Label-free Quantitative Mass Spectrometry Research Articles

Stoichiometry of protein complexes in plant photosynthetic membranes

Hetero-oligomeric membrane protein complexes form the electron transport chain (ETC) of oxygenic photosynthesis. The ETC complexes undertake the light-driven vectorial electron and proton transport reactions, which generate energy-rich ATP and electron-rich NADPH molecules for carbon fixation. The rate of photosynthetic electron transport depends on the availability of photons and the relative abundance of electron transport complexes. The relative abundance of the two photosystems, critical for the quantum efficiency of photosynthesis in changing light quality conditions, has been determined successfully by optical methods. Due to the lack of spectroscopic signatures, however, relatively little is known about the stoichiometry of other non-photosystem complexes in plant photosynthetic membrane. Here we determine the ratios of all major thylakoid-bound ETC complexes in Arabidopsis by a label-free quantitative mass spectrometry technique. The calculated stoichiometries are consistent with known subunit composition of complexes and current estimates of photosystem and cytochrome b6f concentrations. The implications of these stoichiometries for photosynthetic light harvesting and the partitioning of electrons between the linear and cyclic electron transport pathways of photosynthesis are discussed.

Identification of Prognostic Biomarkers in the Urinary Peptidome of the Small Renal Mass

Renal cell carcinoma (RCC) is often diagnosed incidentally as a small renal mass (SRM; pT1a, ≤4 cm). Increasing concerns surrounding the overtreatment of patients with benign or clinically silent SRMs has resulted in a recent shift in treatment recommendations, especially in elderly and infirm patients. There are currently no biomarkers that can predict progression. We used a quantitative label-free liquid chromatography-tandem mass spectrometry peptidomics approach and targeted parallel-reaction monitoring to identify early, noninvasive diagnostic and prognostic biomarkers for early-stage RCC-SRMs. In total, 115 urine samples, including 33 renal oncocytoma (≤4 cm) cases, 30 progressive and 26 nonprogressive clear cell RCC-SRM cases, and 26 healthy controls were evaluated. Nine endogenous peptides that displayed significantly elevated expression in clear cell RCC-SRMs relative to healthy controls were identified. Peptides NVINGGSHAGNKLAMQEF, VNVDEVGGEALGRL, and VVAGVANALAHKYH showed significantly elevated expression in clear cell RCC-SRMs relative to renal oncocytoma. Additionally, peptides SHTSDSDVPSGVTEVVVKL and IVDNNILFLGKVNRP displayed significantly elevated expression in progressive relative to nonprogressive clear cell RCC-SRMs. Peptide SHTSDSDVPSGVTEVVVKL showed the most significant discriminatory utility (area under the curve, 0.76; 95% CI, 0.62-0.90; P=0.0027). Patients with elevated SHTSDSDVPSGVTEVVVKL expression had significantly shorter overall survival (hazard ratio, 4.13; 95% CI, 1.09-15.65; P=0.024) compared to patients with low expression. Pretreatment characterization of urinary peptides can provide insight into early RCC progression and may aid clinical decision-making and improve disease management.

Proteomics analysis of the matrisome from MC38 experimental mouse liver metastases.

Dissemination of primary tumors to distant anatomical sites has a substantial negative impact on patient prognosis. The liver is a common site for metastases from colorectal cancer, and patients with hepatic metastases have generally much shorter survival, raising a need to develop and implement novel strategies for targeting metastatic disease. The extracellular matrix (ECM) is a meshwork of highly crosslinked, insoluble high-molecular-mass proteins maintaining tissue integrity and establishing cell-cell interactions. Emerging evidence identifies the importance of the ECM in cancer cell migration, invasion, intravasation, and metastasis. Here, we isolated the ECM from MC38 mouse liver metastases using our optimized method of mild detergent solubilization followed by biochemical enrichment. The matrices were subjected to label-free quantitative mass spectrometry analysis, revealing proteins highly abundant in the metastatic matrisome. The resulting list of proteins upregulated in the ECM significantly predicted survival in patients with colorectal cancer but not other cancers with strong involvement of the ECM component. One of the proteins upregulated in liver metastatic ECM, annexin A1, was not previously studied in the context of cancer-associated matrisome. Here, we show that annexin A1 was markedly upregulated in colon cancer cell lines compared with cancer cells of other origin and also over-represented in human primary colorectal lesions, as well as hepatic metastases, compared with their adjacent healthy tissue counterparts. In conclusion, our study provides a comprehensive ECM characterization of MC38 experimental liver metastases and proposes annexin A1 as a putative target for this disease.NEW & NOTEWORTHY Here, the authors provide an extensive proteomics characterization of murine colorectal cancer liver metastasis matrisome (the ensemble of all extracellular matrix molecules). The findings presented in this study may enable identification of therapeutic targets or biomarkers of hepatic metastases.

