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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. Here we report a novel strategy for the rapid detection of SARS-CoV-2 based on an enrichment approach exploiting the affinity between the virus and cellulose sulfate ester functional groups, hot acid hydrolysis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Virus samples were enriched using cellulose sulfate ester microcolumns. Virus peptides were prepared using the hot acid aspartate-selective hydrolysis and characterized by MALDI-TOF MS. Collected spectra were processed with a peptide fingerprint algorithm, and searching parameters were optimized for the detection of SARS-CoV-2. These peptides provide high sequence coverage for nucleocapsid (N protein) and allow confident identification of SARS-CoV-2. Peptide markers contributing to the detection were rigorously identified using bottom-up proteomics. The approach demonstrated in this study holds the potential for developing a rapid assay for COVID-19 diagnosis and detecting virus variants from a variety of sources, such as sewage and nasal swabs.

A top-down proteomic strategy with semiautomated analysis of data sets has proven successful for the global identification of truncated proteins without the use of chemical derivatization, enzymatic manipulation, immunoprecipitation, or other enrichment. This approach provides the reliable identification of internal polypeptides formed from precursor gene products by proteolytic cleavage of both the N- and C-termini, as well as truncated proteoforms that retain one or the other termini. The strategy has been evaluated by application to the immunosuppressive extracellular vesicles released by myeloid-derived suppressor cells. More than 1000 truncated proteoforms have been identified, from which binding motifs are derived to allow characterization of the putative proteases responsible for truncation.

Rapid identification of Bacillus spores in the environment has depended primarily on a family of small acid soluble proteins (SASPs) as biomarkers. However, SASP sequences and molecular masses are similar or identical in some critical cases. For example, some strains of B. subtilis, and B. thuringiensis cannot be distinguished from strains of B. anthracis based on SASPs. Consequently, additional or alternative biomarkers should be sought. In this study microwave-assisted hot acid hydrolysis was coupled with mass spectrometry as a potentially powerful approach to the rapid automatable characterization of Bacillus spores. Hot acid provides lysis of the spores, Asp-selective hydrolysis of proteins, and peptides compatible with automated analysis of either peptide fingerprints or tandem mass spectra. Peptide biomarkers are compared here for a selection of Bacillus spores, and peptides unique to each spore type are identified.

A strategy for top-down analysis of branched proteins has been reported earlier, which relies on electron transfer dissociation assisted by collisional activation, and software designed for graphic interpretation of tandem mass spectra and adapted for branched proteins. In the present study, the strategy is applied to identify unknown and novel products of reactions in which rationally mutated proteoforms of Rub1 are used to probe the selectivity of E1 and E2 enzymes normally active in ubiquitination. To test and demonstrate this application, components and attachment sites of three branched dimers are deduced and the mutations are confirmed.

Collaborative research is reviewed in which mass spectrometry-based proteomics and next generation sequencing were used qualitatively and quantitatively to interrogate proteins and RNAs carried in intact myeloid-derived suppressor cells (MDSC) and exosomes shed in vitro by MDSC. In aggregate exosomes more than 4000 proteins were identified, including annexins and immunosuppressive mediators. Bioassays showed that exosomes induce MDSC chemotaxis dependent on S100A8 and S100A9 in their cargo. Surface selective chemistry identified glycoproteins on MDSC and exosome surfaces, including CD47 and thrombospondin 1, which both facilitate exosome-catalyzed chemotaxis. Large numbers of mRNAs and microRNAs were identified in aggregate exosomes, whose potential functions in receptor cells include angiogenesis, and proinflammatory and immunosuppressive activities. Inflammation was found to have asymmetric effects on MDSC and exosomal cargos. Collectively, our findings indicate that the exosomes shed by MDSC provide divergent and complementary functions that support the immunosuppression and tumor promotion activities of MDSC.

Identification of antigens is important for vaccine production. We tested extraction protocols using cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS) to formulate surfactant vesicles (SVs) containing components from Neisseria gonorrhoeae. Carbohydrate and protein assays demonstrated that protein and carbohydrates were incorporated into the vesicle leaflet. Depending on the extraction protocol utilized, 100&ndash, 400 &micro, g of protein/mL of SVs solution was obtained. Gel electrophoresis followed by silver staining demonstrated that SV extracts contained lipooligosaccharide and a subset of bacterial proteins and lipoproteins. Western blotting and mass spectral analysis indicated that the majority of the proteins were derived from the outer membrane. Mass spectrometric and bioinformatics analysis of SVs identified 29 membrane proteins, including porin and opacity-associated protein. Proteins embedded in the SVs leaflet could be degraded by the addition of trypsin or proteinase K. Our data showed that the incorporation of CTAT and SDBS into vesicles eliminated their toxicity as measured by a THP-1 killing assay. Incorporation of gonococcal cell surface components into SVs reduced toxicity as compared to the whole cell extracts, as measured by cytokine induction, while retaining the immunogenicity. This process constitutes a general method for extracting bacterial surface components and identification of antigens that might be included in vaccines.

