Targeted Quantification of Proteoforms in Complex Samples by Proteoform Reaction Monitoring.

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.

Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms.

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.

Improved Label-Free Quantification of Intact Proteoforms Using Field Asymmetric Ion Mobility Spectrometry.

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.

Multifunctional Nanogenerator-Integrated Metamaterial Concrete Systems for Smart Civil Infrastructure.

Creating 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. These advanced materials are composed of reinforcement auxetic polymer lattices with snap-through buckling behavior fully embedded inside a conductive cement matrix. We rationally design their composite structures to induce contact-electrification between the layers under mechanical excitations/triggering. The conductive cement enhanced with graphite powder serves as the electrode in the proposed systems, while providing the desired mechanical performance. Experimental studies are conducted to investigate the mechanical and electrical properties of the designed prototypes. The metamaterial concrete systems are tuned to achieve up to 15% compressibility under cycling loading. The power output of the nanogenerator-integrated metamaterial concrete prototypes reaches 330 µW. Furthermore, the self-powered sensing functionality of the nanogenerator concrete systems for distributed health monitoring of large-scale concrete structures is demonstrated. The metamaterial concrete paradigm can possibly enable the design of smart civil infrastructure systems with a broad range of advanced functionalities.

Mass spectrometry characterization of antibodies at the intact and subunit levels: from targeted to large-scale analysis.

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.

Comprehensive characterization of the Hsp70 interactome reveals novel client proteins and interactions mediated by posttranslational modifications.

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.

Structural and Functional Characterization of a Novel Scorpion Toxin that Inhibits NaV1.8 via Interactions With the DI Voltage Sensor and DII Pore Module.

Voltage-gated sodium channel NaV1.8 regulates transmission of pain signals to the brain. While NaV1.8 has the potential to serve as a drug target, the molecular mechanisms that shape NaV1.8 gating are not completely understood, particularly mechanisms that couple activation to inactivation. Interactions between toxin producing animals and their predators provide a novel approach for investigating NaV structure-function relationships. Arizona bark scorpions produce Na+ channel toxins that initiate pain signaling. However, in predatory grasshopper mice, toxins inhibit NaV1.8 currents and block pain signals. A screen of synthetic peptide toxins predicted from bark scorpion venom showed that peptide NaTx36 inhibited Na+ current recorded from a recombinant grasshopper mouse NaV1.8 channel (OtNaV1.8). Toxin NaTx36 hyperpolarized OtNaV1.8 activation, steady-state fast inactivation, and slow inactivation. Mutagenesis revealed that the first gating charge in the domain I (DI) S4 voltage sensor and an acidic amino acid (E) in the DII SS2 - S6 pore loop are critical for the inhibitory effects of NaTx36. Computational modeling showed that a DI S1 - S2 asparagine (N) stabilizes the NaTx36 - OtNaV1.8 complex while residues in the DI S3 - S4 linker and S4 voltage sensor form electrostatic interactions that allow a toxin glutamine (Q) to contact the first S4 gating charge. Surprisingly, the models predicted that NaTx36 contacts amino acids in the DII S5 - SS1 pore loop instead of the SS2 - S6 loop; the DII SS2 - S6 loop motif (QVSE) alters the conformation of the DII S5 - SS1 pore loop, enhancing allosteric interactions between toxin and the DII S5 - SS1 pore loop. Few toxins have been identified that modify NaV1.8 gating. Moreover, few toxins have been described that modify sodium channel gating via the DI S4 voltage sensor. Thus, NaTx36 and OtNaV1.8 provide tools for investigating the structure-activity relationship between channel activation and inactivation gating, and the connection to alternative pain phenotypes.

Identification and Characterization of Novel Proteins from Arizona Bark Scorpion Venom That Inhibit Nav1.8, a Voltage-Gated Sodium Channel Regulator of Pain Signaling.

The 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 Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure-activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a "CXCE" motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

Sequential Ion-Ion Reactions for Enhanced Gas-Phase Sequencing of Large Intact Proteins in a Tribrid Orbitrap Mass Spectrometer.

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.

Proteomic Profiling of Hsp90 Inhibitors.

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.

Emerging roles of sumoylation in the regulation of actin, microtubules, intermediate filaments, and septins.

Sumoylation 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. However, knowledge of the mechanisms by which this signal transduction system controls the cytoskeleton is still in its infancy. One story that is beginning to unfold is that SUMO may regulate the microtubule motor protein dynein by modification of its adaptor Lis1. In other instances, cytoskeletal elements can both bind to SUMO non-covalently and also be conjugated by it. The molecular mechanisms for many of these new functions are not yet clear, but are under active investigation. One emerging model links the function of MAP sumoylation to protein degradation through SUMO-targeted ubiquitin ligases, also known as STUbL enzymes. Other possible functions for cytoskeletal sumoylation are also discussed.