Pilot-Scale Study Of Human Plasma Proteomics Identifies ApoE And IL33 As Markers In Atopic Asthma.

BackgroundThe pathobiology of atopic asthma is complex and the symptoms similar to other respiratory diseases. As such, identification of biomarkers of atopic asthma is of prime importance for better diagnosis and control of the disease.ObjectivesWe sought to study the changes in plasma proteome and cytokine-expression profile across healthy and atopic asthmatics for identifying biomarkers and exploring aberrant pathways for atopic asthma.MethodsA pilot-scale study in humans was performed to identify differentially expressed proteins in blood plasma of healthy controls (n=5) and treatment-naïve atopic asthma patients (n=5) using quantitative label-free liquid chromatography–tandem mass spectrometry proteomics and ELISA.ResultsMass spectrometry–based proteomic analysis revealed ApoE to be significantly downregulated in atopic asthmatics compared to healthy volunteers. Decreased expression of ApoE in atopic asthmatics was validated by immunoblotting (50.74% decrease). Comparison with atopic asthmatics and COPD patients showed that ApoE was decreased (36.33%) in atopic asthma compared to COPD. IL33 was significantly upregulated in atopic asthmatics compared to healthy subjects (3.84-fold).ConclusionApoE was downregulated and IL33 upregulated in atopic asthma patients compared to healthy volunteers. These two proteins' profiles were distinct in atopic asthma from healthy and COPD plasma samples. Differential expression of these proteins could serve as a probable candidate for a two-protein classifier–based prognostic biomarker of atopic asthma.

Comprehensive Landscape of Active Deubiquitinating Enzymes Profiled by Advanced Chemoproteomics.

Enzymes that bind and process ubiquitin, a small 76-amino-acid protein, have been recognized as pharmacological targets in oncology, immunological disorders, and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition to the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation, and more potent/selective ubiquitin active-site probes with propargylic-based electrophiles to profile 74 DUBs including distinguishable isoforms for 5 DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents and pan-DUB inhibitors combined with probe labeling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39, and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small-molecule DUB selectivity profiling.

An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium.

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.

Redundancy and Complementarity between ERAP1 and ERAP2 Revealed by their Effects on the Behcet's Disease-associated HLA-B*51 Peptidome*[S

The endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 trim peptides to be loaded onto HLA molecules, including the main risk factor for Behçet's disease HLA-B*51. ERAP1 is also a risk factor among HLA-B*51-positive individuals, whereas no association is known with ERAP2. This study addressed the mutual relationships between both enzymes in the processing of an HLA-bound peptidome, interrogating their differential association with Behçet's disease. CRISPR/Cas9 was used to generate knock outs of ERAP1, ERAP2 or both from transfectant 721.221-HLA-B*51:01 cells. The surface expression of HLA-B*51 was reduced in all cases. The effects of depleting each or both enzymes on the B*51:01 peptidome were analyzed by quantitative label-free mass spectrometry. Substantial quantitative alterations of peptide length, subpeptidome balance, N-terminal residue usage, affinity and presentation of noncanonical ligands were observed. These effects were often different in the presence or absence of the other enzyme, revealing their mutual dependence. In the absence of ERAP1, ERAP2 showed similar and significant processing of B*51:01 ligands, indicating functional redundancy. The high overlap between the peptidomes of wildtype and double KO cells indicates that a large majority of B*51:01 ligands are present in the ER even in the absence of ERAP1/ERAP2. These results indicate that both enzymes have distinct, but complementary and partially redundant effects on the B*51:01 peptidome, leading to its optimization and maximal surface expression. The distinct effects of both enzymes on the HLA-B*51 peptidome provide a basis for their differential association with Behçet's disease and suggest a pathogenetic role of the B*51:01 peptidome.