Rapid identification of Bacillus spores in the environment has depended primarily on a family of small acid soluble proteins (SASPs) as biomarkers. However, SASP sequences and molecular masses are similar or identical in some critical cases. For example, some strains of B. subtilis, and B. thuringiensis cannot be distinguished from strains of B. anthracis based on SASPs. Consequently, additional or alternative biomarkers should be sought. In this study microwave-assisted hot acid hydrolysis was coupled with mass spectrometry as a potentially powerful approach to the rapid automatable characterization of Bacillus spores. Hot acid provides lysis of the spores, Asp-selective hydrolysis of proteins, and peptides compatible with automated analysis of either peptide fingerprints or tandem mass spectra. Peptide biomarkers are compared here for a selection of Bacillus spores, and peptides unique to each spore type are identified.

Ricin is a protein toxin of considerable interest in forensics. A novel strategy is reported here for rapid detection of ricin based on microwave-assisted hot acid digestion and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Ricin samples are subjected to aspartate-selective hydrolysis, and biomarker peptide products are characterized by mass spectrometry. Spectra are obtained using post source decay and searched against a protein database. Several advantages are offered by chemical hydrolysis, relative to enzymatic hydrolysis, notably speed, robustness, and the production of heavier biomarkers. Agglutinin contamination is reliably recognized, as is the disulfide bond strongly characteristic of ricin.

Post-translational modifications by the covalent attachment of Rub1 (NEDD8), ubiquitin, SUMO, and other small signaling proteins have profound impacts on the functions and fates of cellular proteins. Investigations of the relationship of these bioactive structures and their functions are limited by analytical methods that are scarce and tedious. A novel strategy is reported here for the analysis of branched proteins by top-down mass spectrometry and illustrated by application to four recombinant proteins and one synthetic peptide modified by covalent bonds with ubiquitin or Rub1. The approach allows an analyte to be recognized as a branched protein; the participating proteins to be identified; the site of conjugation to be defined; and other chemical, native, and recombinant modifications to be characterized. In addition to the high resolution and high accuracy provided by the mass spectrometer, success is based on sample fragmentation by electron-transfer dissociation assisted by collisional activation and on software designed for graphic interpretation and adapted for branched proteins. The strategy allows for structures of unknown, two-component branched proteins to be elucidated directly the first time and can potentially be extended to more complex systems.

Ubiquitinated proteins carried by the extracellular vesicles (EV) released by myeloid-derived suppressor cells (MDSC) have been investigated using proteomic strategies to examine the effect of tumor-associated inflammation. EV were collected from MDSC directly following isolation from tumor-bearing mice with low and high inflammation. Among the 1092 proteins (high inflammation) and 925 proteins (low inflammation) identified, more than 50% were observed as ubiquitinated proteoforms. More than three ubiquitin-attachment sites were characterized per ubiquitinated protein, on average. Multiple ubiquitination sites were identified in the pro-inflammatory proteins S100 A8 and S100 A9, characteristic of MDSC and in histones and transcription regulators among other proteins. Spectral counting and pathway analysis suggest that ubiquitination occurs independently of inflammation. Some ubiquitinated proteins were shown to cause the migration of MDSC, which has been previously connected with immune suppression and tumor progression. Finally, MDSC EV are found collectively to carry all the enzymes required to catalyze ubiquitination, and the hypothesis is presented that a portion of the ubiquitinated proteins are produced in situ.

The small protein ubiquitin and its multiple polymers are encountered free in cells and as post-translational modifications on all proteins. Different polyubiquitin three dimensional structures are shown to correlate uniquely with different cellular functions as part of the diverse ubiquitin signaling. At the same time, this multiplicity of structures provides serious challenges to the analytical biochemist. Globally applicable strategies are presented here for the analyses of polyubiquitins and of ubiquitinated proteins, which take advantage of the speed, specificity and sensitivity of top-down tandem mass spectrometry. Particular attention is given to the supervised interpretation of fragmentation as revealed in the MS/MS spectra of these branched proteins. The strategy is compatible with any MS activation technology, is applicable to all polyubiquitin linkage and chain types, can be extended to ubiquitin-like proteins, and will be compatible with and enhanced by continuing advances in LC-MS/MS instrumentation and interpretation software.

