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Despite being orthogonal to reverse-phase separation and valuable for posttranslational modification (PTM) pre-enrichment, hydrophilic interaction liquid chromatography (HILIC) has not been widely adopted for large-scale proteomic applications. Here, we first evaluated the performance of HILIC in comparison with the popular high-pH reverse-phase (HPRP) separation, as the first dimension for tryptic peptide fractionation in a shotgun workflow to characterize the complex 293T cell proteome. The data indicated that the complementary nature of HILIC and HPRP for peptide separation was mainly due to different hydrophobicity preferences. Realizing that uncaptured components from one mode can be resolved in the other mode, we then designed and compared two multidimensional separation schemes using HILIC and HPRP in tandem for peptide prefractionation, in terms of identification efficiency and coverage at peptide, protein, and PTM levels. A total of 22,604 and 23,566 peptides corresponding to 4481 and 4436 proteins from 293T cell lysate were detected using HILIC-HPRP- and HPRP-HILIC-based shotgun proteomics workflow, respectively. In addition, without assistance of enrichment techniques, the tandem fractionation methods aided to identify 46 different PTMs from over 10,000 of spectra using blind modification search algorithm. We concluded that HILIC is a valuable alternative option for peptide prefractionation in a large-scale proteomic study, but can be further augmented with the use of a secondary HPRP separation. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer is the most deadly gynecological cancer around the world, with high morbidity in industrialized countries. Early diagnosis is key in reducing its morbidity rate. Yet, robust biomarkers, diagnostics, and animal models are still limited for ovarian cancer. This calls for broader omics and systems science oriented diagnostics strategies. In this vein, the domestic chicken has been used as an ovarian cancer animal model, owing to its high rate of developing spontaneous epithelial ovarian tumors. Chicken blood has thus been considered a surrogate reservoir from which cancer biomarkers can be identified. However, the presence of highly abundant proteins in chicken blood has compromised the applicability of proteomics tools to study chicken blood owing to a lack of immunodepletion methods. Here, we demonstrate that a combinatorial peptide ligand library (CPLL) can efficiently remove highly abundant proteins from chicken blood samples, consequently doubling the number of identified proteins. Using an integrated CPLL-1DGE-LC-MSMS workflow, we identified a catalog of 264 unique proteins. Functional analyses further suggested that most proteins were coagulation and complement factors, blood transport and binding proteins, immune- and defense-related proteins, proteases, protease inhibitors, cellular enzymes, or cell structure and adhesion proteins. Semiquantitative spectral counting analysis identified 10 potential biomarkers from the present chicken ovarian cancer model. Additionally, many human homologs of chicken blood proteins we have identified have been independently suggested as diagnostic biomarkers for ovarian cancer, further triangulating our novel observations reported here. In conclusion, the CPLL-assisted proteomic workflow using the chicken ovarian cancer model provides a feasible platform for translational research to identify ovarian cancer biomarkers and understand ovarian cancer biology. To the best of our knowledge, we report here the most comprehensive survey of the chicken blood proteome to date. [ABSTRACT FROM AUTHOR]

Abstract: We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. Although our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semiquantitative method when used in large-scale phosphoproteomics studies in complex backgrounds. [Copyright &y& Elsevier]

Abstract: Protein phosphorylation has been widely documented as a key regulatory and signaling mechanism associated with many cardiac diseases. Recent advances in phosphoproteomic technologies such as phosphopeptide enrichment, novel mass spectrometry applications, and bioinformatic tools have resulted in high-throughput identification and quantitation of protein phosphorylation in a global manner. This review summarizes mainstream phosphoproteomic workflows and highlights the most recent applications of phosphoproteomics used in a range of molecular cardiology research. [Copyright &y& Elsevier]

Abstract: The soy-derived phytoestrogen genistein (GEN) has received attention for its potential benefits on the cardiovascular system by providing direct protection to cardiomyocytes against pathophysiological stresses. Here, we employed a proteomic approach to study the concentration-dependent effects of GEN treatments on cardiomyocytes. Cultured HL-1 cardiomyocytes were treated with low (1μM) and high (50μM) concentrations of GEN. Proteins were pre-fractionated by sequential hydrophilic/hydrophobic extraction and both protein fractions from each treatment group were separated by 2D gel electrophoresis (2DE). Overall, approximately 2,700 spots were visualized on the 2D gels. Thirty-nine and 99 spots changed in volume relative to controls (p<0.05) following the low- and high-concentration GEN treatments, respectively. From these spots, 25 and 62 protein species were identified by ESI-MS/MS and Mascot database searching, respectively. Identified proteins were further categorized according to their functions and possible links to cardioprotection were discussed. MetaCore gene ontology analysis suggested that 1μM GEN significantly impacted the anti-apoptosis process, and that both the low and high concentrations of GEN influenced the glucose catabolic process and regulation of ATPase activity. This proteomics study provides the first global insight into the molecular events triggered by GEN treatment in cardiomyocytes. [Copyright &y& Elsevier]

