Proteomic Methodologies Research Articles

Absolute quantification of proteins using element mass spectrometry and generic standards

Elemental mass spectrometry is a powerful analytical technique widely established in inorganic analysis. However, despite its quantitative capabilities, it is not yet fully integrated or considered in Life Sciences fields like proteomics. Whereas it is true that ICP-MS has suffered from several instrumental and analytical limitations that have hindered its applicability in protein analysis, significant developments during the last decades have turned ICP-MS into an interesting and, in our opinion, a powerful tool to consider for accurate protein quantification without recourse to specific protein standards. Herein we will try to discuss how these traditional limitations in ICP-MS have been overcome, what further improvements are yet necessary (some of which are shared with MS-based proteomics platforms) and enlighten some of the already existing and potential applications of ICP-MS in absolute quantitative proteomics. SignificanceICP-MS has the potential to become a complementary tool to help molecular mass spectrometry cope with existing limitations, especially those related to standardization and accuracy, in the absolute proteomics field. It can provide absolute quantification of diverse proteoforms using a single generic compound containing sulfur and/or another target element (e.g., phosphorous). Moreover, its applications in quantitative proteomics are no longer limited to protein standards certification or quantification of simple or purified mixtures. Interestingly, absolute quantification of proteins using ICP-MS is favored when carried out at the intact level, making it very compatible with top-down proteomics approaches. Recent instrumental and methodological advances enable synergic combination of ICP-MS with stablished LC-MS proteomics methodologies, setting the basis for its implementation in quantitative proteomics workflows.

Urine Proteomics for Noninvasive Monitoring of Biomarkers in Bronchopulmonary Dysplasia

Introduction: Current techniques to diagnose and/or monitor critically ill neonates with bronchopulmonary dysplasia (BPD) require invasive sampling of body fluids, which is suboptimal in these frail neonates. We tested our hypothesis that it is feasible to use noninvasively collected urine samples for proteomics from extremely low gestational age newborns (ELGANs) at risk for BPD to confirm previously identified proteins and biomarkers associated with BPD. Methods: We developed a robust high-throughput urine proteomics methodology that requires only 50 μL of urine. We utilized the methodology with a proof-of-concept study validating proteins previously identified in invasively collected sample types such as blood and/or tracheal aspirates on urine collected within 72 h of birth from ELGANs (gestational age [26 ± 1.2] weeks) who were admitted to a single Neonatal Intensive Care Unit (NICU), half of whom eventually developed BPD (n = 21), while the other half served as controls (n = 21). Results: Our high-throughput urine proteomics approach clearly identified several BPD-associated changes in the urine proteome recapitulating expected blood proteome changes, and several urinary proteins predicted BPD risk. Interestingly, 16 of the identified urinary proteins are known targets of drugs approved by the Food and Drug Administration. Conclusion: In addition to validating numerous proteins, previously found in invasively collected blood, tracheal aspirate, and bronchoalveolar lavage, that have been implicated in BPD pathophysiology, urine proteomics also suggested novel potential therapeutic targets. Ease of access to urine could allow for sequential proteomic evaluations for longitudinal monitoring of disease progression and impact of therapeutic intervention in future studies.

Proteomics in Thrombosis and Hemostasis.

Proteomics, the simultaneous study of all proteins in a given cell, tissue or organism, is an innovative approach used to identify novel markers for diagnosis, prognosis and the pathophysiological mechanisms associated with diseases. Proteomic methodologies have been used in a variety of contexts such as investigating changes in protein abundance that may occur with disease presence, the response to therapeutic treatments as well as the impacts of age on the plasma proteome.Over the last decade, significant technological advancements in proteomic techniques have resulted in an increase in the use of proteomics in thrombosis and hemostasis research, particularly in order to identify relevant and novel clinical markers associated with bleeding and thrombosis. This mini-review explores the use of proteomics in the setting of thrombosis and hemostasis from 2010-2020, across five main domains (platelets, blood clot composition, stroke, venous thromboembolism, and therapeutics), as well as provides insights into key considerations for conducting proteomic studies.

Hsp40 Affinity to Identify Proteins Destabilized by Cellular Toxicant Exposure.

