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Background: The anatomical spatial distribution of microencapsulated islets transplanted into the peritoneal cavity of large animals remains a relatively unexplored area of study. In this study, we developed a new implantation approach using laparoscopy in order to avoid microcapsule amalgamation. This approach constitutes a clinically relevant method, which can be used to evaluate the distribution and in vivo biocompatibility of various types of transplanted microcapsules in the future. Materials and Methods: Two healthy baboons were implanted intraperitoneally with microencapsulated islets through mini-laparotomy and observed at 76 d after implantation. Nine baboons underwent laparoscopic implantation of approximately 80,000 empty microcapsules. Microcapsule distribution was observed by laparoscopic camera during and after implantation at 1, 2, and 4 wk. At each time point, microcapsules were retrieved and evaluated with brightfield microscopy and histologic analysis. Results: Mini-laparotomic implantation resulted in microcapusle aggregation in both baboons. In contrast, laparoscopic implantation resulted in even distribution of microcapsules throughout the peritoneum without sedimentation to the Douglas space in all animals. In eight out of nine animals, retrieved microcapsules were evenly distributed in the peritoneal cavity and presented with no pericapsular overgrowth and easily washed out during laparoscopic procedure. The one exception was attributed to microcapsule contamination with blood from the abdominal wall following trocar insertion. Conclusions: Laparoscopic implantation of microcapsules in non-human primates can be successfully performed and prevents microcapsule aggregation. Given the current widespread clinical application of laparoscopy, we propose that this presented laparoscopy technique could be applied in future clinical trials of microencapsulated islet transplantation. [ABSTRACT FROM AUTHOR]

Defining the subcellular distribution of all human proteins and their remodeling across cellular states remains a central goal in cell biology. Here, we present a high-resolution strategy to map subcellular organization using organelle immunocapture coupled to mass spectrometry. We apply this workflow to a cell-wide collection of membranous and membraneless compartments. A graph-based analysis assigns the subcellular localization of over 7,600 proteins, defines spatial networks, and uncovers interconnections between cellular compartments. Our approach can be deployed to comprehensively profile proteome remodeling during cellular perturbation. By characterizing the cellular landscape following HCoV-OC43 viral infection, we discover that many proteins are regulated by changes in their spatial distribution rather than by changes in abundance. Our results establish that proteome-wide analysis of subcellular remodeling provides key insights for elucidating cellular responses, uncovering an essential role for ferroptosis in OC43 infection. Our dataset can be explored at organelles.czbiohub.org., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell size is tightly controlled in healthy tissues and single-celled organisms, but it remains unclear how cell size influences physiology. Increasing cell size was recently shown to remodel the proteomes of cultured human cells, demonstrating that large and small cells of the same type can be compositionally different. In the present study, we utilize the natural heterogeneity of hepatocyte ploidy and yeast genetics to establish that the ploidy-to-cell size ratio is a highly conserved determinant of proteome composition. In both mammalian and yeast cells, genome dilution by cell growth elicits a starvation-like phenotype, suggesting that growth in large cells is restricted by genome concentration in a manner that mimics a limiting nutrient. Moreover, genome dilution explains some proteomic changes ascribed to yeast aging. Overall, our data indicate that genome concentration drives changes in cell composition independently of external environmental cues., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Consistently collecting high-quality liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) data is a time-consuming hurdle for untargeted workflows. Analytical controls such as internal and biological standards are commonly included in high-throughput workflows, helping researchers recognize low-integrity specimens regardless of their biological source. However, evaluating these standards as data are collected has remained a considerable bottleneck─in both person-hours and accuracy. Here we present Rapid QC-MS, an automated, interactive dashboard for assessing LC-MS/MS data quality. Minutes after a new data file is written, a browser-viewable dashboard is updated with quality control results spanning multiple performance dimensions such as instrument sensitivity, in-run retention time shifts, and mass accuracy drift. Rapid QC-MS provides interactive visualizations that help users recognize acute deviations in these performance metrics, as well as gradual drifts over periods of hours, days, months, or years. Rapid QC-MS is open-source, simple to install, and highly configurable. By integrating open-source Python libraries and widely used MS analysis software, it can adapt to any LC-MS/MS workflow. Rapid QC-MS runs locally and offers optional remote quality control by syncing with Google Drive. Furthermore, Rapid QC-MS can operate in a semiautonomous fashion, alerting users to specimens with potentially poor analytical integrity via frequently used messaging applications. Initially developed for integration with Thermo Orbitrap workflows, Rapid QC-MS offers a fast, straightforward approach to help users collect high-quality untargeted LC-MS/MS data by eliminating many of the most time-consuming steps in manual data curation. Download for free: https://github.com/czbiohub-sf/Rapid-QC-MS.

