Comprehensive Proteomic Analysis Research Articles

Characterization of four subtypes in morphologically normal tissue excised proximal and distal to breast cancer

Widespread mammographic screening programs and improved self-monitoring allow for breast cancer to be detected earlier than ever before. Breast-conserving surgery is a successful treatment for select women. However, up to 40% of women develop local recurrence after surgery despite apparently tumor-free margins. This suggests that morphologically normal breast may harbor early alterations that contribute to increased risk of cancer recurrence. We conducted a comprehensive transcriptomic and proteomic analysis to characterize 57 fresh-frozen tissues from breast cancers and matched histologically normal tissues resected proximal to (<2 cm) and distant from (5–10 cm) the primary tumor, using tissues from cosmetic reduction mammoplasties as baseline. Four distinct transcriptomic subtypes are identified within matched normal tissues: metabolic; immune; matrisome/epithelial–mesenchymal transition, and non-coding enriched. Key components of the subtypes are supported by proteomic and tissue composition analyses. We find that the metabolic subtype is associated with poor prognosis (p < 0.001, HR6.1). Examination of genes representing the metabolic signature identifies several genes able to prognosticate outcome from histologically normal tissues. A subset of these have been reported for their predictive ability in cancer but, to the best of our knowledge, these have not been reported altered in matched normal tissues. This study takes an important first step toward characterizing matched normal tissues resected at pre-defined margins from the primary tumor. Unlocking the predictive potential of unexcised tissue could prove key to driving the realization of personalized medicine for breast cancer patients, allowing for more biologically-driven analyses of tissue margins than morphology alone.

Extracellular Vesicles from Cancer-Associated Fibroblasts Containing Annexin A6 Induces FAK-YAP Activation by Stabilizing β1 Integrin, Enhancing Drug Resistance.

Extracellular vesicles (EV) from cancer-associated fibroblasts (CAF) are composed of diverse payloads. Although CAFs impact the aggressive characteristics of gastric cancer cells, the contribution of CAF-EV to gastric cancer progression has not been elucidated. Here, we investigated the molecular mechanism of the changes in gastric cancer characteristics induced by CAF-EV. CAF abundance in gastric cancer tissues was associated with poor prognosis of patients with gastric cancer receiving chemotherapy. Moreover, CAF-EV induced tubular network formation and drug resistance of gastric cancer cells in the extracellular matrix (ECM). Comprehensive proteomic analysis of CAF-EV identified that Annexin A6 plays a pivotal role in network formation and drug resistance of gastric cancer cells in the ECM via activation of β1 integrin-focal adhesion kinase (FAK)-YAP. A peritoneal metastasis mouse model revealed that CAF-EV induced drug resistance in peritoneal tumors, and inhibition of FAK or YAP efficiently attenuated gastric cancer drug resistance in vitro and in vivo. These findings demonstrate that drug resistance is conferred by Annexin A6 in CAF-EV and provide a potential avenue for overcoming gastric cancer drug resistance through the inhibition of FAK-YAP signaling in combination with conventional chemotherapeutics. SIGNIFICANCE: This study elucidates a novel molecular mechanism through which Annexin A6 in CAF-EV activates FAK-YAP by stabilizing β1 integrin at the cell surface of gastric cancer cells and subsequently induces drug resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/16/3222/F1.large.jpg.

Abstract 2856: An intelligent boosting to amplify signal with isobaric labeling (iBASIL) strategy for precise quantitative single-cell proteomics analysis

Abstract Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection (e.g., our recent boosting to amplify signal with isobaric labeling (BASIL) approach) has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have systematically evaluated and optimized the BASIL approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of single cells. This improved, intelligent BASIL (iBASIL) approach enables comprehensive and quantitative single-cell proteomics analysis by carefully controlling the boosting-to-sample ratio and optimizing MS automatic gain control and ion injection time settings. Using standard LC-MS platforms, iBASIL enabled precise quantification of 800-1,200 proteins from single FACS-isolated cells (depending on the cell type). Moreover, coupling iBASIL to nanoscale fractionation enabled identification of &amp;gt;3,000 proteins and reliable quantification of &amp;gt;2,000 proteins while readily separating single cells from 3 different acute myeloid leukemia cell lines. We believe iBASIL has broad utility for comprehensive and precise quantitative single-cell proteomics for systems biology and biomedical research, as well as for comprehensive proteomic analysis of size-limited clinical specimens that cannot be readily accessed by regular proteomics platforms. It also has the potential to be adapted for phosphoproteome analysis of as few as 100 cells. Citation Format: Chia-Feng Tsai, Rui Zhao, Ronald Moore, Sarah Williams, Kendall Schultz, Paul Piehowski, Ljiljana Pasa-Tolic, Karin Rodland, Richard Smith, Tujin Shi, Ying Zhu, Ying Zhu, Tao Liu. An intelligent boosting to amplify signal with isobaric labeling (iBASIL) strategy for precise quantitative single-cell proteomics analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2856.

