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Fast charging and discharging are keen focus areas of electric vehicles (EVs) in order to reduce vehicle down time and support the variable load requirement. In EVs, mainly lithium batteries with various chemistry such as NCA (nickel cobalt aluminum oxides), LTO (lithium titanate oxide), LFP (lithium iron phosphate), LNO (lithium nickel oxide) and NMC (nickel manganese cobalt oxides) are used as energy storage system. Performance of lithium batteries varies with the chemistry and temperature of batteries along with surrounding conditions. More heat is generated during fast charging and discharging of batteries which lead to high temperature rise and further impact the performance, life and safety of batteries. Thus, it’s essential to study the thermal behaviour for fast charging and discharging of various lithium batteries to provide desired thermal management system for safety and better performance. In this paper, the thermal characteristics of various 18650 lithium batteries including NCA, NMC and LFP are investigated experimentally and numerically from slow charging and discharging loading rate of 0.5C to fast charging and discharging loading rates of 1.5C and 2.5C at different surrounding temperature of 27°C and 45°C. In the numerical investigation, the internal resistance of the batteries is first measured experimentally at various SOCs and battery temperatures, and then the battery surface temperature is determined using an appropriate numerical method for solving the energy balance equation. From slow to fast loading rates at varying ambient temperatures, the numerical study approach presented in this work estimates the battery surface temperature with at least 90% accuracy for the whole duration of the load cycle. The thermal assessment of NCA, NMC, and LFP batteries in this work can help to determine battery management system operating strategies and, ultimately, to develop an appropriate thermal management system.

With growing concerns over climate change due to automotive emissions, fossil fuel depletion, and the increasing price of crude oil, electric vehicles have gained more interest as a mode of transportation. Various electric vehicles have been developed in recent years, including pure electric vehicles (EVs), hybrid electric vehicles (HEVs), and plug-in hybrid electric vehicles (PHEVs). One of the keen focus area in the ongoing development of EVs is their energy storage systems. Mostly lithium-ion batteries are being used as an energy storage system in EVs. Various complicated reaction occurs during charging/discharging of batteries and the thermal behavior of the batteries are coupled to these reactions. Electrochemical reactions affect the heat generation rate, and higher temperatures further increase the speed of the electrochemical reactions. Adverse operating temperatures can impact battery performance, degradation, and safety. Hence it is become very important to investigate the thermal behavior of the lithium-ion batteries. Thermal investigation of cylindrical lithium-ion batteries of different chemistry and shape factor is conducted for different charging/discharging rate and atmospheric conditions using numerical technique. The numerical technique includes the determination of surface temperature of battery by solving the energy balance equation using suitable numerical method. The accuracy of numerical techniques is validated using the experimental techniques. Thermal investigation conducted in this research can help in deciding the operating strategies of battery management system and further in designing the proper thermal management system.

Supplementary Table S4. The complete list of identified phosphopeptides after (pY) enrichment and TMT-based quantitative phosphoproteomics analysis in KRASG12C mutant lung cancer cell lines (n=8) with mean-centered log abundance values.

Supplementary Fig. S1. Differential response (2D and 3D culture) of KRASG12C specific covalent inhibitors (ARS-1620 and AMG510) in KRASG12C lung cancer cell lines and signaling rebound following KRASG12C inhibition. Supplementary Fig. S2. MetaCore literature network after ARS-1620 treatment in H358 cells. Supplementary Fig. S3. TMT design for expression and pY comparison in 8 KRASG12C lung cancer cell lines and two group comparison of phosphosites Supplementary Fig. S4. Analysis of CCLE data set for EMT markers and ERBB3 gene expression; and PCA representing 105 probeset TGFβ-EMT signatures (Gordian et al. 2019) translated into proteins expression to describe EMT activity in KRASG12C lung cancer cell lines. Supplementary Fig. S5. Impact of SHP2 inhibitors (SHP-099 and RMC-4550) on signaling in H358 cells and cell viability analysis following treatment with SHP2 inhibitors or in the combination with ARS-1620 in H358 and H1792 cells. Supplementary Fig. S6. Literature-based signaling network after KRASG12C inhibition in H1792 cells and cell viability analysis following treatment with ARS-1620 (1µM), AZD-4547 (1µM) and their combination in NSCLCG12C mutant cell lines. Supplementary Fig. S7. Effect of TGFβ stimulation on cell viability in response to KRASG12C inhibition and different combination strategies. Supplementary Fig. S8. Heatmap showing mean-centered log abundance expression values for indicated phosphosites/protein expression in KRASG12C mutant cell lines and E vs M 2-group comparison performed for FGFR1 gene expression in CCLE data set. Supplementary Fig. S9. Literature-based signaling network after KRASG12C inhibition in Calu1 cells and impact on signaling following treatment with ARS-1620 plus drugs known to target AXL Receptor.

