bims-prodis 2018-04-22 papers

bims-prodis

Biomed News

on Proteomics in disease

Issue of 2018–04–22
twenty papers selected by
Nancy Gough, Bioserendipity

Myristic acid induces proteomic and secretomic changes associated with steatosis, cytoskeleton remodeling, endoplasmic reticulum stress, protein turnover and exosome release in HepG2 cells.
Proteomics for Biomarker Identification and Clinical Application in Kidney Disease.
Analysis of Human Nuclear Protein Complexes by Quantitative Mass Spectrometry Profiling.
Comparative proteomic analysis of human malignant ascitic fluids for the development of gastric cancer biomarkers.
Putative salivary biomarkers useful to differentiate patients with fibromyalgia.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of 36 blood group alleles among 396 Thai samples reveals region-specific variants.
Identification of pyruvate kinase as a novel allergen in whiteleg shrimp (Litopenaeus vannamei) by specific-IgE present in patients with shrimp allergy.
Advancing systems immunology through data-driven statistical analysis.
Proteomic investigating the cooperative lethal effect of EGFR and MDM2 inhibitors on ovarian carcinoma.
Role of MSC-derived galectin 3 in the AML microenvironment.
Alisma orientale extract exerts the reversing cholestasis effect by activation of farnesoid X receptor.
Mass cytometry: a powerful tool for dissecting the immune landscape.
Proteomic approach and expression analysis revealed the differential expression of predicted leptospiral proteases capable of ECM degradation.
Tumor classification with MALDI-MSI data of tissue microarrays: a case study.
Epoxygenase inactivation exacerbates diet and aging-associated metabolic dysfunction resulting from impaired adipogenesis.
Systematic analysis of the cerebrospinal fluid proteome of fibromyalgia patients.
Urinary Extracellular Vesicle Biomarkers in Urological Cancers: From Discovery Towards Clinical Implementation.
A Library of Phosphoproteomic and Chromatin Signatures for Characterizing Cellular Responses to Drug Perturbations.
Proteomic analysis of sperm proteins in infertile men with high levels of reactive oxygen species.
Investigation of the therapy targets of Yi-Qi-Yang-Yin-Hua-Tan-Qu-Yu recipe on type 2 diabetes by serum proteome labeled with iTRAQ.

J Proteomics. 2018 Apr 12. pii: S1874-3919(18)30167-2. [Epub ahead of print]

Myristic acid induces proteomic and secretomic changes associated with steatosis, cytoskeleton remodeling, endoplasmic reticulum stress, protein turnover and exosome release in HepG2 cells.

Giulia Speziali, Laura Liesinger, Juergen Gindlhuber, Christina Leopold, Bettina Pucher, Jessica Brandi, Annalisa Castagna, Tamara Tomin, Petra Krenn, Gerhard G Thallinger, Oliviero Olivieri, Nicola Martinelli, Dagmar Kratky, Matthias Schittmayer, Ruth Birner-Gruenberger, Daniela Cecconi.

  Myristic acid, the 14-carbon saturated fatty acid (C14:0), is associated to an increased cardiovascular disease risk. Since it is found in low concentration in cells, its specific properties have not been fully analyzed. The aim of this study was to explore the cell response to this fatty acid to help explaining clinical findings on the relationship between C14:0 and cardiovascular disease. The human liver HepG2 cell line was used to investigate the hepatic response to C14:0 in a combined proteomic and secretomic approach. A total of 47 intracellular and 32 secreted proteins were deregulated after treatments with different concentrations of C14:0. Data are available via ProteomeXchange (PXD007902). In addition, C14:0 treatment of primary murine hepatocytes confirmed that C14:0 induces lipid droplet accumulation and elevates perilipin-2 levels. Functional enrichment analysis revealed that C14:0 modulates lipid droplet formation and cytoskeleton organization, induce ER stress, changes in exosome and extracellular miRNA sorting in HepG2cells. Our data provide for the first time a proteomic profiling of the effects of C14:0 in human hepatoma cells and contribute to the elucidation of molecular mechanisms through which this fatty acid may cause adverse health effects.
BIOLOGICAL SIGNIFICANCE: Myristic acid is correlated with an increase in plasma cholesterol and mortality due to cardiovascular diseases. This study is the first example of an integration of proteomic and secretomic analysis of HepG2 cells to investigate the specific properties and functional roles of myristic acid on hepatic cells. Our analyses will lead to a better understanding of the myristic acid induced effects and can elicit new diagnostic and treatment strategies based on altered proteins.