Population-scale proteome variation in human induced pluripotent stem cells.

Human disease phenotypes are driven primarily by alterations in protein expression and/or function. To date, relatively little is known about the variability of the human proteome in populations and how this relates to variability in mRNA expression and to disease loci. Here, we present the first comprehensive proteomic analysis of human induced pluripotent stem cells (iPSC), a key cell type for disease modelling, analysing 202 iPSC lines derived from 151 donors, with integrated transcriptome and genomic sequence data from the same lines. We characterised the major genetic and non-genetic determinants of proteome variation across iPSC lines and assessed key regulatory mechanisms affecting variation in protein abundance. We identified 654 protein quantitative trait loci (pQTLs) in iPSCs, including disease-linked variants in protein-coding sequences and variants with trans regulatory effects. These include pQTL linked to GWAS variants that cannot be detected at the mRNA level, highlighting the utility of dissecting pQTL at peptide level resolution.

Comprehensive Proteomic Analysis of Lysine Ubiquitination in Seedling Leaves of Nicotiana tabacum.

Lysine ubiquitination, a widely studied posttranslational modification, plays vital roles in various biological processes in eukaryotic cells. Although several studies have examined the plant ubiquitylome, no such research has been performed in tobacco, a model plant for molecular biology. Here, we comprehensively analyzed lysine ubiquitination in tobacco (Nicotiana tabacum) using LC–MS/MS along with highly sensitive immune-affinity purification. In total, 964 lysine-ubiquitinated (Kub) sites were identified in 572 proteins. Extensive bioinformatics studies revealed the distribution of these proteins in various cellular locations, including the cytoplasm, chloroplast, nucleus, and plasma membrane. Notably, 25% of the Kub proteins were located in the chloroplast of which 21 were enzymatically involved in important pathways, that is, photosynthesis and carbon fixation. Western blot analysis indicated that TMV infection can cause changes in ubiquitination levels. This is the first comprehensive proteomic analysis of lysine ubiquitination in tobacco, illustrating the vital role of ubiquitination in various physiological and biochemical processes and representing a valuable addition to the existing landscape of lysine ubiquitination.

Comprehensive transcriptomic and proteomic analyses of antroquinonol biosynthetic genes and enzymes in Antrodia camphorata

Antroquinonol (AQ) has several remarkable bioactivities in acute myeloid leukaemia and pancreatic cancer, but difficulties in the mass production of AQ hamper its applications. Currently, molecular biotechnology methods, such as gene overexpression, have been widely used to increase the production of metabolites. However, AQ biosynthetic genes and enzymes are poorly understood. In this study, an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) were used to identify AQ synthesis-related genes and enzymes in Antrodia camphorata during coenzyme Q0-induced fermentation (FM). The upregulated genes related to acetyl-CoA synthesis indicated that acetyl-CoA enters the mevalonate pathway to form the farnesyl tail precursor of AQ. The metE gene for an enzyme with methyl transfer activity provided sufficient methyl groups for AQ structure formation. The CoQ2 and ubiA genes encode p-hydroxybenzoate polyprenyl transferase, linking coenzyme Q0 and the polyisoprene side chain to form coenzyme Q3. NADH is transformed into NAD+ and releases two electrons, which may be beneficial for the conversion of coenzyme Q3 to AQ. Understanding the biosynthetic genes and enzymes of AQ is important for improving its production by genetic means in the future.

Comprehensive Transcriptome and Proteome Analyses Reveal the Modulation of Aflatoxin Production by Aspergillus flavus on Different Crop Substrates.