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Purpose:Covalent inhibitors of KRASG12C specifically target tumors driven by this form of mutant KRAS, yet early studies show that bypass signaling drives adaptive resistance. Although several combination strategies have been shown to improve efficacy of KRASG12C inhibitors (KRASi), underlying mechanisms and predictive strategies for patient enrichment are less clear.Experimental Design:We performed mass spectrometry–based phosphoproteomics analysis in KRASG12C cell lines after short-term treatment with ARS-1620. To understand signaling diversity and cell type–specific markers, we compared proteome and phosphoproteomes of KRASG12C cells. Gene expression patterns of KRASG12C cell lines and lung tumor tissues were examined.Results:Our analysis suggests cell type–specific perturbation to ERBB2/3 signaling compensates for repressed ERK and AKT signaling following ARS-1620 treatment in epithelial cell type, and this subtype was also more responsive to coinhibition of SHP2 and SOS1. Conversely, both high basal and feedback activation of FGFR or AXL signaling were identified in mesenchymal cells. Inhibition of FGFR signaling suppressed feedback activation of ERK and mTOR, while AXL inhibition suppressed PI3K pathway. In both cell lines and human lung cancer tissues with KRASG12C, we observed high basal ERBB2/3 associated with epithelial gene signatures, while higher basal FGFR1 and AXL were observed in cells/tumors with mesenchymal gene signatures.Conclusions:Our phosphoproteomic study identified cell type–adaptive responses to KRASi. Markers and targets associated with ERBB2/3 signaling in epithelial subtype and with FGFR1/AXL signaling in mesenchymal subtype should be considered in patient enrichment schemes with KRASi.

SARS-CoV-2 infection has become a major public health burden and affects many organs including lungs, kidneys, the liver, and the brain. Although the virus is readily detected and diagnosed using nasopharyngeal swabs by reverse transcriptase polymerase chain reaction (RT-PCR), detection of its presence in body fluids is fraught with difficulties. A number of published studies have failed to detect viral RNA by RT-PCR methods in urine. Although microbial identification in clinical microbiology using mass spectrometry is undertaken after culture, here we undertook a mass spectrometry-based approach that employed an enrichment step to capture and detect SARS-CoV-2 nucleocapsid protein directly from urine of COVID-19 patients without any culture. We detected SARS-CoV-2 nucleocapsid protein-derived peptides from 13 out of 39 urine samples. Further, a subset of COVID-19 positive and COVID-19 negative urine samples validated by mass spectrometry were used for the quantitative proteomics analysis. Proteins with increased abundance in urine of SARS-CoV-2 positive individuals were enriched in the acute phase response, regulation of complement system, and immune response. Notably, a number of renal proteins such as podocin (NPHS2), an amino acid transporter (SLC36A2), and sodium/glucose cotransporter 5 (SLC5A10), which are intimately involved in normal kidney function, were decreased in the urine of COVID-19 patients. Overall, the detection of viral antigens in urine using mass spectrometry and alterations of the urinary proteome could provide insights into understanding the pathogenesis of COVID-19.

SARS-CoV-2, a novel human coronavirus, has created a global disease burden infecting > 100 million humans in just over a year. RT-PCR is currently the predominant method of diagnosing this viral infection although a variety of tests to detect viral antigens have also been developed. In this study, we adopted a SISCAPA-based enrichment approach using anti-peptide antibodies generated against peptides from the nucleocapsid protein of SARS-CoV-2. We developed a targeted workflow in which nasopharyngeal swab samples were digested followed by enrichment of viral peptides using the anti-peptide antibodies and targeted parallel reaction monitoring (PRM) analysis using a high-resolution mass spectrometer. This workflow was applied to 41 RT-PCR-confirmed clinical SARS-CoV-2 positive nasopharyngeal swab samples and 30 negative samples. The workflow employed was highly specific as none of the target peptides were detected in negative samples. Further, the detected peptides showed a positive correlation with the viral loads as measured by RT-PCR Ct values. The SISCAPA-based platform described in the current study can serve as an alternative method for SARS-CoV-2 viral detection and can also be applied for detecting other microbial pathogens directly from clinical samples. Supplementary Information The online version contains supplementary material available at 10.1186/s12014-021-09331-z.

With growing concerns over climate change due to automotive emissions and fossil fuel depletion, electric vehicles (EVs) have gained more interest as a mode of transportation. Mostly, lithium-ion batteries are being used as an energy storage system in EVs. Temperature plays a major role in lithium-ion battery performance, degradation, and safety. Thermal investigation of cylindrical lithium-ion batteries of different chemistry and shape factors (18650 NMC and 21700 NCA) is conducted for different charging/discharging rates (0.5 C, 1 C, 1.5 C) and surrounding temperatures (26 °C and 45 °C) using numerical and experimental techniques. The numerical technique involves the determination of the surface temperature of the battery by solving the energy balance equation using a suitable numerical method. The numerical technique proposed in this study is validated using the experimental technique and can predict the temperature with at least 90% accuracy during the entire charging and discharging cycle from a lower to higher loading rate and different surrounding temperatures. The impact of different battery internal and external parameters on battery temperature is carried out using validated numerical technique. Thermal investigation conducted in this research can help in deciding the operating strategies of the battery management system and further in designing the proper thermal management system.

Since the recent outbreak of COVID-19, there have been intense efforts to understand viral pathogenesis and host immune response to combat SARS-CoV-2. It has become evident that different host alterations can be identified in SARS-CoV-2 infection based on whether infected cells, animal models or clinical samples are studied. Although nasopharyngeal swabs are routinely collected for SARS-CoV-2 detection by RT-PCR testing, host alterations in the nasopharynx at the proteomic level have not been systematically investigated. Thus, we sought to characterize the host response through global proteome profiling of nasopharyngeal swab specimens. A mass spectrometer combining trapped ion mobility spectrometry (TIMS) and high-resolution QTOF mass spectrometer with parallel accumulation-serial fragmentation (PASEF) was deployed for unbiased proteome profiling. First, deep proteome profiling of pooled nasopharyngeal swab samples was performed in the PASEF enabled DDA mode, which identified 7723 proteins that were then used to generate a spectral library. This approach provided peptide level evidence of five missing proteins for which MS/MS spectrum and mobilograms were validated with synthetic peptides. Subsequently, quantitative proteomic profiling was carried out for 90 individual nasopharyngeal swab samples (45 positive and 45 negative) in DIA combined with PASEF, termed as diaPASEF mode, which resulted in a total of 5023 protein identifications. Of these, 577 proteins were found to be upregulated in SARS-CoV-2 positive samples. Functional analysis of these upregulated proteins revealed alterations in several biological processes including innate immune response, viral protein assembly, and exocytosis. To the best of our knowledge, this study is the first to deploy diaPASEF for quantitative proteomic profiling of clinical samples and shows the feasibility of adopting such an approach to understand mechanisms and pathways altered in diseases.