The characterization of polyubiquitin chains has been an analytical challenge for several decades. It has been shown that anchored and unanchored polyubiquitin chains with different isopeptide linkages and lengths exhibit a wide range of profoundly different cellular functions. However, structure function studies have been hindered by the difficulty of characterizing these complex chain structures. This report presents a broadly applicable workflow to characterize ubiquitin tetramers without the need for genetic mutations or reiterative immunoprecipitations. We use a top-down proteomic strategy that exploits ETciD activation on an orbitrap Fusion Lumos and manual interpretation aided by graphical interpretation of mass shifts to facilitate characterization of chain topography and lysine linkage sites. Our workflow differentiates all topological features of the numerous isomers of tetraubiquitin, which have molecular masses in excess of 34 000 Da and identifies linkage sites in these branched proteins. Copyright © 2016 John Wiley & Sons, Ltd.

The profound effects of ubiquitination on the movement and processing of cellular proteins depend exquisitely on the structures of monoubiquitin and polyubiquitin modifications. Unconjugated polyubiquitins also have a variety of intracellular functions. Structures and functions are not well correlated yet, because the structures of polyubiquitins and polyubiquitin modifications of proteins are difficult to decipher. We are moving towards a robust strategy to provide that structural information. In this report electron transfer dissociation mass spectra of six synthetic ubiquitin trimers (multiply branched proteins with molecular masses exceeding 25 600 Da) are examined using an Orbitrap Fusion Lumos instrument to determine how top-down mass spectrometry can characterize the chain topology and linkage sites in a single, facile workflow. The efficacy of this method relies on the formation, detection, and interpretation of extensive fragmentation. Copyright © 2016 John Wiley & Sons, Ltd.

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

Enrichment of the plasma membrane represents one valuable method to characterize the surfaceome, along with other plasma membrane and structural proteins. Currently, the overlapping densities of many subcellular organelles hinder enrichment of the plasma membrane by centrifugation. However, external access to the plasma membrane of intact cells allows the attachment of a nanoparticle pellicle to enhance its density and facilitate enrichment. We describe the synthesis of iron oxide nanoparticles, attachment of the pellicle to suspended cells, and recovery of plasma membrane proteins for proteomic analysis.

Top-down analysis is reported for a portion of the protein cargo of exosomes shed by myeloid-derived suppressor cells that participate in intracellular signaling in the tumor microenvironment. Instrument mass resolution limited the study to proteins of molecular masses below 30 kDa. A two-step fractionation strategy was used, including open tubular gel electrophoresis and C3 reverse phase high performance liquid chromatography. Twenty-one unique proteins were identified among more than 200 proteoforms, and comprising primarily two functionally important protein families: the S100 proinflammatory mediators and an abundance of histones. Fifty-six percent of the total protein in these exosomes was determined to comprise histones, of which H2B variants contribute 42 %.

Multiple studies demonstrate that ubiquitination of proteins codes for regulation of cell differentiation, apoptosis, endocytosis and many other cellular functions. There is great interest in and considerable effort being given to defining the relationships between the structures of polyubiquitin modifications and the fates of the modified proteins. Does each ubiquitin modification achieve a specific effect, much like phosphorylation, or is ubiquitin like glycosylation, where there is heterogeneity and redundancy in the signal? The sensitive analytical tools needed to address such questions readily are not yet mature. To lay the foundation for mass spectrometry (MS)-based studies of the ubiquitin code, we have assembled seven isomeric diubiquitins with all-native sequences and isopeptide linkages. Using these compounds as standards enables the development and testing of a new MS-based strategy tailored specifically to characterize the number and sites of isopeptide linkages in polyubiquitin chains. Here, we report the use of Asp-selective acid cleavage, separation by reverse phase high-performance liquid chromatography and characterization by tandem MS to distinguish and characterize all seven isomeric lysine-linked ubiquitin dimers. Copyright © 2014 John Wiley & Sons, Ltd.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the circulation and the tumor microenvironment of most cancer patients. There, MDSC suppress both adaptive and innate immunity, hindering immunotherapies. The inflammatory milieu often present in cancers facilitates MDSC suppressive activity, causing aggressive tumor progression and metastasis. MDSC from tumor-bearing mice release exosomes, which carry biologically active proteins and mediate some of the immunosuppressive functions characteristic of MDSC. Studies on other cell types have shown that exosomes may also carry RNAs which can be transferred to local and distant cells, yet the mRNA and microRNA cargo of MDSC-derived exosomes has not been studied to date. Here, the cargo of MDSC and their exosomes was interrogated with the goal of identifying and characterizing molecules that may facilitate MDSC suppressive potency. Because inflammation is an established driving force for MDSC suppressive activity, we used the well-established 4T1 mouse mammary carcinoma system, which includes "conventional" as well as "inflammatory" MDSC. We provide evidence that MDSC-derived exosomes carry proteins, mRNAs, and microRNAs with different quantitative profiles than those of their parental cells. Several of these molecules have known or predicted functions consistent with MDSC suppressive activity, suggesting a potential mechanistic redundancy.