Quantitative Proteomics Analysis Reveals Unique But Overlapping Protein Signatures in HIV Infection

Human immunodeficiency virus-1 (HIV-1) exploits human host factors to complete its life cycle. Hence, it is very important to identify HIV-regulated host proteins for better understanding of the virus life cycle and its contribution to pathogenesis. This can potentially lead to identifying core target molecules that can be used as diagnostic and prognostic markers and/or as targets for potential therapeutic intervention. We present here a first holistic attempt to apply global plasma proteomics analysis of three closely related study populations including: 1) patients with HIV type 1 (HIV-1), 2) HIV-1 elite controllers (HIV-1 EC), and 3) patients infected with HIV type 2 (HIV-2). Peripheral blood plasma (PBP) samples from these groups of patients, together with healthy donors, were subjected to expression proteomics using label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS). Over 314 unique PBP protein species were identified of which 100 (approx. 32%) were significantly differentially expressed (≥ 2 to ∞ - fold change; p < 0.05) between HIV-1, HIV-2 and HIV-1 EC control subjects. A total of 91 of the 100 proteins were significantly differentially expressed between pairs of HIV-1 versus HIV-1 EC, while 83 of the 100 proteins differed significantly between HIV-2 and HIV-1 EC. Interestingly, 76 proteins (87.5%) overlap between the two data sets indicating that majority of these proteins share similar expression changes between the three sample groups. Two proteins, XRCC5 and PSME1, were implicated in the early phase of the pathway network for HIV life cycle. Other identified proteins were involved in infectious disease and disease of signal transduction. Among them were MAP2K1, RPL23A, RPS3, RPS8, CALR, PRDX1, SOD2, LMNB1, LMNA, PHB, FGA, and FGB. Interestingly, despite high degree of similarity in protein profiles of HIV-1 and HIV-2, we identified only six proteins with significant expression changes (P<0.05). These include ETFB, PHB2, S100A9, LMO2, PPP3R1 and Vif, a fragment of virion infectivity factor of HIV-1. In conclusion, we have identified HIV-related protein expression changes and these proteins once validated in large sample cohort might potentially be capable of early diagnosis and prognosis of HIV diseases and other related infectious diseases. Funding: We would like to acknowledge King Abdulaziz City of Science and Technology for funding this project, advanced Strategic Program, National plan for Science, Technology and Innovation (10-BIO 1340-46). Declaration of Interest: The authors declare no competing interests. Ethical Approval: This study was conducted in accordance with the Helsinki Declaration and approved by the Research Ethics Committee and Research Advisory Council (ORA # 2110 001). All participants provided written and signed informed consents that were obtained by the institutional (KFSHRC) IRB approval.

The Farmed Atlantic Salmon (Salmo salar) Skin-Mucus Proteome and Its Nutrient Potential for the Resident Bacterial Community.

Norway is the largest producer and exporter of farmed Atlantic salmon (Salmo salar) worldwide. Skin disorders correlated with bacterial infections represent an important challenge for fish farmers due to the economic losses caused. Little is known about this topic, thus studying the skin–mucus of Salmo salar and its bacterial community depict a step forward in understanding fish welfare in aquaculture. In this study, we used label free quantitative mass spectrometry to investigate the skin–mucus proteins associated with both Atlantic salmon and bacteria. In particular, the microbial temporal proteome dynamics during nine days of mucus incubation with sterilized seawater was investigated, in order to evaluate their capacity to utilize mucus components for growth in this environment. At the start of the incubation period, the largest proportion of proteins (~99%) belonged to the salmon and many of these proteins were assigned to protecting functions, confirming the defensive role of mucus. On the contrary, after nine days of incubation, most of the proteins detected were assigned to bacteria, mainly to the genera Vibrio and Pseudoalteromonas. Most of the predicted secreted proteins were affiliated with transport and metabolic processes. In particular, a large abundance and variety of bacterial proteases were observed, highlighting the capacity of bacteria to degrade the skin–mucus proteins of Atlantic salmon.

