Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability ...of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the Saccharomyces cerevisiae (budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.
Antibodies are one of the most formidable molecular weapons available to our immune system. Their high specificity against a target (antigen) and capability of triggering different immune responses ...(e.g., complement system activation and antibody-dependent cell-mediated cytotoxicity) make them ideal drugs to fight many different human diseases. Currently, both monoclonal antibodies and more complex molecules based on the antibody scaffold are used as biologics. Naturally, such highly heterogeneous molecules require dedicated analytical methodologies for their accurate characterization. Mass spectrometry (MS) can define the presence and relative abundance of multiple features of antibodies, including critical quality attributes. The combination of small and large variations within a single molecule can only be determined by analyzing intact antibodies or their large (25 to 100 kDa) subunits. Hence, top-down (TD) and middle-down (MD) MS approaches have gained popularity over the last decade. In this Young Scientist Feature we discuss the evolution of TD and MD MS analysis of antibodies, including the new frontiers that go beyond biopharma applications. We will show how this field is now moving from the "quality control" analysis of a known, single antibody to the high-throughput investigation of complex antibody repertoires isolated from clinical samples, where the ultimate goal is represented by the complete gas-phase sequencing of antibody molecules without the need of any a priori knowledge.
The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0–30 kDa mass range extracted from whole cell or tissue lysates. ...Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of Escherichia coli by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography–tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF.
Obtaining extensive sequencing of an intact protein is essential in order to simultaneously determine both the nature and exact localization of chemical and genetic modifications which distinguish ...different proteoforms arising from the same gene. To effectively achieve such characterization, it is necessary to take advantage of the analytical potential offered by the top-down mass spectrometry approach to protein sequence analysis. However, as a protein increases in size, its gas-phase dissociation produces overlapping, low signal-to-noise fragments. The application of advanced ion dissociation techniques such as electron transfer dissociation (ETD) and ultraviolet photodissociation (UVPD) can improve the sequencing results compared to slow-heating techniques such as collisional dissociation; nonetheless, even ETD- and UVPD-based approaches have thus far fallen short in their capacity to reliably enable extensive sequencing of proteoforms ≥30 kDa. To overcome this issue, we have applied proton transfer charge reduction (PTCR) to limit signal overlap in tandem mass spectra (MS2) produced by ETD (alone or with supplemental ion activation, EThcD). Compared to conventional MS2 experiments, following ETD/EThcD MS2 with PTCR MS3 prior to m/z analysis of deprotonated product ions in the Orbitrap mass analyzer proved beneficial for the identification of additional large protein fragments (≥10 kDa), thus improving the overall sequencing and in particular the coverage of the central portion of all four analyzed proteins spanning from 29 to 56 kDa. Specifically, PTCR-based data acquisition led to 39% sequence coverage for the 56 kDa glutamate dehydrogenase, which was further increased to 44% by combining fragments obtained via HCD followed by PTCR MS3.
Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide ...additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples. Clinical laboratories have been slow to adopt top-down analysis, also due to higher sample handling requirements. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on the depletion of albumin and immunoglobulins, followed by simplified protein fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography–tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to further simplify serum proteoform mixtures. This LC-FAIMS-MS2 method led to the identification of over 1000 serum proteoforms < 30 kDa, outperforming traditional LC-MS2 data acquisition and more than doubling the number of proteoforms identified in previous studies.
Existing mass spectrometric assays used for sensitive and specific measurements of target proteins across multiple samples, such as selected/multiple reaction monitoring (SRM/MRM) or parallel ...reaction monitoring (PRM), are peptide-based methods for bottom-up proteomics. Here, we describe an approach based on the principle of PRM for the measurement of intact proteoforms by targeted top-down proteomics, termed proteoform reaction monitoring (PfRM). We explore the ability of our method to circumvent traditional limitations of top-down proteomics, such as sensitivity and reproducibility. We also introduce a new software program, Proteoform Finder (part of ProSight Native), specifically designed for the easy analysis of PfRM data. PfRM was initially benchmarked by quantifying three standard proteins. The linearity of the assay was shown over almost 3 orders of magnitude in the femtomole range, with limits of detection and quantification in the low femtomolar range. We later applied our multiplexed PfRM assay to complex samples to quantify biomarker candidates in peripheral blood mononuclear cells (PBMCs) from liver-transplanted patients, suggesting their possible translational applications. These results demonstrate that PfRM has the potential to contribute to the accurate quantification of protein biomarkers for diagnostic purposes and to improve our understanding of disease etiology at the proteoform level.