Environmental toxins and toxicants can damage proteins and threaten cellular proteostasis. Most current methodologies to identify misfolded proteins in cells survey the entire proteome for sites of changed reactivity. We describe and apply a quantitative proteomics methodology to identify destabilized proteins based on their binding to the human Hsp40 chaperone DNAJB8. These protein targets are validated by an orthogonal limited proteolysis assay using parallel reaction monitoring. We find that a brief exposure of HEK293T cells to meta-arsenite increases the affinity of two dozen proteins to DNAJB8, including known arsenite-sensitive proteins. In particular, arsenite treatment destabilizes both the pyruvate dehydrogenase complex E1 subunit and several RNA-binding proteins. This platform can be used to explore how environmental toxins impact cellular proteostasis and to identify the susceptible proteome.

Bacterial Extracellular Vesicles in Gastrointestinal Tract Cancer: An Unexplored Territory.

Simple SummaryMicrobial dysbiosis has been credited as one of the contributing factors to the development and progression of gastrointestinal tract cancer. The altered microbiota influences carcinogenesis through the induction of instability and damage to genetic material, modulation of host metabolic and inflammatory pathways, production of carcinogenic metabolites, and suppression of host antitumor response. These microbes secrete extracellular vesicles that are possibly carrying carcinogenic bioactive metabolites within their cargo. Studies have illustrated the ability of bacterial extracellular vesicles to cross the intestinal epithelial barrier and selectively accumulate near intestinal tumor cells. The purpose of this systemic review was to highlight the possible role of gut bacterial vesicles in the development, progression, and pathogenesis of gastrointestinal tract cancer and their possible involvement in the modulation of the tumor microenvironment. An infinitesimal amount of research has been carried out on the impact of bacterial extracellular vesicles on oncogenesis and tumor progression. This review aimed to encourage more investigations on this subject.Bacterial extracellular vesicles are membrane-enclosed, lipid bi-layer nanostructures that carry different classes of biomolecules, such as nucleic acids, lipids, proteins, and diverse types of small molecular metabolites, as their cargo. Almost all of the bacteria in the gut secrete extracellular vesicles to assist them in competition, survival, material exchange, host immune modulation, infection, and invasion. The role of gut microbiota in the development, progression, and pathogenesis of gastrointestinal tract (GIT) cancer has been well documented. However, the possible involvement of bacterial extracellular vesicles (bEVs) in GIT cancer pathophysiology has not been given due attention. Studies have illustrated the ability of bEVs to cross physiological barriers, selectively accumulate near tumor cells, and possibly alter the tumor microenvironment (TME). A systematic search of original published works related to bacterial extracellular vesicles on gastrointestinal cancer was performed for this review. The current systemic review outlines the possible impact of gut microbiota derived bEVs in GIT cancer in light of present-day understanding. The necessity of using advanced sequencing technologies, such as genetic, proteomic, and metabolomic investigation methodologies, to facilitate an understanding of the interrelationship between cancer-associated bacterial vesicles and gastrointestinal cancer is also emphasized. We further discuss the clinical and pharmaceutical potential of bEVs, along with future efforts needed to understand the mechanism of interaction of bEVs in GIT cancer pathogenesis.

ITGA2 protein is associated with rheumatoid arthritis in Chinese and affects cellular function of T cells

BackgroundWe identified proteins significant for rheumatoid arthritis (RA) in peripheral blood mononuclear cells (PBMCs), and to clarify mechanisms mediated by underlying proteins that may involve in the pathogenesis of RA. MethodsProteome-wide protein expressions were profiled by employing label-free quantitative proteomics methodology (Easy-nLC1000 and Q-exactive). The t-test was applied to identify differentially expressed proteins (DEP, p ≤ 0.05) between RA case and control samples. Gene Ontology enrichment analyses and Protein-Protein Interaction analyses were performed to annotate functions of DEPs. The selected DEP was validated in independent samples using Simple Western assay. Plasma protein level of α2 component of integrin (ITGA2) was measured by using ELISA. The DEP, ITGA2, was assessed for its effects on T cell proliferation, cell cycle, apoptosis, and inflammatory cytokine expression. ResultsSixty-four DEPs (p < 0.05) were identified in PBMCs. The selected ITGA2 (Fold Change, FC = 2.20, p = 1.49E-02) was validated to be up-regulated (FC = 12.33, p = 4.90E-2) with RA, and plasma ITGA2 protein level significantly elevated in RA patients vs. controls. Over-expression of ITGA2 could promote proliferation and inhibit apoptosis of Jurkat T cell, and induce IL-8, IFN-γ and TNF-α expression in Jurkat T cells. ConclusionsITGA2 protein was significantly over-expressed in PBMCs in RA patients, and affects T cell growth and pro-inflammatory cytokine expression in T cells.