As a natural severe contaminant of stored grains and other crops worldwide, Aspergillus flavus can produce aflatoxins (AFs), the most powerful naturally producing toxic and hepatocarcinogenic compounds. AFs production is regulated by diverse factors including AFs cluster genes, transcription factors, regulators, and environmental factors. Among them, crop substrate is one of the most important factors. Here, we found that AFB1 production was significantly higher in maize and rice broth than in peanut broth. To clarify the mechanisms involved, complementary transcriptomic and proteomic analyses were performed to identify changes in A. flavus incubated in the three crop substrates. The results indicated that fewer genes and proteins were differentially expressed between maize and rice substrates, whereas more differentially expressed genes were observed between maize/rice broth and peanut broth. In particular, the genes involved in the initial step of AFs biosynthesis (aflA, aflB, and aflC) and the ACCase-encoding gene accA were significantly upregulated on the maize and rice substrates. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses indicated that carbon-metabolism-related genes were obviously enriched in the maize broth, and the genes involved in acetyl-CoA accumulation and consumption were up- and downregulated, respectively. Several genes involved in the regulation of AFs biosynthesis, including veA, ppoB, snf1, and the G-protein-coupled receptor (GPCR) genes, were differentially expressed on the three substrates, suggesting that these genes may be also involved in sugar signal sensing, transfer, and regulation. Interestingly, by the correlation analyses of transcriptome and proteome, trehalose metabolism genes, aldehyde dehydrogenase gene, and tryptophan synthase gene were found to be relevant with the regulation of AFs production on different crop substrates. Taken together, the differential expressions of the AFs cluster genes, several regulatory genes, and carbon metabolism genes were involved in the comprehensive modulation of AFs production on different crop substrates.

LC-MS/MS-based quantitative proteomic and phosphoproteomic analysis of CHO-K1 cells adapted to growth in glutamine-free media.

This study aims to provide insights into the molecular mechanisms underlying adaptation of CHO-K1 cells to growth in glutamine-free media and potentially identifying critical signalling proteins and pathways involved in this phenotype. A CHO-K1 cell line adapted to growth in glutamine-free media was established using a straightforward one-step glutamine reduction strategy. The adapted cell line had a comparable phenotype to the parental cells in terms of cell growth and viability. Global quantitative proteomic and phosphoproteomic analysis was carried out to compare the cells adapted to growth in glutamine-free media to parental cells grown in media containing 8 mM L-glutamine. The adaptation process was accompanied by changes in proteins associated with cytoskeleton rearrangement and mRNA splicing as evidenced via functional analysis of 194 differentially expressed proteins between the two cell lines. 434 phosphoproteins with altered abundance were also identified as a result of adaptation to L-glutamine-free conditions with an associated enrichment of pathways associated with MAPK and calcium signalling. This work provides a comprehensive proteomic and phosphoproteomic analysis of protein expression changes after adaptation to glutamine-free growth conditions highlighting critical pathways to consider in the rational design of improved feeding strategies or in cell line engineering to improve bioprocess phenotypes.

Proteomic analysis of extracellular vesicles released from heat-stroked hepatocytes reveals promotion of programmed cell death pathway

Liver injury is a common complication of severe heat stroke (HS). Extracellular vesicles (EVs) are part of a novel pathway mediating intercellular communication. Whether EVs are involved in the pathogenesis underlying HS-induced liver injury remains unknown. Here, we explored the role of hepatocyte EVs in HS-induced liver injury and their protein regulation patterns after HS induction. Isobaric tags for relative and absolute quantification technology (iTRAQ) and liquid chromatography-tandem mass spectrometry analysis identified changes in the proteomic profiles of hepatocyte-derived heat-stroked EVs, and we identified 53 up-regulated proteins. Bioinformatics analysis verified that the regulation of programmed cell death was the most significant altered pathway. To clarify the effect of HS hepatocyte-derived EVs in inducing hepatocyte-programmed death and injury, they were added to recipient hepatocytes and injected into mice. This treatment significantly induced the synthesis of apoptosis (caspase-3/8) and necroptosis-associated proteins [receptor-interacting protein 1 (RIP1), RIP3, and mixed lineage kinase domain-like protein]; moreover, it increased the numbers of apoptotic and necroptotic cells in hepatocytes and liver tissues and increased the levels of biochemical liver injury markers (alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase). Our study is the first comprehensive analysis of the hepatocyte-derived heat-stroked EV proteome confirming the induction of liver injury by Evs. We provide a novel explanation for the pathological mechanism underlying HS-induced liver injury.