Malaria transmission begins when infected female Anopheles mosquitos deposit Plasmodium parasites into the mammalian host's skin during a bloodmeal. The salivary gland-resident sporozoite parasites migrate to the bloodstream, subsequently invading and replicating within hepatocytes. As Anopheles mosquitos are more active at night, with a 24-hour rhythm, we investigated whether their salivary glands are under circadian control, anticipating bloodmeals and modulating sporozoite biology for host encounters. Here we show that approximately half of the mosquito salivary gland transcriptome, particularly genes essential for efficient bloodmeals such as anti-blood clotting factors, exhibits circadian rhythmic expression. Furthermore, we demonstrate that mosquitoes prefer to feed during nighttime, with the amount of blood ingested varying cyclically throughout the day. Notably, we show a substantial subset of the sporozoite transcriptome cycling throughout the day. These include genes involved in parasite motility, potentially modulating the ability to initiate infection at different times of day. Thus, although sporozoites are typically considered quiescent, our results demonstrate their transcriptional activity, revealing robust daily rhythms of gene expression. Our findings suggest a circadian evolutionary relationship between the vector, parasite and mammalian host that together modulate malaria transmission.

Accurate measurements of the molecular composition of single cells will be necessary for understanding the relationship between gene expression and function in diverse cell types. One of the most important phenotypes that differs between cells is their size, which was recently shown to be an important determinant of proteome composition in populations of similarly sized cells. We, therefore, sought to test if the effects of the cell size on protein concentrations were also evident in single-cell proteomics data. Using the relative concentrations of a set of reference proteins to estimate a cell's DNA-to-cell volume ratio, we found that differences in the cell size explain a significant amount of cell-to-cell variance in two published single-cell proteome data sets.