Mass spectrometry assays demonstrate that Hsp90 inhibitors alter the expression of approximately one-quarter of the assayable proteome in mammalian cells. These changes are extraordinarily robust and ...reproducible, making "proteomics profiling" the gold standard for validating the effects of new Hsp90 inhibitors on cultured cells. Proteomics assays can also suggest novel hypotheses regarding drug mechanisms. To assist investigators in adopting this approach, this Chapter provides detailed protocols for conducting simple proteomics assays of Hsp90 inhibition. The protocols present a robust label-free approach that utilizes pre-fractionation of protein samples by SDS-PAGE, thereby providing reasonably good penetration into the proteome while addressing common issues with sample quality. The actual programming and operation of liquid chromatography-tandem mass spectrometers is not covered, but expectations for achievable performance are discussed, as are alternative approaches, common challenges, and software for data analysis.
While being interviewed in September 2018, Jeff Bezos, Founder and CEO of Amazon, discussed the interplay between corporations' desire to expand and governments' efforts to stifle growth in order to ...foster competition. Despite heading one of the largest and most valuable companies in the world, he conceded, "all big institutions of any kind will be and should be scrutinized... It's not personal. It's kind of what we want to have as a society happen." This Note examines how countries should balance the delicate issue of promoting mergers and allowing business owners to make their own decisions while simultaneously preventing any corporation from cornering too large a portion of any multimedia market.
The Terumo aortic (TA) Treo device (Terumo, Somerset, NJ) is an endograft with unique features that lends itself to fenestrated endovascular aneurysm repair (FEVAR), including a low device profile, a ...wide amplitude stent design, and an increased interstent distance. We have described our initial experience with the Treo device for FEVAR to treat short neck and juxtarenal abdominal aortic aneurysms.
As part of an ongoing physician-sponsored investigational device exemption clinical trial (ClinicalTrials.gov identifier, NCT01538056), subjects were prospectively enrolled and underwent elective FEVAR using a variety of devices. Demographic and procedural details were collected. The data from subjects treated specifically with the Treo device from November 3, 2016 to May 2, 2019 were collected and analyzed.
Of a cohort of 161 patients who had undergone elective FEVAR, 46 had been treated with the TA Treo device. Most patients were men (70%), with a mean age of 75 years and high rates of hypertension (74%), hyperlipidemia (83%), coronary artery disease (33%), and chronic obstructive pulmonary disease (33%). The mean aneurysm size was 66 mm, the mean preoperative infrarenal neck length was 5 mm, and the mean final seal zone length was 45 mm. The average hospital and intensive care unit lengths of stay were 2.4 and 1.5 days, respectively. A total of 129 fenestrations were created for 44 superior mesenteric and 85 renal arteries (2.8 fenestrations per patient). Technical success, defined as successful implantation of the device with all target vessels preserved, was 98% (45 of 46), with only one renal artery not successfully preserved. The mean follow-up period was 598 days. During the study period, 18 endoleaks were detected (17 type II and 1 type III), with one patient with a type III endoleak requiring reintervention. Three subjects had died within 30 days, one of intracranial hemorrhage, one of respiratory failure, and one of ischemic colitis. The graft modification times for the TA Treo were significantly shorter (43 minutes) than those for other commercially available devices (Cook Zenith, 55 minutes; Medtronic Endurant, 54 minutes; P < .0001).
Our institution has reported exclusive worldwide experience using the TA Treo device for FEVAR. This device provides for a highly efficient and technically successful procedure for most patients. The procedural and fluoroscopy times were low even in the setting of high complexity. The technical success rates and simplification of the FEVAR procedure have made this approach a preferred technique for most patients at our institution.
Humans are inextricably linked to each other and our natural world, and microorganisms lie at the nexus of those interactions. Microorganisms form genetically flexible, taxonomically diverse, and ...biochemically rich communities, i.e., microbiomes that are integral to the health and development of macroorganisms, societies, and ecosystems. Yet engagement with beneficial microbiomes is dictated by access to public resources, such as nutritious food, clean water and air, safe shelter, social interactions, and effective medicine. In this way, microbiomes have sociopolitical contexts that must be considered. The Microbes and Social Equity (MSE) Working Group connects microbiology with social equity research, education, policy, and practice to understand the interplay of microorganisms, individuals, societies, and ecosystems. Here, we outline opportunities for integrating microbiology and social equity work through broadening education and training; diversifying research topics, methods, and perspectives; and advocating for evidence-based public policy that supports sustainable, equitable, and microbial wealth for all.
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