Keywords:  HepG2 cells; Myristic acid; Primary murine hepatocytes; Proteome; Secretome

DOI:  https://doi.org/10.1016/j.jprot.2018.04.008
Adv Clin Chem. 2018 ;pii: S0065-2423(18)30005-2. [Epub ahead of print]85 91-113

Proteomics for Biomarker Identification and Clinical Application in Kidney Disease.

Lin Chen, Wei Su, Hua Chen, Dan-Qian Chen, Ming Wang, Yan Guo, Ying-Yong Zhao.

  Treatment effectiveness for kidney disease is limited by lack of accuracy, sensitivity, specificity of diagnostic, prognostic, and therapeutic biomarkers. The gold standard test renal biopsy along with serum creatinine and proteinuria is often necessary to establish a diagnosis, particularly in glomerular disease. Proteomics has become a powerful tool for novel biomarker discovery in kidney disease. Novel proteomics offer earlier and more accurate diagnosis of renal pathology than possible with traditional biomarkers such as serum creatinine and urine protein. In addition, proteomic biomarkers could also be useful to choose the most suitable therapeutic targets. This review focuses on the current status of proteomic biomarkers from animal models (5/6 nephrectomy, unilateral ureteral obstruction, and diabetic nephropathy) and human studies (chronic kidney disease, glomerular diseases, transplantation, dialysis, acute and drug-induced kidney injury) to assess relevant findings and clinical usefulness. Current issues and problems related to the discovery, validation, and clinical application of proteomic biomarkers are discussed. We also describe several proteomic strategies highlighting technologic advancements, specimen selection, data processing and analysis. This review might provide help in future proteomic studies to improve the diagnosis and management of kidney disease.

Keywords:  Biomarker; Chronic kidney disease; Mass spectrometry; Nephropathy; Patients; Proteomics

DOI:  https://doi.org/10.1016/bs.acc.2018.02.005
Proteomics. 2018 Apr 14. e1700427

Analysis of Human Nuclear Protein Complexes by Quantitative Mass Spectrometry Profiling.

Katelyn E Connelly, Victoria Hedrick, Tiago Jose Paschoal Sobreira, Emily C Dykhuizen, Uma K Aryal.

  Analysis of protein complexes provides insights into how the ensemble of expressed proteome is organized into functional units. While there have been advances in techniques for proteome-wide profiling of cytoplasmic protein complexes, information about human nuclear protein complexes are very limited. To close this gap, we combined native size exclusion chromatography (SEC) with label-free quantitative mass spectrometry profiling to characterize hundreds of nuclear protein complexes isolated from human glioblastoma multiforme (GBM) T98G cells. We identified 1794 proteins that overlapped between two biological replicates of which 1244 proteins were characterized as existing within stably associated putative complexes. Co-IP experiments confirmed the interaction of PARP1 with Ku70/Ku80 proteins and HDAC1 and CHD4. HDAC1/2 also co-migrated with various SIN3A and NuRD components in SEC fractionation including SIN3A, SAP30, RBBP4, RBBP7, and NCOR1. Co-elution of HDAC1/2/3 with both the KDM1A and RCOR1 further confirmed that these proteins are integral components of human deacetylase complexes. Our approach also demonstrated the ability to identify potential moonlighting complexes and novel complexes containing uncharacterized proteins. Overall, the results demonstrated the utility of SEC fractionation and LC-MS analysis for system-wide profiling of proteins to predict the existence of distinct forms of nuclear protein complexes. This article is protected by copyright. All rights reserved.