SerThr-PhosphoProteome of Brain from Aged PINK1-KO+A53T-SNCA Mice Reveals pT1928-MAP1B and pS3781-ANK2 Deficits, as Hub between Autophagy and Synapse Changes

Hereditary Parkinson’s disease (PD) can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) as stressor or the autosomal recessive deficiency of PINK1 Serine/Threonine-phosphorylation activity as stress-response. We demonstrated the combination of PINK1-knockout with overexpression of SNCAA53T in double mutant (DM) mice to exacerbate locomotor deficits and to reduce lifespan. To survey posttranslational modifications of proteins underlying the pathology, brain hemispheres of old DM mice underwent quantitative label-free global proteomic mass spectrometry, focused on Ser/Thr-phosphorylations. As an exceptionally strong effect, we detected >300-fold reductions of phosphoThr1928 in MAP1B, a microtubule-associated protein, and a similar reduction of phosphoSer3781 in ANK2, an interactor of microtubules. MAP1B depletion is known to trigger perturbations of microtubular mitochondria trafficking, neurite extension, and synaptic function, so it was noteworthy that relevantly decreased phosphorylation was also detected for other microtubule and microfilament factors, namely MAP2S1801, MARK1S394, MAP1AT1794, KIF1AS1537, 4.1NS541, 4.1GS86, and ADD2S528. While the MAP1B heavy chain supports regeneration and growth cones, its light chain assists DAPK1-mediated autophagy. Interestingly, relevant phosphorylation decreases of DAPK2S299, VPS13DS2429, and VPS13CS2480 in the DM brain affected regulators of autophagy, which are implicated in PD. Overall, significant downregulations were enriched for PFAM C2 domains, other kinases, and synaptic transmission factors upon automated bioinformatics, while upregulations were not enriched for selective motifs or pathways. Validation experiments confirmed the change of LC3 processing as reflection of excessive autophagy in DM brain, and dependence of ANK2/MAP1B expression on PINK1 levels. Our new data provide independent confirmation in a mouse model with combined PARK1/PARK4/PARK6 pathology that MAP1B/ANK2 phosphorylation events are implicated in Parkinsonian neurodegeneration. These findings expand on previous observations in Drosophila melanogaster that the MAP1B ortholog futsch in the presynapse is a primary target of the PARK8 protein LRRK2, and on a report that MAP1B is a component of the pathological Lewy body aggregates in PD patient brains. Similarly, ANK2 gene locus variants are associated with the risk of PD, ANK2 interacts with PINK1/Parkin-target proteins such as MIRO1 or ATP1A2, and ANK2-derived peptides are potent inhibitors of autophagy.

Analysis of the Zika and Japanese Encephalitis Virus NS5 Interactomes.

Mosquito-borne flaviviruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), are major human pathogens. Among the flaviviral proteins, the nonstructural protein 5 (NS5) is the largest, most conserved, and major enzymatic component of the viral replication complex. Disruption of the common key NS5-host protein-protein interactions critical for viral replication could aid in the development of broad-spectrum antiflaviviral therapeutics. Hundreds of NS5 interactors have been identified, but these are mostly DENV-NS5 interactors. To this end, we sought to investigate the JEV- and ZIKV-NS5 interactomes using EGFP immunoprecipitation with label-free quantitative mass spectrometry analysis. We report here a total of 137 NS5 interactors with a significant enrichment of spliceosomal and spliceosomal-associated proteins. The transcription complex Paf1C and phosphatase 6 were identified as common NS5-associated complexes. PAF1 was shown to play opposite roles in JEV and ZIKV infections. Additionally, we validated several NS5 targets and proposed their possible roles in infection. These include lipid-shuttling proteins OSBPL9 and OSBPL11, component of RNAP3 transcription factor TFIIIC, minichromosome maintenance, and cochaperone PAQosome. Mining this data set, our study expands the current interaction landscape of NS5 and uncovers several NS5 targets that are new to flavivirus biology.

Waking the sleeping dragon: gene expression profiling reveals adaptive strategies of the hibernating reptile Pogona vitticeps