Systemic juvenile idiopathic arthritis is a chronic pediatric inflammatory disease of unknown etiology, characterized by fever, rash, hepatosplenomegaly, serositis, and arthritis. We hypothesized that intercellular communication, mediated by extracellular vesicles, contributes to systemic juvenile idiopathic arthritis pathogenesis and that the number and cellular sources of extracellular vesicles would differ between inactive and active states of systemic juvenile idiopathic arthritis and healthy controls. We evaluated plasma from healthy pediatric controls and patients with systemic juvenile idiopathic arthritis with active systemic flare or inactive disease. We isolated extracellular vesicles by size exclusion chromatography and determined total extracellular vesicle abundance and size distribution using microfluidic resistive pulse sensing. Cell-specific extracellular vesicle subpopulations were measured by nanoscale flow cytometry. Isolated extracellular vesicles were validated using a variety of ways, including nanotracking and cryo-electron microscopy. Extracellular vesicle protein content was analyzed in pooled samples using mass spectrometry. Total extracellular vesicle concentration did not significantly differ between controls and patients with systemic juvenile idiopathic arthritis. Extracellular vesicles with diameters <200 nm were the most abundant, including the majority of cell-specific extracellular vesicle subpopulations. Patients with systemic juvenile idiopathic arthritis had significantly higher levels of extracellular vesicles from activated platelets, intermediate monocytes, and chronically activated endothelial cells, with the latter significantly more elevated in active systemic juvenile idiopathic arthritis relative to inactive disease and controls. Protein analysis of isolated extracellular vesicles from active patients showed a proinflammatory profile, uniquely expressing heat shock protein 47, a stress-inducible protein. Our findings indicate that multiple cell types contribute to altered extracellular vesicle profiles in systemic juvenile idiopathic arthritis. The extracellular vesicle differences between systemic juvenile idiopathic arthritis disease states and healthy controls implicate extracellular vesicle-mediated cellular crosstalk as a potential driver of systemic juvenile idiopathic arthritis disease activity., Competing Interests: Conflict of interest. Elizabeth Mellins has received grant support from Novartis. The funding sponsor had no role in the design, execution, interpretation, or writing of the study., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The high diversity and complexity of the microbial community make it a formidable challenge to identify and quantify the large number of proteins expressed in the community. Conventional metaproteomics approaches largely rely on accurate identification of the MS/MS spectra to their corresponding short peptides in the digested samples, followed by protein inference and subsequent taxonomic and functional analysis of the detected proteins. These approaches are dependent on the availability of protein sequence databases derived either from sample-specific metagenomic data or from public repositories. Due to the incompleteness and imperfections of these protein sequence databases, and the preponderance of homologous proteins expressed by different bacterial species in the community, this computational process of peptide identification and protein inference is challenging and error-prone, which hinders the comparison of metaproteomes across multiple samples., Results: We developed metaSpectraST, an unsupervised and database-independent metaproteomics workflow, which quantitatively profiles and compares metaproteomics samples by clustering experimentally observed MS/MS spectra based on their spectral similarity. We applied metaSpectraST to fecal samples collected from littermates of two different mother mice right after weaning. Quantitative proteome profiles of the microbial communities of different mice were obtained without any peptide-spectrum identification and used to evaluate the overall similarity between samples and highlight any differentiating markers. Compared to the conventional database-dependent metaproteomics analysis, metaSpectraST is more successful in classifying the samples and detecting the subtle microbiome changes of mouse gut microbiomes post-weaning. metaSpectraST could also be used as a tool to select the suitable biological replicates from samples with wide inter-individual variation., Conclusions: metaSpectraST enables rapid profiling of metaproteomic samples quantitatively, without the need for constructing the protein sequence database or identification of the MS/MS spectra. It maximally preserves information contained in the experimental MS/MS spectra by clustering all of them first and thus is able to better profile the complex microbial communities and highlight their functional changes, as compared with conventional approaches. tag the videobyte in this section as ESM4 Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Increasing cell size drives changes to the proteome, which affects cell physiology. As cell size increases, some proteins become more concentrated while others are diluted. As a result, the state of the cell changes continuously with increasing size. In addition to these proteomic changes, large cells have a lower growth rate (protein synthesis rate per unit volume). That both the cell's proteome and growth rate change with cell size suggests they may be interdependent. To test this, we used quantitative mass spectrometry to measure how the proteome changes in response to the mTOR inhibitor rapamycin, which decreases the cellular growth rate and has only a minimal effect on cell size. We found that large cell size and mTOR inhibition, both of which lower the growth rate of a cell, remodel the proteome in similar ways. This suggests that many of the effects of cell size are mediated by the size-dependent slowdown of the cellular growth rate. For example, the previously reported size-dependent expression of some senescence markers could reflect a cell's declining growth rate rather than its size per se . In contrast, histones and other chromatin components are diluted in large cells independently of the growth rate, likely so that they remain in proportion with the genome. Finally, size-dependent changes to the cell's growth rate and proteome composition are still apparent in cells continually exposed to a saturating dose of rapamycin, which indicates that cell size can affect the proteome independently of mTORC1 signaling. Taken together, our results clarify the dependencies between cell size, growth, mTOR activity, and the proteome remodeling that ultimately controls many aspects of cell physiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zatulovskiy, Lanz, Zhang, McCarthy, Elias and Skotheim.)