Objective: This study investigated the proteomic characteristics of cerebrospinal fluid (CSF) in patients with varicella zoster virus (VZV) meningitis to understanding the pathogenesis of central nervous system (CNS) infection by reactivated VZV. Method: We used data‐independent acquisition model to analyze the CSF proteomic differences of 28 patients with VZV meningitis and 11 herpes zoster (HZ) patients. According to the clinical manifestations at discharge, 28 VZV meningitis patients were divided into favorable outcome group and unfavorable outcome (UO) group and their differences in CSF proteome were also analyzed. Results: Compared with the HZ group, the proteins (CXCL10, ELANE, IL‐1RN, MPO, PRTN3, etc.) related to inflammation and immune cell activation were significantly upregulated in the VZV meningitis group (p <.01). The protein related to the nerve function and energy metabolism (CKMT1B, SLITRK3, Synaptotagmin‐3, KIF5B, etc.) were significantly downregulated (p <.05). The levels of a pro‐inflammatory factor, IL‐18, in CSF were significantly higher in patients in the UO group as compared to patients with favorable prognosis (p <.05). Conclusion: Inflammatory immune response is an important pathophysiological mechanism of CNS infection by VZV, and the CSF IL‐18 levels might be a potential prognostic indicator of the outcomes of VZV meningitis. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) has become a major health challenge worldwide. However, the precise etiological and pathophysiological factors involved in IBD remain unclear. Proteomics can be used for large-scale protein identification analysis. In the current study, using tandem mass tag- (TMT-) based shotgun proteomics, proteomic differences between intestinal tissue from health controls, patients with Crohn's disease (CD), and patients with ulcerative colitis (UC) were compared. Proteins with fold change >2 or <0.5 and P value < 0.05 between groups were considered differentially expressed. ProteinAtlas was used to analyze the tissue specificity of differentially expressed proteins (DEPs). Reactome pathway analysis was applied to cluster functional pathways. A total of 4786 proteins were identified, with 59 proteins showing higher levels and 43 showing lower levels in patients with IBD than in controls. Seventeen proteins, including angiotensin converting enzyme 2 (ACE2) and angiotensin converting enzyme 1 (ACE), showed higher levels in CD than in UC. Several novel proteins such as CD38, chitinase 3-like 1 (CHI3L1), olfactomedin 4 (OLFM4), and intelectin 1 were screened out between patients with IBD and controls. When proteins with fold change >1.2 or <0.84 and P value < 0.05 between groups were considered differentially expressed, the expression of 10 proteins, including CD38, involved in the nicotinamide adenine dinucleotide (NAD) metabolism and signaling pathway showed significant changes in IBD. Using the NCBI GEO database, we confirmed increased CD38 mRNA expression in patients with UC and in mouse colitis models. Protein CD38 expression was higher in CD and UC than in normal controls. CD38 expression was higher in inflamed tissues than in noninflamed tissues, and CD38 was located in F4/80-positive cells. Our study may provide novel insights into the molecular pathogenesis of IBD. Further studies are required on the role of NAD metabolism and CD38 in intestinal inflammation. [ABSTRACT FROM AUTHOR]

Abstract: Swine influenza viruses (SIV) are zoonotic pathogens that pose a potential threat to human health. In this study, we analyzed the differential mitochondrial proteomes of H3N2 SIV-infected human lung A549 cells using two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) analysis. In the comparative analysis, 24 altered proteins (13 upregulated and 11 downregulated) were identified in the mitochondria of H3N2 SIV-infected cells; these proteins were involved in cell-to-cell signaling and interaction, cellular movement, and post-translational modification. Moreover, the transcriptional profiles of 16 genes corresponding to the identified proteins were estimated by real time RT-PCR. IPA analysis suggested that the differentially expressed proteins were clustered primarily into the mammalian target of rapamycin (mTOR) and d-glucose signaling pathways. In addition, oxidative phosphorylation and integrin signaling appeared to be major pathways modulated in the mitochondria of infected cells. We further demonstrated that apolipoprotein L2 was upregulated in the cytoplasm and translocated to mitochondria during virus infection. These results were verified by Western blot analysis coupled with confocal microscopy. Collectively, the mitochondrial proteome data provide insights to further understand the underlying mechanisms of H3N2 SIV cross-species infection. Biological significance: In recent years, proteomics has emerged as an indispensable tool to unveil the complex molecular events in virology. we firstly perform mitochondrial proteomic profiles of human cells infected with H3N2 subtype SIV to understand virus–host interactions, and 24 differentially expressed proteins in mitochondrial proteomes were identified in SIV-infected cells. The proteins that were identified to have differential expression were involved in cell-to-cell signaling and interaction, post-translational modification, cell morphology, cellular assembly, cell death, and energy production. Furthermore, Western blot analysis and a confocal assay further demonstrated that the cellular protein APOL2 partially co-localized with mitochondria after virus infection. This is a very important discovery in the underlying replication and pathogenesis of SIV which provides a potential target clue for the design of anti-SIV drugs. Our results will inspire basic study on SIV infection and drive the understanding for replication and pathogenesis of SIV to control this disease. [Copyright &y& Elsevier]