A proteomic analysis of the effect of ocean acidification on the haemocyte proteome of the South African abalone Haliotis midae

As a result of increasing CO2 emissions and the prevalence of global climate change, ocean acidification (OA) is becoming more pervasive, affecting many trophic levels, particularly those that rely on succinctly balanced ocean chemistry. This ultimately threatens community structures, as well as the future sustainability of the fishing/aquaculture industry. Understanding the molecular stress response of key organisms will aid in predicting their future survivability under changing environmental conditions. This study sought to elucidate the molecular stress response of the South African abalone, Haliotis midae, an understudied organism with high economic value, utilising a high throughput iTRAQ-based proteomics methodology. Adult abalone were exposed to control (pH 7.9) and experimental (pH 7.5) conditions for 12, 72 and 168 h, following which protein was isolated from sampled haemocytes and subsequently processed. iTRAQ-labelled peptides were analysed using mass spectrometry, while an array of bioinformatics tools was utilised for analysing the proteomic data. COG analysis identified “Cytoskeleton”, “Translation, ribosomal structure and biogenesis”, “Post-translational modification, protein turnover, chaperones”, and “Intracellular trafficking, secretion and vesicular transport” to be the most enriched functional classes, while statistical analysis identified a total of 33 up-regulated and 23 down-regulated effectors of OA stress in abalone. Several of the up-regulated proteins that were identified function in central metabolism (ENO1, PGK, DUOX1, GPD2), the stress/immune response (CAMKI, HSPA5/GRP78, MAPKI), and cytoskeleton, protein sorting and signal transduction (IQGAP1, MYO9B, TLN1, RDX, TCP-1/CCT, SNX6, CHMP1a, VPS13a). Protein-protein interactions were predicted using STRING DB, Cytoscape and Ingenuity Pathway Analysis, providing a model of the effects of OA on the H. midae haemocyte proteome. The data indicated that H. midae underwent a metabolic shift under OA conditions to utilize more energy-efficient mechanisms of ATP generation, while attempts at restoring haemocyte stabilisation and homeostasis were reflected by up-regulation of oxidative stress and cytoskeletal proteins. Our results support other molluscan studies that report a complex array of overlapping functions of both the stress and immune response systems. This interplay of the mounted stress and immune response is maintained and observed through the up-regulation of proteins involved in protein synthesis and turnover, as well as intracellular signalling and transport. The data presented in this study highlight the value of employing sensitive and robust -omics technologies for assessing the effects of changing environmental conditions on marine organisms.

Nanoparticle-Aided Nanoreactor for Nanoproteomics.

Large-scale bottom-up proteomics of few even single cells is crucial for a better understanding of the roles played by cell-to-cell heterogeneity in disease and development. Novel proteomic methodologies with extremely high sensitivity are required for few even single-cell proteomics. Sample processing with high recovery and no contaminants is one key step. Here we developed a nanoparticle-aided nanoreactor for nanoproteomics (Nano3) technique for processing low-nanograms of mammalian cell proteins for proteome profiling. The Nano3 technique employed nanoparticles packed in a capillary channel to form a nanoreactor (≤30 nL) for concentrating, cleaning, and digesting proteins originally in a lysis buffer containing sodium dodecyl sulfate (SDS), followed by nanoRPLC-MS/MS analysis. The Nano3 method identified a 40-times higher number of proteins based on MS/MS from 2-ng mouse brain protein samples compared to the SP3 (single-pot solid-phase-enhanced sample preparation) method, which performed the sample processing using the nanoparticles in a 10 μL solution in an Eppendorf tube. The data indicates a drastically higher sample recovery of the Nano3 compared to the SP3 method for processing mass-limited proteome samples. In this pilot study, the Nano3 method was further applied in processing 10-1000 HeLa cells for bottom-up proteomics, producing 441 ± 263 (n = 4) (MS/MS) and 983 ± 292 (n = 4) [match between runs (MBR)+MS/MS] protein identifications from only 10 HeLa cells using a Q-Exactive HF mass spectrometer. The preliminary results render the Nano3 method a useful approach for processing few mammalian cells for proteome profiling.