Keywords:  glioblastoma multiforme; label free quantitation; mass spectrometry; protein complex; size exclusion chromatography

DOI:  https://doi.org/10.1002/pmic.201700427
Clin Biochem. 2018 Apr 11. pii: S0009-9120(17)31256-0. [Epub ahead of print]

Comparative proteomic analysis of human malignant ascitic fluids for the development of gastric cancer biomarkers.

Jonghwa Jin, Minsoo Son, Hyeyoon Kim, Hyeyeon Kim, Seong-Ho Kong, Hark Kyun Kim, Youngsoo Kim, Dohyun Han.

   OBJECTIVES: Malignant ascites is a sign of peritoneal seeding, which is one of the most frequent forms of incurable distant metastasis. Because the development of malignant ascites is associated with an extremely poor prognosis, determining whether it resulted from peritoneal seeding has critical clinical implications in diagnosis, choice of treatment, and active surveillance. At present, the molecular characterizations of malignant ascites are especially limited in case of gastric cancer. We aimed to identify malignant ascites-specific proteins that may contribute to the development of alternative methods for diagnosis and therapeutic monitoring and also increase our understanding of the pathophysiology of peritoneal seeding.
DESIGN & METHODS: First, comprehensive proteomic strategies were employed to construct an in-depth proteome of ascitic fluids. Label-free quantitative proteomic analysis was subsequently performed to identify candidates that can differentiate between malignant ascitic fluilds of gastric cancer patients from benign ascitic fluids. Finally, two candidate proteins were verified by ELISA in 84 samples with gastric cancer or liver cirrhosis.
RESULTS: Comprehensive proteome profiling resulted in the identification of 5347 ascites proteins. Using label-free quantification, we identified 299 proteins that were differentially expressed in ascitic fluids between liver cirrhosis and stage IV gastric cancer patients. In addition, we identified 645 proteins that were significantly expressed in ascitic fluids between liver cirrhosis and gastric cancer patients with peritoneal seeding. Finally, Gastriscin and Periostin that can distinguish malignant ascites from benign ascites were verified by ELISA.
CONCLUSIONS: This study identified and verified protein markers that can distinguish malignant ascites with or without peritoneal seeding from benign ascites. Consequently, our results could be a significant resource for gastric cancer research and biomarker discovery in the diagnosis of malignant ascites.

Keywords:  Gastric cancer; Label-free quantification; Malignant ascites; Peritoneal seeding; Quantitative proteomics

DOI:  https://doi.org/10.1016/j.clinbiochem.2018.04.003
J Proteomics. 2018 Apr 11. pii: S1874-3919(18)30172-6. [Epub ahead of print]

Putative salivary biomarkers useful to differentiate patients with fibromyalgia.

Federica Ciregia, Camillo Giacomelli, Laura Giusti, Claudia Boldrini, Isabella Piga, Pasquale Pepe, Arianna Consensi, Sara Gori, Antonio Lucacchini, Maria R Mazzoni, Laura Bazzichi.