Background The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time. Methods Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data. Findings The optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922–0.997) (86/88) sensitivity and 100% (95% CI = 0.958–1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method. Interpretation Our results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures. Funding This study was supported by DBT/Wellcome Trust India Alliance Margdarshi Fellowship grant IA/M/15/1/502023 awarded to AP and the generosity of Eric and Wendy Schmidt.

Background We evaluated the analytical sensitivity and specificity of 4 rapid antigen diagnostic tests (Ag RDTs) for severe acute respiratory syndrome coronavirus 2, using reverse transcription quantitative PCR (RT-qPCR) as the reference method and further characterizing samples using droplet digital quantitative PCR (ddPCR) and a mass spectrometric antigen test. Methods Three hundred fifty (150 negative and 200 RT-qPCR positive) residual PBS samples were tested for antigen using the BD Veritor lateral flow (LF), ACON LF, ACON fluorescence immunoassay (FIA), and LumiraDx FIA. ddPCR was performed on RT-qPCR–positive samples to quantitate the viral load in copies/mL applied to each Ag RDT. Mass spectrometric antigen testing was performed on PBS samples to obtain a set of RT-qPCR–positive, antigen-positive samples for further analysis. Results All Ag RDTs had nearly 100% specificity compared to RT-qPCR. Overall analytical sensitivity varied from 66.5% to 88.3%. All methods detected antigen in samples with viral load >1 500 000 copies/mL RNA, and detected ≥75% of samples with viral load of 500 000 to 1 500 000 copies/mL. The BD Veritor LF detected only 25% of samples with viral load between 50 000 to 500 000 copies/mL, compared to 75% for the ACON LF device and >80% for LumiraDx and ACON FIA. The ACON FIA detected significantly more samples with viral load Conclusions Ag RDTs differ significantly in analytical sensitivity, particularly at viral load

Gallbladder cancer (GBC) is a rare malignancy, associated with poor disease prognosis with a 5-year survival of only 20%. This has been attributed to late presentation of the disease, lack of early diagnostic markers and limited efficacy of therapeutic interventions. Elucidation of molecular events in GBC can contribute to better management of the disease by aiding in the identification of therapeutic targets. To identify aberrantly activated signaling events in GBC, tandem mass tag-based quantitative phosphoproteomic analysis of five GBC cell lines was carried out. Proline-rich Akt substrate 40 kDa (PRAS40) was one of the proteins found to be hyperphosphorylated in all the invasive GBC cell lines. Tissue microarray-based immunohistochemical labeling of phospho-PRAS40 (T246) revealed moderate to strong staining in 77% of the primary gallbladder adenocarcinoma cases. Regulation of PRAS40 activity by inhibiting its upstream kinase PIM1 resulted in a significant decrease in cell proliferation, colony forming and invasive ability of GBC cells. Our results support the role of PRAS40 phosphorylation in GBC cell survival and aggressiveness. This study also elucidates phospho-PRAS40 as a clinical marker in GBC and the role of PIM1 as a therapeutic target in GBC. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12079-018-00503-5) contains supplementary material, which is available to authorized users.

The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic profiling of human anterior pituitary gland (adenohypophysis) using high-resolution Fourier transform mass spectrometry. A total of 2164 proteins were identified in this study, of which 105 proteins were identified for the first time compared with high-throughput proteomic-based studies from human pituitary glands. In addition, we identified 480 proteins with secretory potential and 187 N-terminally acetylated proteins. These are the first region-specific data that could serve as a vital resource for further investigations on the physiological role of the human anterior pituitary glands and the proteins secreted by them. We anticipate that the identification of previously unknown proteins in the present study will accelerate biomedical research to decipher their role in functioning of the human anterior pituitary gland and associated human diseases.

Purpose: Covalent inhibitors of KRASG12C specifically target tumors driven by this form of mutant KRAS, yet early studies show that bypass signaling drives adaptive resistance. Although several combination strategies have been shown to improve efficacy of KRASG12C inhibitors (KRASi), underlying mechanisms and predictive strategies for patient enrichment are less clear. Experimental Design: We performed mass spectrometry–based phosphoproteomics analysis in KRASG12C cell lines after short-term treatment with ARS-1620. To understand signaling diversity and cell type–specific markers, we compared proteome and phosphoproteomes of KRASG12C cells. Gene expression patterns of KRASG12C cell lines and lung tumor tissues were examined. Results: Our analysis suggests cell type–specific perturbation to ERBB2/3 signaling compensates for repressed ERK and AKT signaling following ARS-1620 treatment in epithelial cell type, and this subtype was also more responsive to coinhibition of SHP2 and SOS1. Conversely, both high basal and feedback activation of FGFR or AXL signaling were identified in mesenchymal cells. Inhibition of FGFR signaling suppressed feedback activation of ERK and mTOR, while AXL inhibition suppressed PI3K pathway. In both cell lines and human lung cancer tissues with KRASG12C, we observed high basal ERBB2/3 associated with epithelial gene signatures, while higher basal FGFR1 and AXL were observed in cells/tumors with mesenchymal gene signatures. Conclusions: Our phosphoproteomic study identified cell type–adaptive responses to KRASi. Markers and targets associated with ERBB2/3 signaling in epithelial subtype and with FGFR1/AXL signaling in mesenchymal subtype should be considered in patient enrichment schemes with KRASi.