Celiac disease (CeD) is an autoimmune disorder induced by consuming gluten proteins from wheat, barley, and rye. Glutens resist gastrointestinal proteolysis, resulting in peptides that elicit inflammation in patients with CeD. Despite well-established connections between glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, impeding our knowledge of potentially diverse peptide species that encounter the immune system. Here, we develop a liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. We detect over 600 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and ~5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differ from controls. Our results provide a new foundation for understanding gluten immunogenicity, improving CeD management, and characterizing the dietary and urinary peptidomes., (© 2022. The Author(s).)

Physical exercise is generally beneficial to all aspects of human and animal health, slowing cognitive ageing and neurodegeneration 1 . The cognitive benefits of physical exercise are tied to an increased plasticity and reduced inflammation within the hippocampus 2-4 , yet little is known about the factors and mechanisms that mediate these effects. Here we show that 'runner plasma', collected from voluntarily running mice and infused into sedentary mice, reduces baseline neuroinflammatory gene expression and experimentally induced brain inflammation. Plasma proteomic analysis revealed a concerted increase in complement cascade inhibitors including clusterin (CLU). Intravenously injected CLU binds to brain endothelial cells and reduces neuroinflammatory gene expression in a mouse model of acute brain inflammation and a mouse model of Alzheimer's disease. Patients with cognitive impairment who participated in structured exercise for 6 months had higher plasma levels of CLU. These findings demonstrate the existence of anti-inflammatory exercise factors that are transferrable, target the cerebrovasculature and benefit the brain, and are present in humans who engage in exercise., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.

The steady-state size of bacterial cells correlates with nutrient-determined growth rate. Here, we explore how rod-shaped bacterial cells regulate their morphology during rapid environmental changes. We quantify cellular dimensions throughout passage cycles of stationary-phase cells diluted into fresh medium and grown back to saturation. We find that cells exhibit characteristic dynamics in surface area to volume ratio (SA/V), which are conserved across genetic and chemical perturbations as well as across species and growth temperatures. A mathematical model with a single fitting parameter (the time delay between surface and volume synthesis) is quantitatively consistent with our SA/V experimental observations. The model supports that this time delay is due to differential expression of volume and surface-related genes, and that the first division after dilution occurs at a tightly controlled SA/V. Our minimal model thus provides insight into the connections between bacterial growth rate and cell shape in dynamic environments.

The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability 1-3 . Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins 4,5 . Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.

Objectives: Previously, we showed that diazoxide (DZ), an effective ischemic preconditioning agent, protected rodent pancreas against ischemia-reperfusion injury. Here, we further investigate whether DZ supplementation to University of Wisconsin (UW) solution during pancreas procurement and islet isolation has similar cytoprotection in a preclinical nonhuman primate model., Methods: Cynomolgus monkey pancreata were flushed with UW or UW + 150 μM DZ during procurement and preserved for 8 hours before islet isolation., Results: First, a significantly higher islet yield was observed in UW + DZ than in UW (57,887 vs 23,574 IEq/pancreas and 5396 vs 1646 IEq/g). Second, the DZ treated islets had significantly lower apoptotic cells per islet (1.64% vs 9.85%). Third, DZ significantly inhibited ROS surge during reperfusion with a dose-response manner. Fourth, DZ improved in vitro function of isolated islets determined by mitochondrial potentials and calcium influx in responses to glucose and KCI. Fifth, the DZ treated islets had much higher cure rate and better glycemia control in diabetic mice transplant model., Conclusions: This study showed a strong mitochondrial protection of DZ on nonhuman primate islets against ischemia-reperfusion injury that provides strong evidence for its clinical application in islet and pancreas transplantation.

Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.

For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur 1 . Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.