  Fibromyalgia (FM) is a chronic pain disorder characterized by widespread pain and associated with unspecific symptoms. So far, no laboratory tests have been validated. The aim of the present study was to investigate the presence in saliva of potential diagnostic and/or prognostic biomarkers which could be useful for the management of FM patients. Specifically, the salivary profile of FM patients was compared with those of healthy subjects, subjects suffering migraine (model of non-inflammatory chronic pain), and patients affected by rheumatoid arthritis (model of inflammatory chronic pain). For proteomics analysis 2-DE and SELDI-TOF-MS were applied. From 2-DE serotransferrin and alpha-enolase were found differentially expressed in FM. Hence, their expression was validated by ELISA together with phosphoglycerate-mutase-I and transaldolase, which were found in a previous work. Moreover, ROC curve was calculated by comparing FM patients versus control subjects (healthy plus migraine) to investigate the discriminative power of biomarkers. The best performance was obtained by combining alpha-enolase, phosphoglycerate-mutase-I and serotransferrin. On the other hand, none of the candidate proteins showed a statistical correlation with clinical features. Finally, preliminary SELDI analysis highlighted two peaks whose identification need to be validated. Overall, these results could be useful in supporting the clinical diagnosis of FM.
SIGNIFICANCE: FM is one of the most common chronic pain condition which is associated with significant disability. The fibromyalgic pain is a peculiar characteristic of this disease and FM patients suffer from reduced quality of life, daily functioning and productivity. Considering the deep complexity of FM, the discovery of more objective markers is crucial for supporting clinical diagnosis. Therefore, the aim of the present study was the selection of biomarkers effectively associated with fibromyalgic pain which will enable clinicians to achieve an unambiguous diagnosis, and to improve approaches to patients' management. We defined a panel of 3 salivary proteins which could be one of the criteria to be taken into account. Consequently, the identification of disease salivary biomarkers could be helpful in detecting FM clusters and targeted treatment. Actually, our future perspective foresees to develop a simple, rapid and not invasive point-of-care testing which will be of use during the diagnostic process. In addition, the present results can offer a clue for shedding light upon the complex entity of such a disease like FM.

Keywords:  Fibromyalgia; biomarkers; proteomics; two-dimensional electrophoresis; whole saliva

DOI:  https://doi.org/10.1016/j.jprot.2018.04.012
Transfusion. 2018 Apr 15.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of 36 blood group alleles among 396 Thai samples reveals region-specific variants.

Philaiphon Jongruamklang, Christoph Gassner, Stefan Meyer, Aksarakorn Kummasook, Marion Darlison, Chayanun Boonlum, Surin Chanta, Beat M Frey, Martin L Olsson, Jill R Storry.

BACKGROUND: Blood group phenotype variation has been attributed to potential resistance to pathogen invasion. Variation was mapped in blood donors from Lampang (northern region) and Saraburi (central region), Thailand, where malaria is endemic. The previously unknown blood group allele profiles were characterized and the data were correlated with phenotypes. The high incidence of the Vel-negative phenotype previously reported in Thais was investigated.
STUDY DESIGN AND METHODS: DNA from 396 blood donors was analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Outliers were investigated by serology and DNA sequencing. Allele discrimination assays for SMIM1 rs1175550A/G and ACKR1 rs118062001C/T were performed and correlated with antigen expression.
RESULTS: All samples were phenotyped for Rh, MNS, and K. Genotyping/phenotyping for RhD, K, and S/s showed 100% concordance. Investigation of three RHCE outliers revealed an e-variant antigen encoded by RHCE*02.22. Screening for rs147357308 (RHCE c.667T) revealed a frequency of 3.3%. MN typing discrepancies in 41 samples revealed glycophorin variants, of which 40 of 41 were due to Mia . Nine samples (2.3%) were heterozygous for FY*01W.01 (c.265C > T), and six samples (1.5%) were heterozygous for JK*02N.01. All samples were wildtype SMIM1 homozygotes with 97% homozygosity for rs1175550A.
CONCLUSIONS: Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry is an efficient method for rapid routine genotyping and investigation of outliers identified novel variation among our samples. The expected high prevalence of the Mi(a+) phenotype was observed from both regions. Of potential clinical relevance in a region where transfusion-dependent thalassemia is common, we identified two RHCE*02 alleles known to encode an e-variant antigen.

DOI: https://doi.org/10.1111/trf.14624
Food Chem. 2018 Aug 30. pii: S0308-8146(18)30521-1. [Epub ahead of print]258 359-365

Identification of pyruvate kinase as a novel allergen in whiteleg shrimp (Litopenaeus vannamei) by specific-IgE present in patients with shrimp allergy.