The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostics including RT-PCR-based assays, antigen detection by lateral flow assays and antibody-based assays have been developed and deployed in a short time. However, many of these assays are lacking in sensitivity and/or specificity. Here, we describe an immunoaffinity purification followed by high resolution mass spectrometry-based targeted assay capable of detecting viral antigen in nasopharyngeal swab samples of SARS-CoV-2 infected individuals. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assays on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was created using fragment ion intensities in the PRM data. This resulted in 97.8% sensitivity and 100% specificity with RT-PCR-based molecular testing as the gold standard. Our results demonstrate that direct detection of infectious agents from clinical samples by mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories.

Signaling plays an important role in regulating all cellular pathways. Altered signaling is one of the hallmarks of cancers. Phosphoproteomics enables interrogation of kinase mediated signaling pathways in biological systems. In cancers, this approach can be utilized to identify aberrantly activated pathways that potentially drive proliferation and tumorigenesis. To identify signaling alterations in head and neck squamous cell carcinoma (HNSCC), we carried out proteomic and phosphoproteomic analysis of HNSCC cell lines using a combination of tandem mass tag (TMT) labeling approach and titanium dioxide-based enrichment. We identified 4,920 phosphosites corresponding to 2,212 proteins in six HNSCC cell lines compared to a normal oral cell line. Our data indicated significant enrichment of proteins associated with splicing. We observed hyperphosphorylation of SRSF protein kinase 2 (SRPK2) and its downstream substrates in HNSCC cell lines. SRPK2 is a splicing kinase, known to phosphorylate serine/arginine (SR) rich domain proteins and regulate splicing process in eukaryotes. Although genome-wide studies have reported the contribution of alternative splicing events of several genes in the progression of cancer, the involvement of splicing kinases in HNSCC is not known. In this study, we studied the role of SRPK2 in HNSCC. Inhibition of SRPK2 resulted in significant decrease in colony forming and invasive ability in a panel of HNSCC cell lines. Our results indicate that phosphorylation of SRPK2 plays a crucial role in the regulation of splicing process in HNSCC and that splicing kinases can be developed as a new class of therapeutic target in HNSCC.

The debilitating disease kala-azar or visceral leishmaniasis is caused by the kinetoplastid protozoan parasite Leishmania donovani. The parasite is transmitted by the hematophagous sand fly vector of the genus Phlebotomus in the old world and Lutzomyia in the new world. The predominant Phlebotomine species associated with the transmission of kala-azar are Phlebotomus papatasi and Phlebotomus argentipes. Understanding the molecular interaction of the sand fly and Leishmania, during the development of parasite within the sand fly gut is crucial to the understanding of the parasite life cycle. The complete genome sequences of sand flies (Phlebotomus and Lutzomyia) are currently not available and this hinders identification of proteins in the sand fly vector. The current study utilizes a three frame translated transcriptomic data of P. papatasi in the absence of genomic sequences to analyze the mass spectrometry data of P. papatasi cell line using a proteogenomic approach. Additionally, we have carried out the proteogenomic analysis of P. papatasi by comparative homology-based searches using related sequenced dipteran protein data. This study resulted in the identification of 1313 proteins from P. papatasi based on homology. Our study demonstrates the power of proteogenomic approaches in mapping the proteomes of unsequenced organisms.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in India. Despite improvements in treatment strategy, the survival rates of HNSCC patients remain poor. Thus, it is necessary to identify biomarkers that can be used for early detection of disease. In this study, we employed iTRAQ-based quantitative mass spectrometry analysis to identify dysregulated proteins from a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. We identified 2468 proteins, of which 496 proteins were found to be dysregulated in at least two out of three HNSCC cell lines compared to immortalized normal oral keratinocytes. We detected increased expression of replication protein A1 (RPA1) and heat shock protein family H (Hsp110) member 1 (HSPH1), in HNSCC cell lines compared to control. The differentially expressed proteins were further validated using parallel reaction monitoring (PRM) and western blot analysis in HNSCC cell lines. Immunohistochemistry-based validation using HNSCC tissue microarrays revealed overexpression of RPA1 and HSPH1 in 15.7% and 32.2% of the tested cases, respectively. Our study illustrates quantitative proteomics as a robust approach for identification of potential HNSCC biomarkers. The proteomic data has been submitted to ProteomeXchange Consortium (http://www.proteomecentral.proteomexchange.org) via the PRIDE public data repository accessible using the data identifier - PXD009241. Keywords: HNSCC, iTRAQ, Parallel reaction monitoring, Mass spectrometry, OKF6/TERT1