Myeloid-derived suppressor cells (MDSC) are a diverse population of immature myeloid cells that have potent immune-suppressive activity. Studies in both mice and humans have demonstrated that MDSC accumulate in most individuals with cancer, where they promote tumor progression, inhibit antitumor immunity, and are an obstacle to many cancer immunotherapies. As a result, there has been intense interest in understanding the mechanisms and in situ conditions that regulate and sustain MDSC, and the mechanisms MDSC use to promote tumor progression. This article reviews the characterization of MDSC and how they are distinguished from neutrophils, describes the suppressive mechanisms used by MDSC to mediate their effects, and explains the role of proinflammatory mediators and the tumor microenvironment in driving MDSC accumulation, suppressive potency, and survival.

Aim: To construct a new robust nanowire-based pellicle for eukaryotic cells, to investigate the interactions between nanowires (NWs) and cell surfaces and the internalization of nanowires, and to demonstrate for isolation of the plasma membrane with improved enrichment of transmembrane proteins. Materials & methods: Silica NWs were coated with alumina to give positive charges on their surface. Multiple myeloma cells were coated with the positively charged NWs by dropping the cells into a buffered suspension of NWs. After the NW-coated cells were lysed, plasma membrane fragments were enriched by differential centrifugation for proteomic studies. Results: Here we demonstrate complete cell coating with positively charged, alumina-coated silica NWs via nonspecific electrostatic interactions, and characterize a robust pellicle and little/no uptake of NWs. Conclusion: Robust pellicles provide a new platform for therapeutic, diagnostic and biochemical interactions of nanostructures with eukaryotic cells. Original submitted 13 December 2012; Revised submitted 29 August 2013

Myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they inhibit natural anti-tumor immunity and are an obstacle to anti-cancer immunotherapies. They mediate immune suppression through their production of proteins and soluble mediators that prevent the activation of tumor-reactive T lymphyocytes, polarize macrophages towards a tumor-promoting phenotype, and facilitate angiogenesis. The accumulation and suppressive potency of MDSC is regulated by inflammation within the tumor microenvironment. Recently exosomes have been proposed to act as intercellular communicators, carrying active proteins and other molecules between sender cells and receiver cells. In this report we describe the proteome of exosomes shed by MDSC induced in BALB/c mice by the 4T1 mammary carcinoma. Using bottom-up proteomics, we have identified 412 proteins. Spectral counting identified 63 proteins whose abundance was altered > 2-fold in the inflammatory environment. The pro-inflammatory proteins S100A8 and S100A9, previously shown to be secreted by MDSC and to be chemotactic for MDSC, are abundant in MDSC-derived exosomes. Bioassays reveal that MDSC-derived exosomes polarize macrophages towards a tumor-promoting type 2 phenotype, in addition to possessing S100A8/A9 chemotactic activity. These results suggest that some of the tumor-promoting functions of MDSC are implemented by MDSC-shed exosomes.

A strategy is presented for enhancing the middle-down analysis of higher mass peptides recovered from complex protein mixtures. Following a 30-min digestion of multiple myeloma cell lysate by an acid cleavage reaction that is selective for aspartic acid, a 3000 Da membrane filter is used to bifurcate the peptide product mixture, and the heavier fraction is subjected to collisional activation with precursor selection that excludes charge states below +4. Filtration and charge state selection are shown to provide significant increases in the number of peptides identified in the mass range above 3000 Da and in information about protein sequences. Copyright © 2013 John Wiley & Sons, Ltd.

Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation, and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular components. Herein, we test the hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis. Multiple myeloma cells were grown and coated in suspension with three different pellicles of three different densities and both pellicle coated and uncoated suspensions analyzed by high-throughput LC-MS/MS. Enrichment was evaluated by the total number and the spectral counts of identified plasma membrane proteins.

Spectral counting is a straightforward label-free quantitation strategy used in bottom-up proteomics workflows. The application of spectral counting in label-free top-down proteomics workflows can be similarly straightforward but has not been applied as widely as quantitation by chromatographic peak areas or peak intensities. In this study, we evaluate spectral counting for quantitative comparisons in label-free top down proteomics workflows by comparison with chromatographic peak areas and intensities. We tested these quantitation approaches by spiking standard proteins into a complex protein background and comparing relative quantitation by spectral counts with normalized chromatographic peak areas and peak intensities from deconvoluted extracted ion chromatograms of the spiked proteins. Ratio estimates and statistical significance of differential abundance from each quantitation technique are evaluated against the expected ratios and each other. In this experiment, spectral counting was able to detect differential abundance of spiked proteins for expected ratios ≥2, with comparable or higher sensitivity than normalized areas and intensities. We also found that while ratio estimates using peak areas and intensities are usually more accurate, the spectral-counting-based estimates are not substantially worse. Following the evaluation and comparison of these label-free top down quantitation strategies using spiked proteins, spectral counting, along with normalized chromatographic peak areas and intensities, were used to analyze the complex protein cargo of exosomes shed by myeloid-derived suppressor cells collected under high and low conditions of inflammation, revealing statistically significant differences in abundance for several proteoforms, including the active pro-inflammatory proteins S100A8 and S100A9.

In this report, we use a proteomic strategy to identify glycoproteins on the surface of exosomes derived from myeloid-derived suppressor cells (MDSCs), and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSCs. We report successful modification of a surface chemistry method for use with exosomes and identify 21 surface N-glycoproteins on exosomes released by mouse mammary carcinoma-induced MDSCs. These glycoprotein identities and functionalities are compared with 93 N-linked glycoproteins identified on the surface of the parental cells. As with the lysate proteomes examined previously, the exosome surface N-glycoproteins are primarily a subset of the glycoproteins on the surface of the suppressor cells that released them, with related functions and related potential as therapeutic targets. The “don’t eat me” molecule CD47 and its binding partners thrombospondin-1 (TSP1) and signal regulatory protein α (SIRPα) were among the surface N-glycoproteins detected. Functional bioassays using antibodies to these three molecules demonstrated that CD47, TSP1, and to a lesser extent SIRPα facilitate exosome-mediated MDSC chemotaxis and migration.

A better understanding of molecular signaling between myeloid-derived suppressor cells (MDSC), tumor cells, T-cells, and inflammatory mediators is expected to contribute to more effective cancer immunotherapies. We focus on plasma membrane associated proteins, which are critical in signaling and intercellular communication, and investigate changes in their abundance in MDSC of tumor-bearing mice subject to heightened versus basal inflammatory conditions. Using spectral counting, we observed statistically significant differential abundances for 35 proteins associated with the plasma membrane, most notably the pro-inflammatory proteins S100A8 and S100A9 which induce MDSC and promote their migration. We also tested whether the peptides associated with canonical pathways showed a statistically significant increase or decrease subject to heightened versus basal inflammatory conditions. Collectively, these studies used bottom-up proteomic analysis to identify plasma membrane associated pro-inflammatory molecules and pathways that drive MDSC accumulation, migration, and suppressive potency.

The characterization of polyubiquitin chains has been an analytical challenge for several decades. It has been shown that anchored and unanchored polyubiquitin chains with different isopeptide linkages and lengths exhibit a wide range of profoundly different cellular functions. However, structure function studies have been hindered by the difficulty of characterizing these complex chain structures. This report presents a broadly applicable workflow to characterize ubiquitin tetramers without the need for genetic mutations or reiterative immunoprecipitations. We use a top-down proteomic strategy that exploits ETciD activation on an orbitrap Fusion Lumos and manual interpretation aided by graphical interpretation of mass shifts to facilitate characterization of chain topography and lysine linkage sites. Our workflow differentiates all topological features of the numerous isomers of tetraubiquitin, which have molecular masses in excess of 34 000 Da and identifies linkage sites in these branched proteins. Copyright © 2016 John WileySons, Ltd.