BackgroundHibernation is a physiological state exploited by many animals exposed to prolonged adverse environmental conditions associated with winter. Large changes in metabolism and cellular function occur, with many stress response pathways modulated to tolerate physiological challenges that might otherwise be lethal. Many studies have sought to elucidate the molecular mechanisms of mammalian hibernation, but detailed analyses are lacking in reptiles. Here we examine gene expression in the Australian central bearded dragon (Pogona vitticeps) using mRNA-seq and label-free quantitative mass spectrometry in matched brain, heart and skeletal muscle samples from animals at late hibernation, 2 days post-arousal and 2 months post-arousal.ResultsWe identified differentially expressed genes in all tissues between hibernation and post-arousal time points; with 4264 differentially expressed genes in brain, 5340 differentially expressed genes in heart, and 5587 differentially expressed genes in skeletal muscle. Furthermore, we identified 2482 differentially expressed genes across all tissues. Proteomic analysis identified 743 proteins (58 differentially expressed) in brain, 535 (57 differentially expressed) in heart, and 337 (36 differentially expressed) in skeletal muscle. Tissue-specific analyses revealed enrichment of protective mechanisms in all tissues, including neuroprotective pathways in brain, cardiac hypertrophic processes in heart, and atrophy protective pathways in skeletal muscle. In all tissues stress response pathways were induced during hibernation, as well as evidence for gene expression regulation at transcription, translation and post-translation.ConclusionsThese results reveal critical stress response pathways and protective mechanisms that allow for maintenance of both tissue-specific function, and survival during hibernation in the central bearded dragon. Furthermore, we provide evidence for multiple levels of gene expression regulation during hibernation, particularly enrichment of miRNA-mediated translational repression machinery; a process that would allow for rapid and energy efficient reactivation of translation from mature mRNA molecules at arousal. This study is the first molecular investigation of its kind in a hibernating reptile, and identifies strategies not yet observed in other hibernators to cope stress associated with this remarkable state of metabolic depression.

Standard-flow LC and thermal focusing ESI elucidates altered liver proteins in late stage Niemann-Pick, type C1 disease.

Aim: Mass spectrometry (MS)-based proteomics, particularly with the development of nano-ESI, have been invaluable to our understanding of altered proteins related to human disease. Niemann-Pick, type C1 (NPC1)disease is a fatal, autosomal recessive,neurodegenerative disorder. The resulting defects include unesterified cholesterol and sphingolipids accumulation in the late endosomal/lysosomal system resulting in organ dysfunction including liver disease. Materials & methods: First, we performed MS analysis of a complex mammalian proteome using both nano- and standard-flow ESI with the intent of developing a differential proteomics platform using standard-flow ESI. Next, we measured the differential liver proteome in the NPC1 mouse model via label-free quantitative MS using standard-flow ESI. Results: Using the standard-flow ESI approach, we found altered protein levels including, increased Limp2 and Rab7a in liver tissue ofNpc1-/- compared to control mice. Conclusion: Standard-flow ESI can be a tool for quantitative proteomic studies when sample amount is not limited. Using this method, we have identified new protein markers of NPC1.

Optimization and Standardization of Thermal Treatment as a Plasma Prefractionation Method for Proteomic Analysis.

Prefractionation is a prerequisite step for deep plasma proteomics. Highly abundant proteins, particularly human serum albumin (HSA) and immunoglobulin G (IgG), typically interfere with investigation of proteins with lower abundance. A relatively simple preparation method based on high temperature can precipitate thermolabile proteins, providing a strategic window to access the thermostable plasma subproteome. This study aimed to optimize thermal treatment as a reliable prefractionation method and to compare it with two commercial kits, including HSA and IgG immunodepletion (IMDP) and combinatorial peptide ligand libraries (CPLL), using untreated plasma as a control condition. By varying the temperature and the incubation period, the optimal condition was found as treatment at 95°C for 20 min, which maintained about 1% recovery yield of soluble proteins. Consistency and reproducibility of thermal treatment-derived plasma subproteome were checked by two-dimensional electrophoresis. The coefficient of variation regarding protein spot numbers was less than 10% among three independent specimens. Highly abundant protein depletion of the thermal treatment was evaluated by immunoblotting against HSA and IgG as compared to the untreated plasma, IMDP, and CPLL. Multidimensional comparison based on 489 unique peptides derived from the label-free quantitative mass spectrometry revealed that the thermal treatment, IMDP, and CPLL provided distinct sets of plasma subproteome compared to untreated plasma, and these appeared to be complementary to each other. Comparing the characteristics of the three procedures suggested that thermal treatment was more cost-effective and less time-consuming than IMDP and CPLL. This study proposes the use of thermal treatment as a reliable and cost-effective method for plasma prefractionation which provides benefits to large-scale proteomic projects and biomarker studies.

The interactome of a family of potential methyltransferases in HeLa cells

Human methytransferase like proteins (METTL) are part of a large protein family characterized by the presence of binding domains for S-adenosyl methionine, a co-substrate for methylation reactions. Despite the fact that members of this protein family were shown or predicted to be DNA, RNA or protein methyltransferases, most METTL proteins are still poorly characterized. Identification of complexes in which these potential enzymes act could help to understand their function(s) and substrate specificities. Here we systematically studied interacting partners of METTL protein family members in HeLa cells using label-free quantitative mass spectrometry. We found that, surprisingly, many of the METTL proteins appear to function outside of stable complexes whereas others including METTL7B, METTL8 and METTL9 have high-confidence interaction partners. Our study is the first systematic and comprehensive overview of the interactome of METTL protein family that can provide a crucial resource for further studies of these potential novel methyltransferases.