Acute ischemic stroke (AIS) is a devastating cerebrovascular disorder that leads to permanent physical and neurological disabilities in adults worldwide. Proteins associated with stroke pathogenesis may appear in the serum of AIS patients due to blood-brain barrier dysfunction, thus permitting the development of blood-based biomarkers for early diagnosis of stroke. These biomarkers could perhaps be an adjunct to the existing imaging modalities and aid in better management and therapeutic intervention during the course of the disease. For this exploratory study, a combination of multiplexed isobaric tagging using iTRAQ reagents and high resolution tandem mass spectrometry was used to identify differentially expressed proteins in serum samples from AIS patients. The quantitative proteomic analysis of serum from both AIS and control subjects revealed 389 high confidence protein identifications and their relative levels. Among them, 60 proteins showed a ≥1.5-fold change in the AIS subjects. We verified the altered serum levels of candidate proteins such as vWF, ADAMTS13, S100A7, and DLG4 through ELISA, and the results also corroborate with the experimental findings. vWF and ADAMTS13 are key players that regulate blood hemostasis, and their altered concentration may contribute to endothelial dysfunction. S100A7 is a novel candidate protein identified in this study that is also known to mediate inflammation, endothelial proliferation, and angiogenesis. The current study provided a potential and novel biomarker panel that may in turn provide diagnostic aid to the existing imaging modalities for the rapid diagnosis of ischemic stroke.

Dysregulation of protein expression is associated with most diseases including cancer. MS-based proteomic analysis is widely employed as a tool to study protein dysregulation in cancers. Proteins that are differentially expressed in head and neck squamous cell carcinoma (HNSCC) cell lines compared to the normal oral cell line could serve as biomarkers for patient stratification. To understand the proteomic complexity in HNSCC, we carried out iTRAQ-based MS analysis on a panel of HNSCC cell lines in addition to a normal oral keratinocyte cell line. LC-MS/MS analysis of total proteome of the HNSCC cell lines led to the identification of 3263 proteins, of which 185 proteins were overexpressed and 190 proteins were downregulated more than twofold in at least two of the three HNSCC cell lines studied. Among the overexpressed proteins, 23 proteins were related to DNA replication and repair. These included high-mobility group box 2 (HMGB2) protein, which was overexpressed in all three HNSCC lines studied. Overexpression of HMGB2 has been reported in various cancers, yet its role in HNSCC remains unclear. Immunohistochemical labeling of HMGB2 in a panel of HNSCC tumors using tissue microarrays revealed overexpression in 77% (54 of 70) of tumors. The HMGB proteins are known to bind to DNA structure resulting from cisplatin-DNA adducts and affect the chemosensitivity of cells. We observed that siRNA-mediated silencing of HMGB2 increased the sensitivity of the HNSCC cell lines to cisplatin and 5-FU. We hypothesize that targeting HMGB2 could enhance the efficacy of existing chemotherapeutic regimens for treatment of HNSCC. All MS data have been deposited in the ProteomeXchange with identifier PXD000737 (http://proteomecentral.proteomexchange.org/dataset/PXD000737).

Despite advances in clinical management, 5-year survival rate in patients with late-stage head and neck squamous cell carcinoma (HNSCC) has not improved significantly over the past decade. Targeted therapies have emerged as one of the most promising approaches to treat several malignancies. Though tyrosine phosphorylation accounts for a minority of total phosphorylation, it is critical for activation of signaling pathways and plays a significant role in driving cancers. To identify activated tyrosine kinase signaling pathways in HNSCC, we compared the phosphotyrosine profiles of a panel of HNSCC cell lines to a normal oral keratinocyte cell line. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) was one of the kinases hyperphosphorylated at Tyr-321 in all HNSCC cell lines. Inhibition of DYRK1A resulted in an increased apoptosis and decrease in invasion and colony formation ability of HNSCC cell lines. Further, administration of the small molecular inhibitor against DYRK1A in mice bearing HNSCC xenograft tumors induced regression of tumor growth. Immunohistochemical labeling of DYRK1A in primary tumor tissues using tissue microarrays revealed strong to moderate staining of DYRK1A in 97.5% (39/40) of HNSCC tissues analyzed. Taken together our results suggest that DYRK1A could be a novel therapeutic target in HNSCC.

Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions,192 novel protein start sites,19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted "noncoding RNAs" to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes. Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India [EMR/2014/000444]; DBT Program Support grant on "Development of infrastructure and a computational framework for analysis of proteomic data" [BT/01/COE/08/05]; Infosys Foundation; pilot grant from the Johns Hopkins Malaria Research Institute; Council of Scientific and Industrial Research, Department of Biotechnology, University Grants Commission; Department of Science and Technology, Government of India; Indian Council of Medical Research This paper is funded by the joint research project to NIMR and IOB entitled "Characterization of Malaria vector Anopheles stephensi Proteome and Transcriptome" (EMR/2014/000444) from the Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India. T.S.K.P. is also supported by the DBT Program Support grant on "Development of infrastructure and a computational framework for analysis of proteomic data" (BT/01/COE/08/05). We also thank Infosys Foundation for financial support to IOB. A.P. and P.S. were funded by a pilot grant from the Johns Hopkins Malaria Research Institute. This paper bears the NIMR publication screening committee approval No. 009/2015. H.G. is a Wellcome Trust-DBT India Alliance Early Career Fellow. We thank the Council of Scientific and Industrial Research, Department of Biotechnology, University Grants Commission, Indian Council of Medical Research and Department of Science and Technology, Government of India for research fellowships to M.K., S.K.S., G.D., R.S.N., S.M.P., A.K. M. (IOB), S.S.M., M.K.G., S.B.D., D.S.K., P.R., N.S., S.D.Y., K.K.D., R.R., A.A.K., A.R., G.J.S., S.C., and R.V. M.D. is funded by the Faculty Improvement Program of Siddaganga Institute of Technology, Tumkur.

