MALDI-TOF Mass Spectrometric Detection of SARS-CoV-2 Using Cellulose Sulfate Ester Enrichment and Hot Acid Treatment.

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.

Molecular cargo in myeloid-derived suppressor cells and their exosomes.

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.

How many human proteoforms are there?

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.

Myeloid-Derived Suppressor Cells: Immune-Suppressive Cells That Impair Antitumor Immunity and Are Sculpted by Their Environment.

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.

Top-Down Proteomic Characterization of Truncated Proteoforms.

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.

Microwave supported hydrolysis prepares Bacillus spores for proteomic analysis.

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.

Ubiquitin Conjugation Probed by Inflammation in Myeloid-Derived Suppressor Cell Extracellular Vesicles.

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.

Differential Content of Proteins, mRNAs, and miRNAs Suggests that MDSC and Their Exosomes May Mediate Distinct Immune Suppressive Functions.

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.

Top-Down Analysis of Branched Proteins Using Mass Spectrometry.

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.

Surface Glycoproteins of Exosomes Shed by Myeloid-Derived Suppressor Cells Contribute to Function.

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.

Peptide-based systems analysis of inflammation induced myeloid-derived suppressor cells reveals diverse signaling pathways.

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.

Evaluation of Spectral Counting for Relative Quantitation of Proteoforms in Top-Down Proteomics.

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.

Extracellular vesicle proteomes reflect developmental phases of Bacillus subtilis.

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 < 5 %. Sixty-two proteins were found to be significantly more abundant in EV shed by vegetative cells. Membrane fusion was shown to take place between these EVs and Gram-positive cells. CONCLUSION: Biogenesis of EV is a continuous process over the entire life cycle of this sporulating bacterium. The formation of EV during sporulation is strongly supported by the delineation of protein content that differs from the proteome of EV formed by vegetative spores.

A Duplicated ESAT-6 Region of ESX-5 Is Involved in Protein Export and Virulence of Mycobacteria.

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.

Unexpected trypsin cleavage at ubiquitinated lysines.

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.

Top-down analysis of low mass proteins in exosomes shed by murine myeloid-derived suppressor cells.

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 %.

Exosomes from myeloid-derived suppressor cells carry biologically active proteins.

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 toward 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 toward 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.

Ubiquitinated proteins in exosomes secreted by myeloid-derived suppressor cells.

We provide evidence at the molecular level that ubiquitinated proteins are present in exosomes shed by myeloid-derived suppressor cells (MDSC). Ubiquitin was selected as a post-translational modification of interest because it is known to play a determinant role in the endosomal trafficking that culminates in exosome release. Enrichment was achieved by two immunoprecipitations, first at the protein level and subsequently at the peptide level. Fifty ubiquitinated proteins were identified by tandem mass spectrometry filtering at a 5% spectral false discovery rate and using the conservative requirement that glycinylglycine-modified lysine residues were observed in tryptic peptides. Thirty five of these proteins have not previously been reported to be ubiquitinated. The ubiquitinated cohort spans a range of protein sizes and favors basic pI values and hydrophobicity. Five proteins associated with endosomal trafficking were identified as ubiquitinated, along with pro-inflammatory high mobility group protein B1 and proinflammatory histones.

Enrichment of plasma membrane proteins using nanoparticle pellicles: comparison between silica and higher density nanoparticles.

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.

Comparative study of workflows optimized for in-gel, in-solution, and on-filter proteolysis in the analysis of plasma membrane proteins.

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.