Proteome analysis revealed the essential functions of protein phosphatase PP2A in the induction of Th9 cells

Proteomic analysis identifies post-translational functions of proteins, which remains obscure in transcriptomics. Given the important functions of Th9 cells in anti-tumor immunity, we performed proteome analysis of Th9 cells to understand the involvement of proteins that might be crucial for the anti-tumor functions of Th9 cells. Here we performed a comprehensive proteomic analysis of murine Th0 and Th9 cells, and identified proteins that are enriched in Th9 cells. Pathway analysis identified an abundance of phosphoproteins in the proteome of Th9 cells as compared to Th0 cells. Among upregulated phosphoproteins, Ppp2ca (catalytic subunit of protein phosphatase, PP2A) was found to be highly enriched in Th9 cells. Although the role of PP2A has been shown to regulate the differentiation and functions of Th1, Th2, Th17 and Tregs, its role in the differentiation and functions of Th9 cells is not identified yet. Here we found that PP2A is required for the induction of Th9 cells, as PP2A inhibition leads to the suppression of IL-9 and expression of key transcription factors of Th9 cells. PP2A inhibition abrogates Th9 cell-mediated anti-tumor immune response in B16-OVA melanoma tumor model. Thus, we report that PP2A is essential for the differentiation and anti-tumor functions of Th9 cells.

Integrative Proteomic Characterization of Human Lung Adenocarcinoma

Genomic studies of lung adenocarcinoma (LUAD) have advanced our understanding of the disease's biology and accelerated targeted therapy. However, the proteomic characteristics of LUAD remain poorly understood. We carried out a comprehensive proteomics analysis of 103 cases of LUAD in Chinese patients. Integrative analysis of proteome, phosphoproteome, transcriptome, and whole-exome sequencing data revealed cancer-associated characteristics, such as tumor-associated protein variants, distinct proteomics features, and clinical outcomes in patients at an early stage or with EGFR and TP53 mutations. Proteome-based stratification of LUAD revealed three subtypes (S-I, S-II, and S-III) related to different clinical and molecular features. Further, we nominated potential drug targets and validated the plasma protein level of HSP 90β as a potential prognostic biomarker for LUAD in an independent cohort. Our integrative proteomics analysis enables a more comprehensive understanding of the molecular landscape of LUAD and offers an opportunity for more precise diagnosis and treatment.

Scoping review of chronic rhinosinusitis proteomics.

Progressive advances in proteomic technology has improved our understanding of the chronic rhinosinusitis (CRS) pathogenesis and endotypes. This scoping review aims to present a comprehensive and descriptive analysis of nasal mucosa and mucus proteome of CRS patients. Studies investigating the proteome of nasal mucosa and mucus from healthy and CRS patients via mass spectrometry were included. Critical appraisal of methodological quality was conducted with extraction of protein lists. Gene set enrichment analysis (GSEA) was performed on studies including CRS patients. 2962 proteins were identified in the 21 studies included in this review. Eleven studies investigated the nasal mucus proteome and ten studies investigated the nasal mucosa proteome. Studies demonstrated heterogeneity in patients, sampling and mass spectrometry methodology. Samples from CRS patients suggested a trend in enrichment of immune system and programmed cell death pathways. Increased expression of proteins involved in cellular components including the cytoskeleton and adherens junctions was also present in CRS. Alterations in the healthy sinonasal proteome may lead to the increased immunological, metabolic and tissue remodeling processes observed in CRS. However, it is difficult to draw significant conclusions from the GSEA due to the heterogeneity present in the limited literature available. These findings allow us to direct further research to better understand CRS pathogenesis and its endotypes.

Proteomics of Brucella: Technologies and Their Applications for Basic Research and Medical Microbiology

Brucellosis is a global zoonosis caused by Gram-negative, facultative intracellular bacteria of the genus Brucella (B.). Proteomics has been used to investigate a few B. melitensis and B. abortus strains, but data for other species and biovars are limited. Hence, a comprehensive analysis of proteomes will significantly contribute to understanding the enigmatic biology of brucellae. For direct identification and typing of Brucella, matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI—TOF MS) has become a reliable tool for routine diagnosis due to its ease of handling, price and sensitivity highlighting the potential of proteome-based techniques. Proteome analysis will also help to overcome the historic but still notorious Brucella obstacles of infection medicine, the lack of safe and protective vaccines and sensitive serologic diagnostic tools by identifying the most efficient protein antigens. This perspective summarizes past and recent developments in Brucella proteomics with a focus on species identification and serodiagnosis. Future applications of proteomics in these fields are discussed.