Among the neglected tropical diseases, leishmaniasis is one of the most devastating, resulting in significant mortality and contributing to nearly 2 million disability-adjusted life years. Cutaneous leishmaniasis is a debilitating disorder caused by the kinetoplastid protozoan parasite Leishmania major, which results in disfiguration and scars. L. major genome was the first to be sequenced within the genus Leishmania. Use of proteomic data for annotating genomes is a complementary approach to conventional genome annotation approaches and is referred to as proteogenomics. We have used a proteogenomics-based approach to map the proteome of L. major and also annotate its genome. In this study, we searched L. major promastigote proteomic data against the annotated L. major protein database. Additionally, we searched the proteomic data against six-frame translated L. major genome. In all, we identified 3613 proteins in L. major promastigotes, which covered 43% of its proteome. We also identified 26 genome search-specific peptides, which led to the identification of three novel genes previously not identified in L. major. We also corrected the annotation of N-termini of 15 genes, which resulted in extension of their protein products. We have validated our proteogenomics findings by RT-PCR and sequencing. In addition, our study resulted in identification of 266 N-terminally acetylated peptides in L. major, one of the largest acetylated peptide datasets thus far in Leishmania. This dataset should be a valuable resource to researchers focusing on neglected tropical diseases.

In line with the aims of the Chromosome-centric Human Proteome Project (C-HPP) to completely annotate proteins of each chromosome and biology/disease driven HPP (B/D-HPP) to decipher their relation to diseases, we have generated a nonredundant catalogue of protein-coding genes for Chromosome 12 (Chr. 12) and further annotated proteins associated with major neurological disorders. Integrating high level proteomic evidence from four major databases (neXtProt, Global Proteome Machine (GPMdb), PeptideAtlas, and Human Protein Atlas (HPA)) along with Ensembl data resource resulted in the identification of 1066 protein coding genes, of which 171 were defined as "missing proteins" based on the weak or complete absence of experimental evidence. With functional annotations using DAVID and GAD, about 40% of the proteins could be grouped as brain related with implications in cancer or neurological disorders. We used published and unpublished high confidence mass spectrometry data from our group and other literature consisting of more than 5000 proteins derived from clinical specimens from patients with human gliomas, Alzheimer's disease, and Parkinson's disease and mapped it onto Chr. 12. We observed a total of 202 proteins mapping to human Chr. 12, 136 of which were differentially expressed in these disease conditions as compared to the normal. Functional grouping indicated their association with cell cycle, cell-to-cell signaling, and other important processes and networks, whereas their disease association analysis confirmed neurological diseases and cancer as the major group along with psycological disorders, with several overexpressed genes/proteins mapping to 12q13-15 amplicon region. Using multiple strategies and bioinformatics tools, we identified 103 differentially expressed proteins to have secretory potential, 17 of which have already been reported in direct analysis of the plasma or cerebrospinal fluid (CSF) from the patients and 21 of them mapped to cancer associated protein (CAPs) database that are amenable to selective reaction monitoring (SRM) assays for targeted proteomic analysis. Our analysis also reveals, for the first time, mass spectrometric evidence for two "missing proteins" from Chr. 12, namely, synaptic vesicle 2-related protein (SVOP) and IQ motif containing D (IQCD). The analysis provides a snapshot of Chr. 12 encoded proteins associated with gliomas and major neurological conditions and their secretability which can be used to drive efforts for clinical applications.

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

Despite advances in clinical management, 5-year survival rate in patients with late-stage head and neck squamous cell carcinoma (HNSCC) has not improved significantly over the past decade. Targeted therapies have emerged as one of the most promising approaches to treat several malignancies. Though tyrosine phosphorylation accounts for a minority of total phosphorylation, it is critical for activation of signaling pathways and plays a significant role in driving cancers. To identify activated tyrosine kinase signaling pathways in HNSCC, we compared the phosphotyrosine profiles of a panel of HNSCC cell lines to a normal oral keratinocyte cell line. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) was one of the kinases hyperphosphorylated at Tyr-321 in all HNSCC cell lines. Inhibition of DYRK1A resulted in an increased apoptosis and decrease in invasion and colony formation ability of HNSCC cell lines. Further, administration of the small molecular inhibitor against DYRK1A in mice bearing HNSCC xenograft tumors induced regression of tumor growth. Immunohistochemical labeling of DYRK1A in primary tumor tissues using tissue microarrays revealed strong to moderate staining of DYRK1A in 97.5% (39/40) of HNSCC tissues analyzed. Taken together our results suggest that DYRK1A could be a novel therapeutic target in HNSCC.

The pineal gland is a neuroendocrine gland located at the center of the brain. It is known to regulate various physiological functions in the body through secretion of the neurohormone melatonin. Comprehensive characterization of the human pineal gland proteome has not been undertaken to date. We employed a high-resolution mass spectrometry-based approach to characterize the proteome of the human pineal gland. A total of 5874 proteins were identified from the human pineal gland in this study. Of these, 5820 proteins were identified from the human pineal gland for the first time. Interestingly, 1136 proteins from the human pineal gland were found to contain a signal peptide domain, which indicates the secretory nature of these proteins. An unbiased global proteomic profile of this biomedically important organ should benefit molecular research to unravel the role of the pineal gland in neuropsychiatric and neurodegenerative diseases.