Urinary biomarkers for the diagnosis of cervical cancer by quantitative label-free mass spectrometry analysis.

Due to the invasive procedure associated with Pap smears for diagnosing cervical cancer and the conservative culture of developing countries, identifying less invasive biomarkers is of great interest. Quantitative label-free mass spectrometry was performed to identify potential biomarkers in the urine samples of patients with cervical cancer. This technique was used to study the differential expression of urinary proteomes between normal individuals and cancer patients. The alterations in the levels of urinary proteomes in normal and cancer patients were analyzed by Progenesis label-free software and the results revealed that 60 proteins were upregulated while 73 proteins were downregulated in patients with cervical cancer. This method could enrich high molecular weight proteins from 100 kDa. The protein-protein interactions were obtained by Search Tool for the Retrieval of Interacting Genes/Proteins analysis and predicted the biological pathways involving various functions including cell-cell adhesion, blood coagulation, metabolic processes, stress response and the regulation of morphogenesis. Two notable upregulated urinary proteins were leucine-rich α-2-glycoprotein (LRG1) and isoform-1 of multimerin-1 (MMRN1), while the 3 notable downregulated proteins were S100 calcium-binding protein A8 (S100A8), serpin B3 (SERPINB3) and cluster of differentiation-44 antigen (CD44). The validation of these 5 proteins was performed by western blot analysis and the biomarker sensitivity of these proteins was analyzed individually and in combination with receiver operator characteristic curve (ROC) analysis. Quantitative mass spectrometry analysis may allow for the identification of urinary proteins of high molecular weight. The proteins MMRN1 and LRG1 were presented, for the first time, to be highly expressed urinary proteins in cervical cancer. ROC analysis revealed that LRG1 and SERPINB3 could be individually used, and these 5 proteins could also be combined, to detect the occurrence of cervical cancer.

Surfaceome of Exosomes Secreted from the Colorectal Cancer Cell Line SW480: Peripheral and Integral Membrane Proteins Analyzed by Proteolysis and TX114.

Exosomes are important bidirectional cell-cell communicators in normal and pathological physiology. Although exosomal surface membrane proteins (surfaceome) enable target cell recognition and are an attractive source of disease marker, they are poorly understood. Here, a comprehensive surfaceome analysis of exosomes secreted by the colorectal cancer cell line SW480 is described. Sodium carbonate extraction/Triton X-114 phase separation and mild proteolysis (proteinase K, PK) of intact exosomes is used in combination with label-free quantitative mass spectrometry to identify 1025 exosomal proteins of which 208 are predicted to be integral membrane proteins (IMPs) according to TOPCONS and GRAVY scores. Interrogation of UniProt database-annotated proteins reveals 124 predicted peripherally-associated membrane proteins (PMPs). Surprisingly, 108 RNA-binding proteins (RBPs)/RNA nucleoproteins (RNPs) are found in the carbonate/Triton X-114 insoluble fraction. Mild PK treatment of SW480-GFP labeled exosomes reveal 58 proteolytically cleaved IMPs and 14 exoplasmic PMPs (e.g., CLU/GANAB/LGALS3BP). Interestingly, 18 RBPs/RNPs (e.g., EIF3L/RPL6) appear bound to the outer exosome surface since they are sensitive to PK proteolysis. The finding that outer surface-localized miRNA Let-7a-5p is RNase A-resistant, but degraded by a combination of RNase A/PK treatment suggests exosomal miRNA species also reside on the outer surface of exosomes bound to RBPs/RNPs.

