Precise prefractionation of proteome samples is a potent method for realizing in-depth analysis in top-down proteomics. PEPPI-MS (Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS), a gel-based sample fractionation method, enables high-resolution proteome fractionation based on molecular weight by highly efficient extraction of proteins from polyacrylamide gels after SDS-PAGE separation. Thereafter it is essential to effectively remove contaminants such as CBB and SDS from the PEPPI fraction prior to mass spectrometry.
View Article and Find Full Text PDFProteostasis, the maintenance of cellular protein balance, is essential for cell viability and is highly conserved across all organisms. Newly synthesized proteins, or "clients," undergo sequential processing by Hsp40, Hsp70, and Hsp90 chaperones to achieve proper folding and functionality. Despite extensive characterization of post-translational modifications (PTMs) on Hsp70 and Hsp90, the modifications on Hsp40 remain less understood.
View Article and Find Full Text PDFModern mass spectrometry technology allows for extensive sequencing of the ~ 25 kDa subunits of monoclonal antibodies (mAbs) produced by IdeS proteolysis followed by disulfide bond reduction, an approach known as middle-down mass spectrometry (MD MS). However, the spectral congestion of tandem mass spectra of large polypeptides dramatically complicates fragment ion assignment. Here, we report the development and benchmark of an MD MS strategy based on the combination of different ion fragmentation techniques with proton transfer charge reduction (PTCR) to simplify the gas-phase sequencing of mAb subunits.
View Article and Find Full Text PDFProteostasis, the maintenance of cellular protein balance, is essential for cell viability and is highly conserved across all organisms. Newly synthesized proteins, or "clients," undergo sequential processing by Hsp40, Hsp70, and Hsp90 chaperones to achieve proper folding and functionality. Despite extensive characterization of post-translational modifications (PTMs) on Hsp70 and Hsp90, the modifications on Hsp40 remain less understood.
View Article and Find Full Text PDFAntibodies 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 (, 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.
View Article and Find Full Text PDFExisting 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.
View Article and Find Full Text PDFBlood 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.
View Article and Find Full Text PDFThe 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 by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS).
View Article and Find Full Text PDFCreating multifunctional concrete materials with advanced functionalities and mechanical tunability is a critical step toward reimagining the traditional civil infrastructure systems. Here, the concept of nanogenerator-integrated mechanical metamaterial concrete is presented to design lightweight and mechanically tunable concrete systems with energy harvesting and sensing functionalities. The proposed metamaterial concrete systems are created via integrating the mechanical metamaterial and nano-energy-harvesting paradigms.
View Article and Find Full Text PDFHsp70 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.
View Article and Find Full Text PDFVoltage-gated sodium channel Na1.8 regulates transmission of pain signals to the brain. While Na1.
View Article and Find Full Text PDFThe voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.
View Article and Find Full Text PDFObtaining 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.
View Article and Find Full Text PDFMass 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.
View Article and Find Full Text PDFSumoylation is a powerful regulatory system that controls many of the critical processes in the cell, including DNA repair, transcriptional regulation, nuclear transport, and DNA replication. Recently, new functions for SUMO have begun to emerge. SUMO is covalently attached to components of each of the four major cytoskeletal networks, including microtubule-associated proteins, septins, and intermediate filaments, in addition to nuclear actin and actin-regulatory proteins.
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