The profound effects of ubiquitination on the movement and processing of cellular proteins depend exquisitely on the structures of monoubiquitin and polyubiquitin modifications. Unconjugated polyubiquitins also have a variety of intracellular functions. Structures and functions are not well correlated yet, because the structures of polyubiquitins and polyubiquitin modifications of proteins are difficult to decipher. We are moving towards a robust strategy to provide that structural information. In this report electron transfer dissociation mass spectra of six synthetic ubiquitin trimers (multiply branched proteins with molecular masses exceeding 25,600 Da) are examined using an Orbitrap Fusion Lumos instrument to determine how top-down mass spectrometry can characterize the chain topology and linkage sites in a single, facile workflow. The efficacy of this method relies on the formation, detection, and interpretation of extensive fragmentation.

Proteomics and bioinformatics provide an interpretive approach for the identification of bacteria and other microorganisms, which is faster and more reliable than library matching. Identifications can be made without controlling sample preparation or instrumental variables. One rapidly growing database supports all ionization techniques. In addition, specific biomarkers can be identified for strain identification and forensics. The advantages of proteomic strategies are illustrated in the analysis of components in mixtures, genetic engineering in bacteria, and bacteria with unsequenced genomes.

Post-translational modification of proteins by covalent attachment of ubiquitin or a polyubiquitin chain is involved in myriad of processes in eukaryotic cells. The particular outcome of ubiquitination is directed by the length of the ubiquitin conjugate and its linkage composition. Among seven possible isopeptide linkage sites in ubiquitin, K48 and K63 occur most commonly and act as distinct cellular signals. Strategies are reported here for analysis of linkage sites and complexity of K63-linked polyubiquitin chains, based on rapid chemical proteolysis at aspartate residues combined with immunoprecipitation and mass spectrometry. Rapid chemical proteolysis at aspartate residues results in K63-linked peptides with truncated branches, which enable identification and characterization of stretches of consecutive K63 linkages on generally available instruments. A characteristic cleavage pattern and a characteristic fragmentation pattern allow recognition of K63 oligomers in proteolytic mixtures. Engineered K63-linked polyubiquitin chains of defined lengths were used to evaluate and demonstrate the method. In-gel microwave-supported acid hydrolysis was used to observe peptides specific to K63-linked ubiquitin dimers and trimers. Acid hydrolysis in solution, used in conjunction with linkage-specific immunoprecipitation, allowed more complex K63-linked branches to be characterized. Finally, a substrate protein, UbcH5b, was conjugated to monoubiquitin and to polyubiquitin chains containing only K63 linkages, and the sites of conjugation and chain lengths were characterized.

We present a building-block approach toward functionalized CB[7] derivatives by the condensation of methylene-bridged glycoluril hexamer 1 and glycoluril bis(cyclic ethers) 2 and 12. The CB[7] derivatives Me2CB[7] and CyCB[7] are highly soluble in water (264 mM and 181 mM, respectively). As a result of the high intrinsic solubility of Me2CB[7], it is able to solubilize the insoluble benzimidazole drug albendazole. The reaction of hexamer 1 with glycoluril derivative 12, which bears a primary alkyl chloride group, gives CB[7] derivative 18 in 16% isolated yield. Compound 18 reacts with NaN3 to yield azide-substituted CB[7] 19 in 81% yield, which subsequently undergoes click reaction with propargylammonium chloride (21) to yield CB[7] derivative 20 in 95% yield, which bears a covalently attached triazolyl ammonium group along its equator. The results of NMR spectroscopy ((1)H, variable-temperature, and DOSY) and electrospray mass spectrometry establish that 20 undergoes self-assembly to form a cyclic tetrameric assembly (204) in aqueous solution. CB[7] derivatives bearing reactive functional groups (e.g., N3, Cl) are now available for incorporation into more complex functional systems.

Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. On the basis of the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins.

Microwave assisted acid cleavage was applied directly to intact adenovirus type 5 to achieve denaturation and proteolysis in a single reaction. The speed of the digestion, coupled with the simplicity of MALDI analysis, allowed peptide products to be profiled in less than 5 min. Identification of peptides from a range of proteins by MALDI-TOF confirms that both denaturation and proteolysis were achieved using low concentrations of acetic acid (12.5%) and short incubations (1.5 to 2 min) at high temperatures (140° C). These conditions favor production of peptides that carry Asp on their C-termini. When this cleavage reaction is carried out in highly enriched H(2) (18)O, a single atom of (18)O is introduced site-specifically into the carboxyl terminal. The labeling reaction is applied to label reporter peptides from human adenovirus type 5 harvested from HeLa cells. Small peptide products of endogenous processing were also observed.