Stratification of asthma phenotypes by airway proteomic signatures

Stratification by eosinophil and neutrophil counts increases our understanding of asthma and helps target therapy, but there is room for improvement in our accuracy in prediction of treatment responses and a need for better understanding of the underlying mechanisms. We sought to identify molecular subphenotypes of asthma defined by proteomic signatures for improved stratification. Unbiased label-free quantitative mass spectrometry and topological data analysis were used to analyze the proteomes of sputum supernatants from 246 participants (206 asthmatic patients) as a novel means of asthma stratification. Microarray analysis of sputum cells provided transcriptomics data additionally to inform on underlying mechanisms. Analysis of the sputum proteome resulted in 10 clusters (ie, proteotypes) based on similarity in proteomic features, representing discrete molecular subphenotypes of asthma. Overlaying granulocyte counts onto the 10 clusters as metadata further defined 3 of these as highly eosinophilic, 3 as highly neutrophilic, and 2 as highly atopic with relatively low granulocytic inflammation. For each of these 3 phenotypes, logistic regression analysis identified candidate protein biomarkers, and matched transcriptomic data pointed to differentially activated underlying mechanisms. This study provides further stratification of asthma currently classified based on quantification of granulocytic inflammation and provided additional insight into their underlying mechanisms, which could become targets for novel therapies.

Quantitative label-free mass spectrometry using contralateral and adjacent breast tissues reveal differentially expressed proteins and their predicted impacts on pathways and cellular functions in breast cancer

Proteins play an essential role in the biological processes associated with cancer. Their altered expression levels can deregulate critical cellular pathways and interactive networks. In this study, the mass spectrometry-based label-free quantification followed by functional annotation was performed to investigate the most significant deregulated proteins among tissues of primary breast tumor (PT) and axillary metastatic lymph node (LN) and corresponding non-tumor tissues contralateral (NCT) and adjacent (ANT) from patients diagnosed with invasive ductal carcinoma. A total of 462 proteins was observed as differentially expressed (DEPs) among the groups analyzed. A high level of similarity was observed in the proteome profile of both non-tumor breast tissues and DEPs (n = 12) were mainly predicted in the RNA metabolism. The DEPs among the malignant and non-tumor breast tissues [n = 396 (PTxNCT) and n = 410 (LNxNCT)] were related to pathways of the LXR/RXR, NO, eNOS, eIF2 and sirtuins, tumor-related functions, fatty acid metabolism and oxidative stress. Remarkable similarity was observed between both malignant tissues, which the DEPs were related to metastatic capabilities. Altogether, our findings revealed differential proteomic profiles that affected cancer associated and interconnected signaling processes. Validation studies are recommended to demonstrate the potential of individual proteins and/or pathways as biological markers in breast cancer. SignificanceThe proteomic analysis of this study revealed high similarity in the proteomic profile of the contralateral and adjacent non-tumor breast tissues. Significant differences were identified among the proteome of the malignant and non-tumor tissue groups of the same patients, providing relevant insights into the hallmarks, signaling pathways, biological functions, and interactive protein networks that act during tumorigenesis and breast cancer progression. These proteins are suggested as targets of relevant interest to be explored as potential biological markers related to tumor development and metastatic progression in the breast cancer disease.

The unfolded protein response controls endoplasmic reticulum stress-induced apoptosis of MCF-7 cells via a high dose of vitamin C treatment.

Recent in vitro studies have shown that vitamin C (Vit C) with pro-oxidative properties causes cytotoxicity of breast cancer cells by selective oxidative stress. However, the effect of Vit C in itself at different concentration levels on MCF-7 breast cancer cell line after 24h, has not yet been described. We aimed to examine the effect of Vit C on the viability and signalling response of MCF-7/WT (MCF-7 wild-type) cells that were exposed to various concentrations (0.125-4mM) of Vit C during 24h. The cytotoxic effect of Vit C on MCF-7/VitC (MCF-7/WT after added 2mM Vit C) was observed, resulting in a decrease of cell index after 12h. Also, the cytotoxicity of Vit C (2mM) after 24h was confirmed by flow cytometry, i.e., increase of dead, late apoptotic, and depolarized dead MCF-7/VitC cells compared to MCF-7/WT cells. Moreover, changes in proteomic profile of MCF-7/VitC cells compared to the control group were investigated via label-free quantitative mass spectrometry and post-translational modification. Using bioinformatics assessment (i.e., iPathwayGuide and SPIA R packages), a significantly impacted pathway in MCF-7/VitC was identified, namely the protein processing in endoplasmic reticulum. The semi-quantitative change (log2fold change = 4.5) and autophosphorylation at Thr-446 of protein kinase (PKR) (involved in this pathway) indicates that PKR protein could be responsible for the unfolded protein response and inhibition of the cell translation during endoplasmic reticulum stress, and eventually, for cell apoptosis. These results suggest that increased activity of PKR (Thr-446 autophosphorylation) related to cytotoxic effect of Vit C (2mM) may cause the MCF-7 cells death.