SWATH-MS based quantitative proteomics analysis to evaluate the antileishmanial effect of Commiphora wightii- Guggul and Amphotericin B on a clinical isolate of Leishmania donovani

The present study provides comprehensive proteomics analyses of the response of L. donovani parasite to pharamacological stress in vitro. Identification of differentially expressed proteins with associated molecular functions and metabolic pathways, clearly provides an insight into the potential mechanism of the antileishmanial effects as well as a comparative response of the parasite to Guggul and AmB. Treatment of parasite with AmB results in an enhanced modulatory mechanism to counteract the drug induced stress which may have contributed to relapse. In the case of Guggul treatment, an effective antipromastigote activity was observed, which is being reported for the first time. Thus, a deeper understanding of the molecular pathways in the Leishmania parasite in response to pharmacological stress would help in designing novel and effective strategies in targeting the key molecules essential for parasite survival. It will also help in screening of new lead molecules targeting these vital pathways which could be used as an adjunct therapy along with the limited repertoire of antileishmanial drugs.

An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general <100×) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ∼2500 proteins and precise quantification of ∼1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.

A Comprehensive Proteomic and Phosphoproteomic Analysis of Retinal Pigment Epithelium Reveals Multiple Pathway Alterations in Response to the Inflammatory Stimuli.

Overwhelming and persistent inflammation of retinal pigment epithelium (RPE) induces destructive changes in the retinal environment. However, the precise mechanisms remain unclear. In this study, we aimed to investigate RPE-specific biological and metabolic responses against intense inflammation and identify the molecular characteristics determining pathological progression. We performed quantitative analyses of the proteome and phosphoproteome of the human-derived RPE cell line ARPE-19 after treatment with lipopolysaccharide (LPS) for 45 min or 24 h using the latest isobaric tandem-mass tags (TMTs) labeling approach. This approach led to the identification of 8984 proteins, of which 261 showed a 1.5-fold change in abundance after 24 h of treatment with LPS. A parallel phosphoproteome analysis identified 20,632 unique phosphopeptides from 3207 phosphoproteins with 3103 phosphorylation sites. Integrated proteomic and phosphoproteomic analyses showed significant downregulation of proteins related to mitochondrial respiration and cell cycle checkpoint, while proteins related to lipid metabolism, amino acid metabolism, cell-matrix adhesion, and endoplasmic reticulum (ER) stress were upregulated after LPS stimulation. Further, phosphorylation events in multiple pathways, including MAPKK and Wnt/β-catenin signalings, were identified as involved in LPS-triggered pathobiology. In essence, our findings reveal multiple integrated signals exerted by RPE under inflammation and are expected to give insight into the development of therapeutic interventions for RPE disorders.

Comprehensive proteomic analysis of murine terminal erythroid differentiation

Murine-based cellular models have provided and continue to provide many useful insights into the fundamental mechanisms of erythropoiesis, as well as insights into the pathophysiology of inherited and acquired red cell disorders. Although detailed information on many aspects of these cell models is available, comprehensive proteomic data are lacking. This is a critical knowledge gap, as proteins are effectors of most biologic processes. To address this critical unmet need, proteomes of the murine cell lines Friend erythroleukemia (MEL), GATA1 erythroid (G1ER), and embryonic stem cell-derived erythroid progenitor (MEDEP) and proteomes of cultured murine marrow-derived erythroblasts at different stages of terminal erythroid differentiation were analyzed. The proteomes of MEDEP cells and primary murine erythroid cells were most similar, whereas those of MEL and G1ER cells were more distantly related. We demonstrated that the overall cellular content of histones does not decrease during terminal differentiation, despite strong chromatin condensation. Comparison of murine and human proteomes throughout terminal erythroid differentiation revealed that many noted transcriptomic changes were significantly dampened at the proteome level, especially at the end of the terminal differentiation process. Analysis of the early events associated with induction of terminal differentiation in MEDEP cells revealed divergent alterations in associated transcriptomes and proteomes. These proteomic data are powerful and valuable tools for the study of fundamental mechanisms of normal and disordered erythropoiesis and will be of broad interest to a wide range of investigators for making the appropriate choice of various cell lines to study inherited and acquired diseases of the erythrocyte.

ITRAQ-based Quantitative Proteomics Analysis Identifies Host Pathways Modulated during Toxoplasma gondii Infection in Swine.