Currently, most MS-based proteomic studies of bacteria and archea match experimental data to known amino acid sequences from the target organism. Top-down studies use a protein's molecular weight along with data gathered from MS/MS experiments to identify proteins by database matching. For Erwinia herbicola and Enterobacter cloacae, studied here, the necessary protein sequences are not available in protein sequence repositories. We apply top-down protein fragmentation, but match the experimental data with homologous proteins from related organisms with sequenced genomes, demonstrating considerable shared protein sequence between closely related bacteria. Using this homology-based approach, we are not only able to identify representative proteins, but are also able to place the two target bacteria in their correct phylogeny. Furthermore, we show that the unexpected mass delta between the experimental precursor and matched protein sequence can often be localized and characterized using accurate-mass precursor and fragment ion measurements. Finally, we demonstrate that proteins identified by top-down workflows provide strong experimental evidence for correct, missing, and misannotated bacterial protein sequences, not only in the analyzed organism, but also for homologous proteins in closely related species.

MALDI mass spectrometry-based systems for rapid characterization of microorganisms in biodefense or medical diagnostics usually detect intact proteins in the 5000-20,000 Da range. To evaluate the reliability of species discrimination, and also for forensic applications, it is important that these biomarker proteins be identified. In the present study we apply high resolution tandem mass analysis on an Orbitrap and top-down bioinformatics to identify major biomarker proteins observed in MALDI spectra of intact bacteria for which little genomic or protein sequence information is available. The strategy depends on recognition of proteins with very high homology in related (sequenced) species, making it possible to place unsequenced organisms in their correct phylogenetic context. We show that this rapid proteomics based approach to phylogenetic characterization produces similar results to the traditional techniques, and may even be applied to target organisms of undetermined taxonomy. We further discuss important issues in combining genomics/proteomics databases and MALDI MS for the rapid characterization of microorganisms.

Butyrophilin 1A1 (BTN1A1) and xanthine oxidoreductase (XOR) are highly expressed in the lactating mammary gland and are secreted into milk associated with the milk fat globule membrane (MFGM). Ablation of the genes encoding either protein causes severe defects in the secretion of milk lipid droplets, suggesting that the two proteins may function in the same pathway. Therefore, we determined whether BTN1A1 and XOR directly interact using protein binding assays, surface plasmon resonance analysis, and gel filtration. Bovine XOR bound with high affinity in a pH- and salt-sensitive manner (KD = 101 ± 31 nm in 10 mm HEPES, 150 mm NaCl, pH 7.4) to the PRY/SPRY/B30.2 domain in the cytoplasmic region of bovine BTN1A1. Binding was stoichiometric, with one XOR dimer binding to either two BTN1A1 monomers or one dimer. XOR bound to BTN1A1 orthologs from mice, humans, or cows but not to the cytoplasmic domains of the closely related human paralogs, BTN2A1 or BTN3A1, or to the B30.2 domain of human RoRet (TRIM 38), a protein in the TRIM family. Analysis of the protein composition of the MFGM of wild type and BTN1A1 null mice showed that most of the XOR in mice lacking BTN1A1 was released from the MFGM in a soluble form when the milk lipid droplets were disrupted to prepare membrane, compared with wild-type mice, in which most of the XOR remained membrane-bound. Thus BTN1A1 functions in vivo to stabilize the association of XOR with the MFGM by direct interactions through the PRY/SPRY/B30.2 domain. The potential significance of BTN1A1/XOR interactions in the mammary gland and other tissues is discussed.

Enzyme-catalyzed 18O2-labeling offers a universal strategy for uniform labeling of all peptides from any kind of proteins, including post-translationally modified proteins. It is applicable to clinical samples with unrivaled sensitivity. This review discusses strengths and limitations, and advocates the separation of proteolysis from the labeling step. Continued advances in software will facilitate widespread use of enzyme-catalyzed 18O2-labeling to determine changes in protein abundances.

This laboratory has introduced a chemical method for residue-specific protein cleavage and has provided a preliminary assessment of the suitability of microwave-accelerated acid cleavage as a proteomic tool. This report is a continuing assessment of the fate of common protein modifications in microwave-accelerated acid cleavage. We have examined the cleavage of ribonuclease A and the related N-linked glycoprotein ribonuclease B, and the O-linked glycoprotein alpha crystallin A chain, using MALDI-TOF and LC–ESI-MS to identify the peptide products. RNase A and B each contains four disulfide bonds, and the addition of a reducing reagent, such as dithiothreitol, was found to be required to achieve efficient acidic proteolysis. The linkage of the glycosidic group to the asparagine side chain in ribonuclease B was found not to be cleaved by brief microwave treatment in 12.5% acetic acid. The distribution of the heterogeneous carbohydrate side chain in the glycopeptide products of acid cleavage was compared to that of the glycopeptide products of tryptic digestion. Hydrolysis within the carbohydrate chain itself is minimal under the conditions used. The O-linked side chain on alpha crystalline A was found to be cleaved during acid cleavage of the protein.