Common variable immunodeficiency (CVID) is the most common form of primary immunodeficiency characterized by antibody deficiency, recurrent bacterial infections, and autoimmunity. Advanced chronic liver disease occurs in a subset of patients with CVID and manifests with various histological features, such as nodular regenerative hyperplasia, inflammation, fibrosis, and cholangiopathy. We present a case series characterizing the outcomes in adult patients transplanted for primary CVID-related liver disease. We discuss the unique transplantation challenges faced in this primary immunodeficiency group including susceptibility to infections and early disease recurrence. There is a statistically significant decrease in 3-year and 5-year survival after liver transplantation in those with CVID-related liver disease (55% at 3 and 5 years) compared with all-comers (89% at 3 years, 81% at 5 years), prompting a need for discussion of suitability of transplantation in this group of patients as well as methods for reducing posttransplantation risk such as scrupulous search for infectious agents and reduction of immunosuppression. Liver Transplantation 24 171-181 2018 AASLD., (© 2017 by the American Association for the Study of Liver Diseases.)

Plasmodium parasites contain several unique membrane compartments in which prenylated proteins may play important roles in pathogenesis. Protein prenylation has also been proposed as an antimalarial drug target because farnesyltransferase inhibitors cause potent growth inhibition of blood-stage Plasmodium However, the specific prenylated proteins that mediate antimalarial activity have yet to be identified. Given the potential for new parasite biology and elucidating drug mechanism-of-action, we performed a large-scale identification of the prenylated proteome in blood-stage P. falciparum parasites using an alkyne-labeled prenyl analog to specifically enrich parasite prenylated proteins. Twenty high-confidence candidates were identified, including several examples of pathogen-specific prenylation activity. One unique parasite prenylated protein was FYVE-containing coiled-coil protein (FCP), which is only conserved in Plasmodium and related Apicomplexan parasites and localizes to the parasite food vacuole. Targeting of FCP to this parasite-specific compartment was dependent on prenylation of its CaaX motif, as mutation of the prenylation site caused cytosolic mislocalization. We also showed that Pf Rab5b, which lacks C -terminal cysteines that are the only known site of Rab GTPase modification, is prenylated. Finally, we show that the THQ class of farnesyltransferase inhibitors abolishes FCP prenylation and causes its mislocalization, providing the first demonstration of a specific prenylated protein disrupted by antimalarial farnesyl transferase inhibitors. Altogether, these findings identify prenylated proteins that reveal unique parasite biology and are useful for evaluating prenyltransferase inhibitors for antimalarial drug development., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4 + T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.

Measuring protein changes over time or following stimuli is one of the important tasks of proteomics. In the past decade, several strategies have been developed for the relative quantification of proteins using mass spectrometry (MS). Isobaric labeling strategies for relative quantitative proteomics allow for parallel multiplexing of quantitative experiments. With this technique, multiple peptide samples are chemically labeled with isobaric chemical tag variants and each variant has the same molecular structure and mass. Each variant, however, is designed to produce a unique "reporter ion" when fragmented inside a mass spectrometer. Once peptide samples are labeled, combined, and analyzed using MS, differentially labeled peptides are indistinguishable in a first, MS spectrum of intact peptides. However, since each tag variant contains a labile component with different mass, "reporter ions" can be generated and recorded in a subsequent MS 2 spectrum. Intensities from each variant are recorded to represent the relative abundances of the peptide in each sample. Isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT) are commercially available reagents for performing this technique. Here, we describe the general workflow of relative quantification of proteins using TMT by MS 2 , or an additional MS 3 spectrum.

Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.

We present a novel pumpless microfluidic array driven by surface tension for studying the physiology of pancreatic islets of Langerhans. Efficient fluid flow in the array is achieved by surface tension-generated pressure as a result of inlet and outlet size differences. Flow properties are characterized in numerical simulation and further confirmed by experimental measurements. Using this device, we perform a set of biological assays, which include real-time fluorescent imaging and insulin secretion kinetics for both mouse and human islets. Our results demonstrate that this system not only drastically simplifies previously published experimental protocols for islet study by eliminating the need for external pumps/tubing and reducing the volume of solution consumption, but it also achieves a higher analytical spatiotemporal resolution due to efficient flow exchanges and the extremely small volume of solutions required. Overall, the microfluidic platform presented can be used as a potential powerful tool for understanding islet physiology, antidiabetic drug development, and islet transplantation.

The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.