Toxoplasma gondii is a leading cause of foodborne illness and consumption of undercooked pig meat is a major risk factor for acquiring toxoplasmosis, which causes a substantial burden on society. Here, we used isobaric tags for relative and absolute quantification (iTRAQ) labelling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify cellular proteins and pathways altered during T. gondii infection in pigs. We also used parallel reaction monitoring-based LC-MS/MS to verify the levels of protein expression of infected spleens and mesenteric lymph nodes (MLNs). At 6 days post-infection (dpi), 156, 391, 170, 292, and 200 differentially expressed proteins (DEPs) were detected in the brain, liver, lung, MLNs and spleen, respectively. At 18 dpi, 339, 351, 483, 388, and 303 DEPs were detected in the brain, liver, lung, MLNs and spleen, respectively. Although proteins involved in immune responses were upregulated in all infected tissues, protein expression signature in infected livers was dominated by downregulation of the metabolic processes. By weighted gene co-expression network analysis, we could further show that all proteins were clustered into 25 co-expression modules and that the pink module significantly correlated with the infection status. We also identified 163 potential anti-T. gondii proteins (PATPs) and provided evidence that two PATPs (HSP70.2 and PDIA3) can reduce T. gondii burden in porcine macrophages in vitro. This comprehensive proteomics analysis reveals new facets in the pathogenesis of T. gondii infection and identifies key proteins that may contribute to the pig’s defense against this infection.

Identification of the cis‑molecular neighbours of the immune checkpoint proteinB7‑H4 in the breast cancer cell‑line SK‑BR‑3 by proteomic proximity labelling.

The immune checkpoint protein B7-H4 plays an important role in the positive as well as the negative regulation of immune T-cell responses. When expressed on cancer cells, B7-H4 inhibits T-cell activity, and numerous types of cancer cells use upregulation of B7-H4 as a survival strategy. Thus, B7-H4 is a potential target for anticancer drug therapy. Unfortunately, the cell biology of this molecule has yet to be fully elucidated. Even basic properties, such as the nature of B7-H4 interactors, are controversial. In particular, the cis-inter-actors of B7-H4 on cancer cell plasma membranes have not been investigated to date. The present study used a proteomic proximity-labelling assay to investigate the molecular neighbours of B7-H4 on the surface of the human breast cancer cells SK-BR-3. By comparison to a comprehensive proteome analysis of SK-BR-3 cells, the proximity method detected a relatively small number of low abundance plasma membrane proteins highly enriched for proteins known to modulate cell adhesion and immune recognition. It may be inferred that these molecules contribute to the immunosuppressive behaviour that is characteristic of B7-H4 on cancer cells.

High‐throughput proteome analysis using 50 cm long micro pillar array columns (μPAC&amp;[trade

The practice of bottom‐up proteomics relies to a large extent on the separation performance that can be achieved with state‐of‐the art nano LC‐MS/MS equipment. Depending on the sample complexity or the instrument time that can be dedicated to a certain sample, different LC columns and corresponding LC‐MS/MS methods are required. When aiming for comprehensive proteome analysis with deep coverage, relatively long columns (lengths up to 75 cm) are typically operated with long and shallow solvent gradients, delivering the highest chromatographic performance. However, daily routine proteome analysis often deals with much less complex samples or demands increased sample throughput, making total analysis times above 120 min undesirable or even impossible. The LC columns typically used for this type of analyses do not exceed 25 cm in length, with a focus on workflow robustness and efficiency rather than on maximizing chromatographic performance or peak capacity (1).In the past few years, micro pillar array column (μPAC™) technology has evolved from a tool to perform fundamental LC research to a powerful alternative for classical nano LC columns. The inherent high permeability and low ‘on‐column’ dispersion obtained by the perfect order of the separation bed makes pillar array based chromatography unique in its kind. The peak dispersion originating from heterogeneous flow paths in the separation bed is drastically reduced and therefore components remain much more concentrated during separation (2). Apart from an improved efficiency, pillar array columns can also be designed with substantially lower flow resistances compared to packed bed columns. The distance between the pillars can be independently controlled from the pillar size, enabling the fabrication of columns over a range of external porosities.Using a 50 cm long reversed phase C18 μPAC™ nano LC column in combination with a Thermo Orbitrap Elite mass spectrometer for detection, we demonstrate extremely robust and high‐throughput proteome analysis at capillary flow rates up to 2 μl/min. At these flow rates, sample throughput can be increased to 20, 30, 60 and even 100 samples per day with corresponding peak capacity values (nC) of respectively 300, 250, 200 and 150. When comparing the chromatographic performance that could be obtained for single protein and cell lysate tryptic digest samples to state‐of‐the art packed bed nano LC columns (packed with sub 2 μm silica particles), average peptide peak widths could be reduced by a factor of 1.6. For complex HeLa cell digest samples, this resulted in an increase in peptide and protein group identifications of respectively 60 and 40%.