Microwave-accelerated proteolysis using acetic acid has been shown to occur specifically on either or both sides of aspartic acid residues. This chemical cleavage has been applied to ovalbumin and several model peptides to test the effect on some of the more common post-translational modifications. No oxidation of methionine or cysteine was observed; however, hydrolysis of phosphate groups proceeds at a detectable rate. Acid cleavage was also extended to the yeast ribosome model proteome, where it provided information on 74% of that proteome. Aspartic acid occurs across the proteome with approximately half the frequency of the combined occurrence of the trypsin residues lysine and arginine, and implications of this are considered.

Forward and reverse 18O labeling are integrated with solution isoelectric focusing and capillary LC-tandem mass spectrometry to evaluate a new strategy for quantitative proteomics, and to study abundance changes in mitochondrial proteins associated with drug resistance in MCF-7 human cancer cells. Galectin-3 binding protein, which is involved in apoptosis, was detected only in the resistant cell line, as a result of reverse labeling. Among 278 proteins identified, twelve were detected with abundances altered at least 2-fold.

Background Extracellular vesicles (EV) are spherical membrane-bound vesicles with nano-scale diameters, which are shed to the extracellular region by most eukaryotic and prokaryotic cells. Bacterial EV are proposed to contribute to intercellular communication, bacterial survival and human pathogenesis as a novel secretion system. EV have been characterized from many Gram-negative species and, more recently, from several vegetative Gram-positive bacteria. Further characterization of EV and their molecular cargos will contribute to understanding bacterial physiology and to developing therapeutic approaches. Results Bacillus subtilis were observed to release EV to a similar extent during sporulation as during the vegetative growth phase. However, the two vesicular cargos show qualitatively and quantitatively different proteomes. Among 193 total proteins identified across both samples, 61 were shown to be significantly more abundant in EV shed by sporulating cells, with (log) ratio of spectral counts RSC > 1 and Fisher-exact test FDR

Unexpected tryptic cleavage has been characterized at modified K48 residues in polyubiquitins. In particular, the tryptic products of all seven of the lysine-linked dimers of ubiquitin and of three trimers-linear Ub-(48)Ub-(48)Ub, linear Ub-(63)Ub-(63)Ub, and the branched trimer [Ub]2-(6,48)Ub-have been analyzed. In addition to the peptide products expected under commonly used tryptic conditions, we observe that peptides are formed with an unexpected ε-glycinylglycinyl-Lys carboxyl terminus when the site of linkage is Lys48. Trypsin from three different commercial sources exhibited this aberration. Initial cleavage at R74 is proposed in a distal ubiquitin to produce a glycinylglycinyl-lysine residue which is bound by trypsin.

The ESX-5 secretion system of Mycobacterium tuberculosis is important for bacterial virulence and for the secretion of the large PE/PPE protein family, whose genes constitute 10% of the M. tuberculosis genome. A four-gene region of the ESX-5 system is duplicated three times in the M. tuberculosis genome, but the functions of these duplicates are unknown. Here we investigated one of these duplicates: the region carrying the esxI , esxJ , ppe15 , and pe8 genes (ESX-5a). An ESX-5a deletion mutant in the model system M. marinum background was deficient in the secretion of some members of the PE/PPE family of proteins. Surprisingly, we also identified other proteins that are not members of this family, thus expanding the range of ESX-5 secretion substrates. In addition, we demonstrated that ESX-5a is important for the virulence of M. marinum in the zebrafish model. Furthermore, we showed the role of the M. tuberculosis ESX-5a region in inflammasome activation but not host cell death induction, which is different from the case for the M. tuberculosis ESX-5 system. In conclusion, the ESX-5a region is nonredundant with its ESX-5 paralog and is necessary for secretion of a specific subset of proteins in M. tuberculosis and M. marinum that are important for bacterial virulence of M. marinum . Our findings point to a role for the three ESX-5 duplicate regions in the selection of substrates for secretion via ESX-5, and hence, they provide the basis for a refined model of the molecular mechanism of this type VII secretion system.