Applied Proteomics in 'One Health'.

‘One Health’ summarises the idea that human health and animal health are interdependent and bound to the health of ecosystems. The purpose of proteomics methodologies and studies is to determine proteins present in samples of interest and to quantify changes in protein expression during pathological conditions. The objectives of this paper are to review the application of proteomics technologies within the One Health concept and to appraise their role in the elucidation of diseases and situations relevant to One Health. The paper develops in three sections. Proteomics Applications in Zoonotic Infections part discusses proteomics applications in zoonotic infections and explores the use of proteomics for studying pathogenetic pathways, transmission dynamics, diagnostic biomarkers and novel vaccines in prion, viral, bacterial, protozoan and metazoan zoonotic infections. Proteomics Applications in Antibiotic Resistance part discusses proteomics applications in mechanisms of resistance development and discovery of novel treatments for antibiotic resistance. Proteomics Applications in Food Safety part discusses the detection of allergens, exposure of adulteration, identification of pathogens and toxins, study of product traits and characterisation of proteins in food safety. Sensitive analysis of proteins, including low-abundant ones in complex biological samples, will be achieved in the future, thus enabling implementation of targeted proteomics in clinical settings, shedding light on biomarker research and promoting the One Health concept.

Proteotoxicity in Cardiac Amyloidosis: Amyloidogenic Light Chains Affect the Levels of Iintracellular Proteins in Human Heart Cells

Introduction. AL amyloidosis is characterized by widespread deposition of immunoglobulin light chains (LCs) as amyloid fibrils. Cardiac involvement is frequent and leads to life-threatening cardiomyopathy. In addition to the tissue alteration caused by fibrils, clinical and experimental evidence indicates that cardiac damage is also caused by proteotoxicity of prefibrillar amyloidogenic species. As in other amyloidoses, the damage mechanisms at the cellular level are complex and largely undefined. Herein we sought to characterize the molecular changes occurring in primary human cells (cardiac fibroblasts, hCF) exposed in vitro to soluble amyloidogenic cardiotropic LCs, and control LC from patients with multiple myeloma, by evaluating their proteome.Methods. Monoclonal LCs were purified from patients with AL amyloidosis and severe heart involvement (CardioLCs), and from multiple myeloma patients without amyloidosis (MMLCs, used as controls). The effects of various LCs (4 CardioLCs and 3 MMLCs) on hCF physiology were explored regarding viability and cytotoxicity. A combination of gel-based and label-free shotgun analyses, followed by bioinformatics and data validation studies, was used to characterize the proteome alterations of cultured hCF exposed for 24h to a representative cardiotropic LC (CardioLC1), by comparison with untreated cells and with cells incubated with a representative MMLC (MMLC2). In order to assess the generalizability of the results, the quantitative changes in a subset of proteins have been further assessed by immunoblotting in hCF exposed to each of the LCs employed in the study.Results. Incubation of hCF with the various cardiotropic LCs translates into impaired viability, with apoptosis activation and oxidative stress. These alterations are associated with significant proteome remodeling: combining the results of the two proteomic methodologies, 85 differentially represented proteins were found overall in the comparison between CardioLC1-treated and untreated hCF, and 51 in the CardioLC1-treated versus the MMLC2-treated comparison. The differential proteins are involved in key cellular processes, including cytoskeletal organization, protein synthesis and quality control, mitochondrial activity and metabolism, signal transduction and molecular trafficking. Among the proteins identified as differential by proteomics, 8 species (Talin, Heat shock protein 27, Transgelin, Porin 31HM, Proteasome subunit beta type-2, Cysteine and glycine-rich protein 2, Cullin-associated NEDD8-dissociated protein 1 and Vimentin) have been selected overall for independent validation by western blotting. Selection was based on their fold change, biological role and/or availability of validated antibodies. Changes are being confirmed to occur also in hCF treated with the other cardiotropic LCs.Conclusions. These results support and expand the concept that soluble species of amyloidogenic cardiotropic LCs possess toxicity towards cardiac cells. Overall, our investigation has allowed characterizing the molecular alterations in target cells exposed to an extrinsic proteotoxic stimulus, represented by monoclonal cardiotropic LCs. Quantitative changes in specific proteins may in the future be explored as potential markers of cardiac proteotoxic stress. Dysregulation of species involved in crucial cellular functions as cytoskeletal remodeling, mitochondrial activity and metabolism, protein synthesis, quality control and degradation, may play a causative role in the cellular alterations that lead to the clinical picture, and targeting these altered pathways may be beneficial for counteracting organ damage in AL amyloidosis. DisclosuresPalladini:Prothena: Honoraria, Other: Travel grant; Celgene: Other: Travel grants; Jannsen-Cilag: Membership on an entity's Board of Directors or advisory committees.