Improved understanding of the interplay between host and microbes stands to illuminate new avenues for disease diagnosis, treatment, and prevention. Here, we provide a high-resolution view of the dynamics between host and gut microbiota during antibiotic-induced intestinal microbiota depletion, opportunistic Salmonella typhimurium and Clostridium difficile pathogenesis, and recovery from these perturbed states in a mouse model. Host-centric proteome and microbial community profiles provide a nuanced longitudinal view, revealing the interdependence between host and microbiota in evolving dysbioses. Time- and condition-specific molecular and microbial signatures are evident and clearly distinguished from pathogen-independent inflammatory fingerprints. Our data reveal that mice recovering from antibiotic treatment or C. difficile infection retain lingering signatures of inflammation, despite compositional normalization of the microbiota, and host responses could be rapidly and durably relieved through fecal transplant. These experiments demonstrate insights that emerge from the combination of these orthogonal, untargeted approaches to the gastrointestinal ecosystem., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Genomic technologies have significantly advanced our understanding of the composition and diversity of host-associated microbial populations. However, their spatial organization and functional interactions relative to the host have been more challenging to study. Here we present a pipeline for the assessment of intestinal microbiota localization within immunofluorescence images of fixed gut cross-sections that includes a flexible software package, BacSpace, for high-throughput quantification of microbial organization. Applying this pipeline to gnotobiotic and human microbiota-colonized mice, we demonstrate that elimination of microbiota-accessible carbohydrates (MACs) from the diet results in thinner mucus in the distal colon, increased proximity of microbes to the epithelium, and heightened expression of the inflammatory marker REG3β. Measurements of microbe-microbe proximity reveal that a MAC-deficient diet alters monophyletic spatial clustering. Furthermore, we quantify the invasion of Helicobacter pylori into the glands of the mouse stomach relative to host mitotic progenitor cells, illustrating the generalizability of this approach., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Chronic microangiopathy of portal venules results in idiopathic non-cirrhotic intrahepatic portal hypertension (NCIPH). Recent data suggest a role for vasoactive factors of portal venous origin in the pathogenesis of this 'pure' vasculopathy of the liver. Enteropathies (often silent), are an important 'driver' of this disease. NCIPH is under-recognized and often mis-labeled as cryptogenic cirrhosis. Liver biopsy is needed to prove the diagnosis of NCIPH. In these patients, with advancing disease and increased porto-systemic shunting, the portal venous vasoactive factors bypass the liver filter and contribute to the development of pulmonary vascular endothelial disorders-porto-pulmonary hypertension and hepato-pulmonary syndrome as well as mesangiocapillary glomerulonephritis. Prognosis in NCIPH patients is determined by presence, recognition and management of associated disorders. With better understanding of the pathogenesis of NCIPH, newer treatment options are being explored. Imbalance in ADAMTS 13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13): vWF (von-Willebrand factor) ratio is documented in NCIPH patients and may have a pathogenic role. Therapeutic interventions to correct this imbalance may prove to be important in the management of NCIPH.