Human follicular fluid is a complex body fluid that constitutes the microenvironment of developing follicles in the ovary. Follicular fluid contains a number of proteins that modulate oocyte maturation and ovulation. Information about the protein constituents of follicular fluid may provide a better understanding of ovarian physiology in addition to opening new avenues for investigating ovarian disorders. However, the composition of follicular fluid proteome remains poorly defined. In this study, we carried out SDS-PAGE, OFFGEL and SCX-based separation followed by LC–MS/MS analysis to characterize the proteome of human follicular fluid. We report high confidence identification of 480 proteins, of which 320 have not been described previously in the follicular fluid. The identified proteins belong to diverse functional categories including growth factor and hormones, receptor signaling, enzyme catalysis, defense/immunity and complement activity. Our dataset should serve as a resource for future studies aimed at developing biomarkers for monitoring oocyte and embryo quality, pregnancy outcomes and ovarian disorders. Biological significance Proteome analysis of human follicular fluid by multi-pronged approach of protein peptide fractionation revealed 480 proteins with high confidence. The identified protein may facilitate the understanding of folliculogenesis. This protein dataset should serve as a useful resource for development of biomarkers for oocyte quality, in vitro fertilization techniques and female infertility.

Oral cancer consists of squamous cell carcinoma within the oral cavity or on the lip. The clinical prognosis of this cancer is mostly poor owing to delayed diagnosis and a lack of appropriate early detection biomarkers to identify the disease. In the current study, we investigated the role of the S100A7 calcium-binding protein in oral squamous cell carcinoma as an activator of the p38/MAPK and RAB2A signaling pathway. The aim of the present study was to determine whether S100A7 and RAB2A have a role in tumor progression and to assess their potential as early detection biomarkers for oral cancer. This study elucidated the functional and molecular mechanisms of S100A7 and RAB2A activity in oral cancer, leading us to conclude that S100A7 is the major contributing factor in the occurrence of oral cancer and promotes local tumor progression by activating the MAPK signaling pathway via the RAB2A pathway. We hypothesize that S100A7 affects cell motility and invasion by regulating the RAB2A-associated MAPK signaling cascades. Also, the downregulation of S100A7 expression by RNA interference-mediated silencing inhibits oral cancer cell growth, migration and invasion.

Sample processing protocols that enable compatible recovery of differentially expressed (DE) transcripts and proteins are necessary for integration of the multiomics data applied in the analysis of tumors. In this pilot study, we compared two different isolation methods for extracting RNA and protein from laryngo-pharyngeal tumor tissues and the corresponding adjacent normal sections. In Method 1, RNA and protein were isolated from a single tissue section sequentially and in Method 2, the extraction was carried out using two different sections and two independent and parallel protocols for RNA and protein. RNA and protein from both methods were subjected to RNA-seq and iTRAQ-based LC-MS/MS analysis, respectively. Analysis of data revealed that a higher number of differentially expressed transcripts and proteins were concordant in their regulation trends in Method 1 as compared to Method 2. Cross method comparison of concordant entities revealed that RNA and protein extraction from the same tissue section (Method 1) recovered more concordant entities that are missed in the other extraction method (Method 2) indicating heterogeneity in distribution of these entities in different tissue sections. Method 1 could thus be the method of choice for integrated analysis of transcriptome and proteome data. This article is protected by copyright. All rights reserved

Background Curcumin, derived from the rhizome Curcuma longa, is a natural anti-cancer agent and has been shown to inhibit proliferation and survival of tumor cells. Although the anti-cancer effects of curcumin are well established, detailed understanding of the signaling pathways altered by curcumin is still lacking. In this study, we carried out SILAC-based quantitative proteomic analysis of a HNSCC cell line (CAL 27) to investigate tyrosine signaling in response to curcumin. Results Using high resolution Orbitrap Fusion Tribrid Fourier transform mass spectrometer, we identified 627 phosphotyrosine sites mapping to 359 proteins. We observed alterations in the level of phosphorylation of 304 sites corresponding to 197 proteins upon curcumin treatment. We report here for the first time, curcumin-induced alterations in the phosphorylation of several kinases including TNK2, FRK, AXL, MAPK12 and phosphatases such as PTPN6, PTPRK, and INPPL1 among others. Pathway analysis revealed that the proteins differentially phosphorylated in response to curcumin are known to be involved in focal adhesion kinase signaling and actin cytoskeleton reorganization. Conclusions The study indicates that curcumin may regulate cellular processes such as proliferation and migration through perturbation of the focal adhesion kinase pathway. This is the first quantitative phosphoproteomics-based study demonstrating the signaling events that are altered in response to curcumin. Considering the importance of curcumin as an anti-cancer agent, this study will significantly improve the current knowledge of curcumin-mediated signaling in cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12014-016-9114-0) contains supplementary material, which is available to authorized users.