DIA proteomics analysis through serum profiles reveals the significant proteins as candidate biomarkers in women with PCOS

BackgroundThe aim of this study was to apply proteomic methodology for the analysis of proteome changes in women with polycystic ovary syndrome (PCOS).Material and methodsAll the participators including 31 PCOS patients and 31 healthy female as controls were recruited, the clinical characteristics data was recorded at the time of recruitment, the laboratory biochemical data was detected. Then, a data-independent acquisition (DIA)-based proteomics method was performed to compare the serum protein changes between PCOS patients and controls. In addition, Western blotting was used to validate the expression of identified proteomic biomarkers.ResultsThere were 80 proteins differentially expressed between PCOS patients and controls significantly, including 54 downregulated and 26 upregulated proteins. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that downregulated proteins were enriched in platelet degranulation, cell adhesion, cell activation, blood coagulation, hemostasis, defense response and inflammatory response terms; upregulated proteins were enriched in cofactor catabolic process, hydrogen peroxide catabolic process, antioxidant activity, cellular oxidant detoxification, cellular detoxification, antibiotic catabolic process and hydrogen peroxide metabolic process. Receiver operating characteristic curves analysis showed that the area under curve of Histone H4 (H4), Histone H2A (H2A), Trem-like transcript 1 protein (TLT-1) were all over than 0.9, indicated promising diagnosis values of these proteins. Western blotting results proved that the detected significant proteins, including H4, H2A, TLT-1, Peroxiredoxin-1, Band 3 anion transport protein were all differently expressed in PCOS and control groups significantly.ConclusionThese proteomic biomarkers provided the potentiality to help us understand PCOS better, but future studies comparing systemic expression and exact role of these candidate biomarkers in PCOS are essential for confirmation of this hypothesis.

Optimized Targeted Proteomics Methodology for Normalizing Tissue Protein Abundance Data in Analysis of Placental Markers