Chih-Hung Lee, Chia-Ching Wu, Yu-Chang Tyan, Wei-Tai Yu, Edward S Huang, Hsu-Sheng Yu.

  Food allergy is one of the most important health issues worldwide. In Taiwan, current literature suggests shrimps and crabs are the most common causes of food allergy, and are frequently associated with acute allergic reactions such as urticaria, atopic dermatitis, and asthma. However, knowledge regarding the shrimp allergens remains limited. Thus, there is an urgent need to establish comprehensive information for elucidating underlying triggers for food allergy. In this study, whiteleg shrimp (Litopenaeus vannamei) was used to evaluate the IgE-binding properties of various shrimp proteins to 7 allergic patients' sera by western blot. A 63 kDa protein was found in raw and cooked shrimp bound to specific-IgEs in 7 and 4 patients' sera, respectively. This protein was further identified as pyruvate kinase based on the proteomic mass spectrometry. This study identifies an important shrimp allergen unique to Taiwan and further testing and prevention measures might be implemented in the allergen analysis.

Keywords:  Allergen; Food allergy; Pyruvate kinase; Shrimp; Specific-IgE

DOI:  https://doi.org/10.1016/j.foodchem.2018.03.088
Curr Opin Biotechnol. 2018 Apr 12. pii: S0958-1669(18)30011-9. [Epub ahead of print]52 109-115

Advancing systems immunology through data-driven statistical analysis.

Linda E Fong, Andrés R Muñoz-Rojas, Kathryn Miller-Jensen.

Systems biology provides an effective approach to decipher, predict, and ultimately manipulate the complex and inter-connected networks that regulate the immune system. Advances in high-throughput, multiplexed experimental techniques have increased the availability of proteomic and transcriptomic immunological datasets, and as a result, have also accelerated the development of new data-driven computational algorithms to extract biological insight from these data. This review highlights how data-driven statistical models have been used to characterize immune cell subsets and their functions, to map the signaling and intercellular networks that regulate immune responses, and to connect immune cell states to disease outcomes to generate hypotheses for novel therapeutic strategies. We focus on recent advances in evaluating immune cell responses following viral infection and in the tumor microenvironment, which hold promise for improving vaccines, antiviral and cancer immunotherapy.

DOI: https://doi.org/10.1016/j.copbio.2018.03.009
Arch Biochem Biophys. 2018 Apr 12. pii: S0003-9861(18)30104-8. [Epub ahead of print]647 10-32

Proteomic investigating the cooperative lethal effect of EGFR and MDM2 inhibitors on ovarian carcinoma.

Shing-Jyh Chang, En-Chi Liao, Hsin-Yueh Yeo, Wen-Hong Kuo, Hsin-Yi Chen, Yi-Ting Tsai, Yu-Shan Wei, Ying-Jen Chen, Yi-Shiuan Wang, Ji-Min Li, Chuan-Chi Shih, Chia-Hao Chan, Hsiu-Chuan Chou, Yung-Jen Chuang, Hong-Lin Chan.

  With the concept of precision medicine, combining multiple molecular-targeting therapies has brought new approaches to current cancer treatments. Malfunction of the tumor suppressor protein, p53 is a universal hallmark in human cancers. Under normal conditions, p53 is degraded through an ubiquitin-proteosome pathway regulated by its negative regulator, MDM2. In contrast, cellular stress such as DNA damage will activate p53 to carry out DNA repair, cell cycle arrest, and apoptosis. In this study, we focused on ovarian carcinoma with high EGFR and MDM2 overexpression rate. We assessed the effects of combined inhibition by MDM2 (JNJ-26854165) and EGFR (gefitinib) inhibitors on various ovarian cell lines to determine the importance of these two molecular targets on cell proliferation. We then used a proteomic strategy to investigate the relationship between MDM2 and EGFR inhibition to explore the underlying mechanisms of how their combined signaling blockades work together to exert cooperative inhibition. Our results demonstrated that all four cell lines were sensitive to both individual and combined, MDM2 and EGFR inhibition. The proteomic analysis also showed that gefitinib/JNJ-treated CAOV3 cells exhibited downregulation of proteins involved in nucleotide biosynthesis such as nucleoside diphosphate kinase B (NME2). In conclusion, our study showed that the combined treatment with JNJ and gefitinib exerted synergistic inhibition on cell proliferation, thereby suggesting the potential application of combining MDM2 inhibitors with EGFR inhibitors for enhancing efficacy in ovarian cancer treatment.