Objectives: To identify potential candidate proteins which are secretory in nature and present at a higher abundance in oral cancer patients with tobacco habits. Methods: Conditioned media of tobacco-treated and -untreated non-neoplastic oral keratinocytes were analyzed using iTRAQ-based mass spectrometry. Hypersecreted proteins; SPARC (osteonectin), cwcv and kazal like domains proteoglycan 1 (SPOCK1); prosaposin (PSAP); and protein tyrosine phosphatase, receptor type S (PTPRS) were validated by enzyme-linked immunosorbent assay (ELISA) using saliva samples from oral cancer patients who are tobacco users. Results: Proteomic analysis of tobacco-treated and -untreated cells led to the identification of 2873 proteins. Among these, 378 proteins showed high abundance and 253 proteins showed low abundance (2-fold cutoff) in conditioned-media of tobacco-treated cells. ELISA-based validation showed significantly higher levels of SPOCK1, PSAP, and PTPRS in oral cancer patients with tobacco chewing habits compared to healthy controls. However, PSAP showed low specificity compared to SPOCK1 and PTPRS. Conclusions: This study indicates significantly increased levels of SPOCK1, PSAP, and PTPRS in saliva of oral cancer patients with tobacco habits. These protein biomarkers might be useful to identify tobacco users with high risk of developing oral cancers.

High throughput molecular profiling and integrated data analysis with tumor tissues require overcoming challenges like tumor heterogeneity and tissue paucity. This study is an attempt to understand and optimize various steps during tissue processing and in establishing pipelines essential for integrated analysis. Towards this effort, we subjected laryngo-pharyngeal primary tumors and the corresponding adjacent normal tissues (n=2) to two RNA and protein isolation methods, one wherein RNA and protein were isolated from the same tissue sequentially (Method 1) and second, wherein the extraction was carried out using two independent methods (Method 2). RNA and protein from both methods were subjected to RNA-seq and iTRAQ based LC-MS/MS analysis. Transcript and peptide identification and quantification was followed by both individual -ome and integrated data analysis. As a result of this analysis, we identified a higher number of total, as well as differentially expressed (DE) transcripts (1329 vs 1134) and proteins (799 vs 408) with fold change ≥ 2.0, in Method 1. Among these, 173 and 86 entities were identified by both transcriptome and proteome analysis in Method 1 and 2, respectively, with higher concordance in the regulation trends observed in the former. The significant cancer related pathways enriched with the individual DE transcript or protein data were similar in both the methods. However, the entities mapping to them were different, allowing enhanced view of the pathways identified after integration of the data and subsequent mapping. The concordant DE transcripts and proteins also revealed key molecules of the pathways with important roles in cancer development. This study thus demonstrates that sequential extraction of the RNA and proteins from the same tissue allows for better profiling of differentially expressed entities and a more accurate integrated data analysis.

Protein biomarker discovery for early detection of head and neck squamous cell carcinoma (HNSCC) is a crucial unmet need to improve patient outcomes. Mass spectrometry-based proteomics has emerged as a promising tool for identification of biomarkers in different cancer types. Proteins secreted from cancer cells can serve as potential biomarkers for early diagnosis. In the current study, we have used isobaric tag for relative and absolute quantitation (iTRAQ) labeling methodology coupled with high resolution mass spectrometry to identify and quantitate secreted proteins from a panel of head and neck carcinoma cell lines. In all, we identified 2,472 proteins, of which 225 proteins were secreted at higher or lower abundance in HNSCC-derived cell lines. Of these, 148 were present in higher abundance and 77 were present in lower abundance in the cancer-cell derived secretome. We detected a higher abundance of some previously known markers for HNSCC including insulin like growth factor binding protein 3, IGFBP3 (11-fold) and opioid growth factor receptor, OGFR (10-fold) demonstrating the validity of our approach. We also identified several novel secreted proteins in HNSCC including olfactomedin-4, OLFM4 (12-fold) and hepatocyte growth factor activator, HGFA (5-fold). IHC-based validation was conducted in HNSCC using tissue microarrays which revealed overexpression of IGFBP3 and OLFM4 in 70% and 75% of the tested cases, respectively. Our study illustrates quantitative proteomics of secretome as a robust approach for identification of potential HNSCC biomarkers. This article is part of a Special Issue entitled: An Updated Secretome.

Early events in the development of esophageal squamous cell carcinoma (ESCC) are poorly understood and many of the key molecules involved have not yet been identified. We previously used isobaric tags for a relative and absolute quantitation (iTRAQ)-based quantitative proteomics approach to identify differentially expressed proteins in ESCC tissue as compared to the adjacent normal mucosa. Cornulin was identified as one of the major downregulated molecules in ESCC. Cornulin is a member of the S100 fused-type protein family, which has an EF-hand calcium binding motif and multiple tandem repeats of specific peptide motifs. Cornulin was 5-fold downregulated in ESCC as compared to normal epithelium mirroring our previous findings in a gene expression study of ESCC. In the present study, we performed immunohistochemical validation of cornulin (CRNN) in a larger set of patients with ESCC. Downregulation of cornulin was observed in 89% (n=239) of 266 different ESCC tissues arrayed on tissue microarrays (TMAs). Expression of cornulin was observed in the prickle and functional cell layers of normal esophageal mucosa, localized predominantly in the cytoplasm and perinuclear region. The large majority of ESCC cases had little or no expression of cornulin in the carcinoma or stroma. These findings suggest that cornulin is an important molecule in normal esophageal pathology and is likely lost during the conversion of normal to neoplastic epithelium.

We previously developed NetPath as a resource for comprehensive manually curated signal transduction pathways. The pathways in NetPath contain a large number of molecules and reactions which can sometimes be difficult to visualize or interpret given their complexity. To overcome this potential limitation, we have developed a set of more stringent curation and inclusion criteria for pathway reactions to generate high-confidence signaling maps. NetSlim is a new resource that contains this ‘core’ subset of reactions for each pathway for easy visualization and manipulation. The pathways in NetSlim are freely available at http://www.netpath.org/netslim. Database URL: www.netpath.org/netslim