. Finally, in addition to providing information on solvent accessibility of lysine sites, dead end iqPIR cross-linked products can provide protein abundance and/or lysine site modification level information all from a single in vivo cross-linking experiment.

Increased sensitivity with automated validation of XL-MS cleavable peptide crosslinks.

MOTIVATION: Peptides crosslinked with cleavable chemical crosslinkers are identified with mass spectrometry by independent database search of spectra associated with the two linked peptides. A major challenge is to combine together the evidence of the two peptides into an overall assessment of the two-peptide crosslink. RESULTS: Here, we describe software that models crosslink specific information to automatically validate XL-MS cleavable peptide crosslinks. Using a dataset of crosslinked protein mixtures, we demonstrate that it computes accurate and highly discriminating probabilities, enabling as many as 75% more identifications than was previously possible using only search scores and a predictable false discovery rate. AVAILABILITY AND IMPLEMENTATION: XLinkProphet software is freely available on the web at http://brucelab.gs.washington.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry.

Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I-III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.

Prediction of an Upper Limit for the Fraction of Interprotein Cross-Links in Large-Scale In Vivo Cross-Linking Studies.

Chemical cross-linking and mass spectrometry is of growing use for establishment of distance constraints on protein conformations and interactions. Whereas intraprotein cross-links can arise from proteins in isolation, interprotein cross-links reflect proximity of two interacting proteins in the sample. Prediction of expected ratios of the number of interprotein to intraprotein cross-links is hindered by lacking comprehensive knowledge on the interactome network and global occupancy levels for all interacting complex subunits. Here we determine the theoretical number of possible inter- and intraprotein cross-links in available PDB structures of proteins bound in complexes to predict a maximum expected fraction of interprotein cross-links in large scale in vivo cross-linking studies. We show how the maximum fraction can guide interpretation of reported interprotein fractions with respect to the extent of sample protein binding, comparing whole cell and lysate cross-linked samples as an example. We also demonstrate how an observation of interprotein cross-link fractions greater than the maximum value can result from the presence of false positive cross-links which are predominantly interprotein, their number estimable from the observed surplus fraction of interprotein cross-links.

Tools for 3D Interactome Visualization.

In cells, intra- and intermolecular interactions of proteins confer function, and the dynamic modulation of this interactome is critical to meet the changing needs required to support life. Cross-linking and mass spectrometry (XL-MS) enable the detection of both intra- and intermolecular protein interactions in organelles, cells, tissues, and organs. Quantitative XL-MS enables the detection of interactome changes in cells due to environmental, phenotypic, pharmacological, or genetic perturbations. We have developed new informatics capabilities, the first to enable 3D visualization of multiple quantitative interactome data sets, acquired over time or with varied perturbation levels, to reveal relevant dynamic interactome changes. These new tools are integrated within release 3.0 of our online cross-linked peptide database and analysis tool suite XLinkDB. With the recent rapid expansion in XL-MS for protein structural studies and the extension to quantitative XL-MS measurements, 3D interactome visualization tools are of critical need.

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.