Plasmodium falciparum and Toxoplasma gondii are obligate intracellular apicomplexan parasites that rapidly invade and extensively modify host cells. Protein phosphorylation is one mechanism by which these parasites can control such processes. Here we present a phosphoproteome analysis of peptides enriched from schizont stage P. falciparum and T. gondii tachyzoites that are either "intracellular" or purified away from host material. Using liquid chromatography-tandem mass spectrometry, we identified over 5,000 and 10,000 previously unknown phosphorylation sites in P. falciparum and T. gondii, respectively, revealing that protein phosphorylation is an extensively used regulation mechanism both within and beyond parasite boundaries. Unexpectedly, both parasites have phosphorylated tyrosines, and P. falciparum has unusual phosphorylation motifs that are apparently shaped by its A:T-rich genome. This data set provides important information on the role of phosphorylation in the host-pathogen interaction and clues to the evolutionary forces operating on protein phosphorylation motifs in both parasites., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Proximal tubular cells are most vulnerable to ischemia reperfusion injury (IRI) in renal transplantation. Caspase-3 can be up-regulated by IRI due to a variety of pathogenic processes such as oxidative damage. This study utilized synthetic small interfering RNA (siRNA) to posttranscriptionally silence target gene, caspase-3, may represent a feasible approach to produce transient effects, but avoid side actions caused by viral vectors. The porcine proximal tubular cells (LLC-PK1), with or without the stimulation of hydrogen peroxide (H(2) O(2) , an oxidizer), were transfected with synthetic caspase-3 siRNA using a cationic lipid-based transfection regent. The expression of caspase-3 at mRNA and protein level was assessed at different times posttransfection and its downstream biological events were also monitored. The caspase-3 mRNA was posttranscriptionally silenced by its siRNA up to 50% after 24 h. The active caspase-3 protein was increased by transfection reagent alone and H(2) O(2) in a dose- and time-dependent manner. Both the precursor and active protein of caspase-3 were decreased by siRNA after 48 h and maintained up to 96 h at least, with a consistent change in its activity. Consequently, apoptotic cells and active IL-1β protein expression was reduced by caspase-3 siRNA; cell viability, especially with H(2) O(2) treatment, was also improved. Taken together, caspase-3 and apoptosis are sensitive markers for cellular injury; using synthetic siRNA silencing caspase-3 may provide not only a valid approach for underlying mechanisms of diseases, but also a potential therapeutic intervention for a wide range of acute clinical disorders including IRI in renal transplantation., (Copyright © 2011 Wiley-Liss, Inc.)

The continual destruction and renewal of proteins that maintain cellular homeostasis has been rigorously studied since the late 1930s. Experimental techniques for measuring protein turnover have evolved to measure the dynamic regulation of key proteins and now, entire proteomes. In the past decade, the proteomics field has aimed to discover how cells adjust their proteomes to execute numerous regulatory programs in response to specific cellular and environmental cues. By combining classical biochemical techniques with modern, high-throughput technologies, researchers have begun to reveal the synthesis and degradation mechanisms that shape protein turnover on a global scale. This review examines several recent developments in protein turnover research, emphasizing the combination of metabolic labeling and mass spectrometry., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Accurate and precise methods for estimating incorrect peptide and protein identifications are crucial for effective large-scale proteome analyses by tandem mass spectrometry. The target-decoy search strategy has emerged as a simple, effective tool for generating such estimations. This strategy is based on the premise that obvious, necessarily incorrect "decoy" sequences added to the search space will correspond with incorrect search results that might otherwise be deemed to be correct. With this knowledge, it is possible not only to estimate how many incorrect results are in a final data set but also to use decoy hits to guide the design of filtering criteria that sensitively partition a data set into correct and incorrect identifications.

Protein phosphorylation is essential for numerous cellular processes. Large-scale profiling of phosphoproteins continues to enhance the depth and speed at which we understand these processes. The development of effective phosphoprotein and peptide enrichment techniques and improvements to mass spectrometric instrumentation have intensified phosphoproteomic research in recent years, leading to unprecedented achievements. Here, we describe a large-scale phosphorylation analysis of alpha-factor-arrested yeast. Using a multidimensional separation strategy involving preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and immobilized metal affinity chromatography (IMAC) enrichment of phosphopeptides, followed by LC-MS/MS analysis employing a hybrid LTQ-Orbitrap mass spectrometer, we were able to catalog a substantial portion of the phosphoproteins present in yeast whole-cell lysate. This analysis yielded the confident identification of 2288 nonredundant phosphorylation sites from 985 proteins. The ambiguity score (Ascore) algorithm was utilized to determine the certainty of site localization for the entire data set. In addition, the size of the data set permitted extraction of known and novel kinase motifs using the Motif-X algorithm. Finally, a large number of members of the pheromone signaling pathway were found as phosphoproteins and are discussed.

Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 microm i.d.) approach for RP-LC-MS/MS analysis. More than 162,000 MS/MS spectra were collected with 26,815 matched to yeast peptides (7,537 unique peptides). A total of 1,504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.