Objective Placental tissue analysis provides important information about the in-utero environment that could have an impact on fetal development. Due to the heterogeneity and variability of perfusion in placental tissue, the technical variability during sample collection can confound placental proteomics data analysis. The aim of this study was to develop a targeted proteomics method for selecting housekeeping placental proteins for normalizing placental data. Methods A list of placental housekeeping proteins was selected based on previously reported placental house-keeping genes (mRNA) (1). The surrogate peptides of the housekeeping proteins were selected for targeted analysis based on an optimized strategy (2). Placental tissue (n=5) was collected from women enrolled in the CANDLE (Conditions Affecting Neurocognitive Development and Learning in Early childhood) study. The samples were fractionated into cytosolic and membrane fractions, and the fractions were enriched, reduced, alkylated, and digested based on previously reported methods (2). Targeted proteomics data were acquired on Waters XevoTQ-XS with IonKey and a microflow liquid chromatography in multiple reaction monitoring mode. The results were analyzed using Skyline software. The coefficient of variation (%CV) of the proteins was calculated to determine the technical or inter-individual variability. Results The following surrogate peptides of the housekeeping proteins were reproducibly quantified: VIGGDDLSTLTGK (HPRT); LGANSLLDLVVFGR (SDHA); LGFQVIFTDFK (TBPL1); DSTLIMQLLR (1433Z); DLLHVLAFSK (LEP); GPQLAAQNLGISLANLLLSK (HEM3); VIHDNFGIVEGLMTTVHAITATQK (G3P); SVALAVLALLSLSGLEAIQR (B2MG); NLVQEWLAK (PPB1); VFDEFKPLVEEPQNLIK (ALBUMIN); and IVEIPFNSTNK (AT1A1), with the fragments and peptides correlation (R2) equal to 0.8-0.99 and 0.80-0.96, respectively. HPRT, SDHA, 1433Z, HEM3, G3P, and B2MG were enriched in the cytosolic fraction, whereas only PPB1 and AT1A1 were enriched in the membrane fraction. TBPL1 and LEP were detected in both fractions. The following proteins showed %CV below 20%: HPRT, SDHA, TBPL1, HEM3, and AT1A1. Positive correlation was observed between 1433Z and G3P, and HPRT and B2MG with an R2 equal to 0.98 and 0.86, respectively. HPRT and SDHA were negatively correlated with an R2 equal to 0.95. Discussion We confirmed that housekeeping proteins could be quantified with good correlation and low coefficients of variation. The placenta is an important organ and its research applications in birth cohort-based studies requires consistent data normalization techniques. Proteomics analysis of placental tissue is paramount to understand the placental phenotype since the proteome is closely associated with function. Creating a cocktail method of housekeeping protein markers that can be used to normalize the protein concentration is vital to comparing samples and data within and across laboratories.

O-GlcNAcylation protein disruption by Thiamet G promotes changes on the GBM U87-MG cells secretome molecular signature

Glioblastoma (GBM) is a grade IV glioma highly aggressive and refractory to the therapeutic approaches currently in use. O-GlcNAcylation plays a key role for tumor aggressiveness and progression in different types of cancer; however, experimental evidence of its involvement in GBM are still lacking. Here, we show that O-GlcNAcylation plays a critical role in maintaining the composition of the GBM secretome, whereas inhibition of OGA activity disrupts the intercellular signaling via microvesicles. Using a label-free quantitative proteomics methodology, we identified 51 proteins in the GBM secretome whose abundance was significantly altered by activity inhibition of O-GlcNAcase (iOGA). Among these proteins, we observed that proteins related to proteasome activity and to regulation of immune response in the tumor microenvironment were consistently downregulated in GBM cells upon iOGA. While the proteins IGFBP3, IL-6 and HSPA5 were downregulated in GBM iOGA cells, the protein SQSTM1/p62 was exclusively found in GBM cells under iOGA. These findings were in line with literature evidence on the role of p62/IL-6 signaling axis in suppressing tumor aggressiveness and our experimental evidence showing a decrease in radioresistance potential of these cells. Taken together, our findings provide evidence that OGA activity may regulate the p62 and IL-6 abundance in the GBM secretome. We propose that the assessment of tumor status from the main proteins present in its secretome may contribute to the advancement of diagnostic, prognostic and even therapeutic tools to approach this relevant malignancy.

The impact of mass spectrometry application to screen new proteomics biomarkers in Ophthalmology

In the search for molecular markers that aid in the early diagnosis and treatment of various human diseases, many studies have focused on changes in genes, their transcripts and protein products. Recent advances in proteomic methodologies, such as mass spectrometry (MS), generate new opportunities to obtain relevant information on normal and abnormal processes that occur in many important cell pathways. The human eye is a highly specialized and compartmentalized organ, and the interpretation of molecular biomarkers helps to evaluate its cellular structure, providing a broader molecular understanding that corroborates in the pathophysiology of ophthalmological diseases, with marked improvements in their diagnosis, prognosis and treatment. This review summarizes the most important protein biomarkers in Ophthalmology screened by MS tools. The use of translational medicine techniques (as MS), integrating basic and clinical research, still transforms scientific findings, from laboratory researches to clinical applications, from the bedside into the community.

Discovery and Pharmacological Evaluation of STEAP4 as a Novel Target for HER2 Overexpressing Breast Cancer.

Breast cancer (BC) is a highly heterogeneous disease encompassing multiple subtypes with different molecular and histopathological features, disease prognosis, and therapeutic responses. Among these, the Triple Negative BC form (TNBC) is an aggressive subtype with poor prognosis and therapeutic outcome. With respect to HER2 overexpressing BC, although advanced targeted therapies have improved the survival of patients, disease relapse and metastasis remains a challenge for therapeutic efficacy. In this study the aim was to identify key membrane-associated proteins which are overexpressed in these aggressive BC subtypes and can serve as potential biomarkers or drug targets. We leveraged on the development of a membrane enrichment protocol in combination with the global profiling GeLC-MS/MS technique, and compared the proteomic profiles of a HER2 overexpressing (HCC-1954) and a TNBC (MDA-MB-231) cell line with that of a benign control breast cell line (MCF-10A). An average of 2300 proteins were identified from each cell line, of which approximately 600 were membrane-associated proteins. Our global proteomic methodology in tandem with invigoration by Western blot and Immunofluorescence analysis, readily detected several previously-established BC receptors like HER2 and EPHA2, but importantly STEAP4 and CD97 emerged as novel potential candidate markers. This is the first time that the mitochondrial iron reductase STEAP4 protein up-regulation is linked to BC (HER2+ subtype), while for CD97, its role in BC has been previously described, but never before by a global proteomic technology in TNBC. STEAP4 was selected for further detailed evaluation by the employment of Immunohistochemical analysis of BC xenografts and clinical tissue microarray studies. Results showed that STEAP4 expression was evident only in malignant breast tissues whereas all the benign breast cases had no detectable levels. A functional role of STEAP4 intervention was established in HER2 overexpressing BC by pharmacological studies, where blockage of the STEAP4 pathway with an iron chelator (Deferiprone) in combination with the HER2 inhibitor Lapatinib led to a significant reduction in cell growth in vitro. Furthermore, siRNA mediated knockdown of STEAP4 also suppressed cell proliferation and enhanced the inhibition of Lapatinib in HER2 overexpressing BC, confirming its potential oncogenic role in BC. In conclusion, STEAP4 may represent a novel BC related biomarker and a potential pharmacological target for the treatment of HER2 overexpressing BC.

Proteomic Approaches to Dissect Host SUMOylation during Innate Antiviral Immune Responses.

SUMOylation is a highly dynamic ubiquitin-like post-translational modification that is essential for cells to respond to and resolve various genotoxic and proteotoxic stresses. Virus infections also constitute a considerable stress scenario for cells, and recent research has started to uncover the diverse roles of SUMOylation in regulating virus replication, not least by impacting antiviral defenses. Here, we review some of the key findings of this virus-host interplay, and discuss the increasingly important contribution that large-scale, unbiased, proteomic methodologies are making to discoveries in this field. We highlight the latest proteomic technologies that have been specifically developed to understand SUMOylation dynamics in response to cellular stresses, and comment on how these techniques might be best applied to dissect the biology of SUMOylation during innate immunity. Furthermore, we showcase a selection of studies that have already used SUMO proteomics to reveal novel aspects of host innate defense against viruses, such as functional cross-talk between SUMO proteins and other ubiquitin-like modifiers, viral antagonism of SUMO-modified antiviral restriction factors, and an infection-triggered SUMO-switch that releases endogenous retroelement RNAs to stimulate antiviral interferon responses. Future research in this area has the potential to provide new and diverse mechanistic insights into host immune defenses.

High-Throughput Microbore Ultrahigh-Performance Liquid Chromatography-Ion Mobility-Enabled-Mass Spectrometry-Based Proteomics Methodology for the Exploratory Analysis of Serum Samples from Large Cohort Studies.

The deployment of proteomic analysis in clinical studies represents a significant opportunity to detect and validate biomarkers in translational medicine, improve disease understanding, and provide baseline information on population health. However, comprehensive proteome studies usually employ nanoscale chromatography and often require several hours of analysis/sample. Here, we describe a high-throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) methodology using 1 mm scale chromatography requiring only 15 min/sample, coupled to ion mobility-enabled mass spectrometry. The short run time effected a 6-fold increase in productivity compared with nanoscale LC/MS. The method demonstrated excellent reproducibility with retention time coefficient of variations of less than 0.05% and peak area reproducibility ranging from 5 to 15%. The 1 mm system produced similar chromatographic peak capacity values to the nanoscale miniaturized system, detecting 90% of the Escherichia coli proteins identified by the 75 μm LC/MS system (albeit based on only 75% of the peptides found by the latter). Application to the analysis of serum samples from a human prostate cancer study group resulted in the identification of a total of 533 proteins revealing the differential expression of proteins linked to patients receiving hormone-radiotherapy or undergoing surgery.

IMProv: A Resource for Cross-link-Driven Structure Modeling that Accommodates Protein Dynamics

Proteomics methodology has expanded to include protein structural analysis, primarily through cross-linking mass spectrometry (XL-MS) and hydrogen–deuterium exchange mass spectrometry (HX-MS). However, while the structural proteomics community has effective tools for primary data analysis, there is a need for structure modeling pipelines that are accessible to the proteomics specialist. Integrative structural biology requires the aggregation of multiple distinct types of data to generate models that satisfy all inputs. Here, we describe IMProv, an app in the Mass Spec Studio that combines XL-MS data with other structural data, such as cryo-EM densities and crystallographic structures, for integrative structure modeling on high-performance computing platforms. The resource provides an easily deployed bundle that includes the open-source Integrative Modeling Platform program (IMP) and its dependencies. IMProv also provides functionality to adjust cross-link distance restraints according to the underlying dynamics of cross-linked sites, as characterized by HX-MS. A dynamics-driven conditioning of restraint values can improve structure modeling precision, as illustrated by an integrative structure of the five-membered Polycomb Repressive Complex 2. IMProv is extensible to additional types of data.

Recent technical progress in sample preparation and liquid-phase separation-mass spectrometry for proteomic analysis of mass-limited samples.

Mass spectrometry (MS)-based proteomics has enabled the identification and quantification of thousands of proteins from complex proteomes in a single experiment. However, its performance for mass-limited proteome samples (e.g., single cells and tissue samples from laser capture microdissection) is still not satisfying. The development of novel proteomic methodologies with better overall sensitivity is vital. During the last several years, substantial technical progress has been achieved for the preparation and liquid-phase separation-MS characterization of mass-limited proteome samples. In this review, we summarize recent technological progress of sample preparation, liquid chromatography (LC)-MS, capillary zone electrophoresis (CZE)-MS and MS instrumentation for bottom-up proteomics of trace biological samples, highlight some exciting applications of the novel techniques for single-cell proteomics, and provide a very brief perspective about the field at the end.

Data-independent acquisition mass spectrometry (DIA-MS) for proteomic applications in oncology.

Data-independent acquisition mass spectrometry (DIA-MS) is a next generation proteomic methodology that generates permanent digital proteome maps offering highly reproducible retrospective analysis of cellular and tissue specimens. The adoption of this technology has ushered a new wave of oncology studies across a wide range of applications including its use in molecular classification, oncogenic pathway analysis, drug and biomarker discovery and unravelling mechanisms of therapy response and resistance. In this review, we provide an overview of the experimental workflows commonly used in DIA-MS, including its current strengths and limitations versus conventional data-dependent acquisition mass spectrometry (DDA-MS). We further summarise a number of key studies to illustrate the power of this technology when applied to different facets of oncology. Finally we offer a perspective of the latest innovations in DIA-MS technology and machine learning-based algorithms necessary for driving the development of high-throughput, in-depth and reproducible proteomic assays that are compatible with clinical diagnostic workflows, which will ultimately enable the delivery of precision cancer medicine to achieve optimal patient outcomes.