Keywords:  DIGE; EGFR; MDM2; Proteomics

DOI:  https://doi.org/10.1016/j.abb.2018.04.004
Biochim Biophys Acta. 2018 Apr 12. pii: S0167-4889(18)30062-4. [Epub ahead of print]1865(7): 959-969

Role of MSC-derived galectin 3 in the AML microenvironment.

Peter P Ruvolo, Vivian R Ruvolo, Jared K Burks, YiHua Qiu, Rui-Yu Wang, Elizabeth J Shpall, Leonardo Mirandola, Numsen Hail, Zhihong Zeng, Teresa McQueen, Naval Daver, Sean M Post, Maurizio Chiriva-Internati, Steven M Kornblau, Michael Andreeff.

  In acute myeloid leukemia (AML), high Galectin 3 (LGALS3) expression is associated with poor prognosis. The role of LGALS3 derived from mesenchymal stromal cells (MSC) in the AML microenvironment is unclear; however, we have recently found high LGALS3 expression in MSC derived from AML patients is associated with relapse. In this study, we used reverse phase protein analysis (RPPA) to correlate LGALS3 expression in AML MSC with 119 other proteins including variants of these proteins such as phosphorylated forms or cleaved forms to identify biologically relevant pathways. RPPA revealed that LGALS3 protein was positively correlated with expression of thirteen proteins including MYC, phosphorylated beta-Catenin (p-CTNNB1), and AKT2 and negatively correlated with expression of six proteins including integrin beta 3 (ITGB3). String analysis revealed that proteins positively correlated with LGALS3 showed strong interconnectivity. Consistent with the RPPA results, LGALS3 suppression by shRNA in MSC resulted in decreased MYC and AKT expression while ITGB3 was induced. In co-culture, the ability of AML cell to adhere to MSC LGALS3 shRNA transductants was reduced compared to AML cell adhesion to MSC control shRNA transductants. Finally, use of novel specific LGALS3 inhibitor CBP.001 in co-culture of AML cells with MSC reduced viable leukemia cell populations with induced apoptosis and augmented the chemotherapeutic effect of AraC. In summary, the current study demonstrates that MSC-derived LGALS3 may be critical for important biological pathways for MSC homeostasis and for regulating AML cell localization and survival in the leukemia microenvironmental niche.

Keywords:  Acute myeloid leukemia; Galectin 3; Integrin beta 3; MYC; Reverse phase protein analysis; Tumor microenvironment

DOI:  https://doi.org/10.1016/j.bbamcr.2018.04.005
Phytomedicine. 2018 Mar 15. pii: S0944-7113(18)30052-7. [Epub ahead of print]42 34-42

Alisma orientale extract exerts the reversing cholestasis effect by activation of farnesoid X receptor.

Xiao-Kui Huo, Jing Liu, Zhen-Long Yu, Yi-Fei Wang, Chao Wang, Xiang-Ge Tian, Jing Ning, Lei Feng, Cheng-Peng Sun, Bao-Jing Zhang, Xiao-Chi Ma.

   BACKGROUND: Cholestasis is a clinical syndrome of liver damage that is caused by accumulation of bile acids in the liver and systemic circulation. Farnesoid X receptor (FXR) can regulate synthesis, metabolism, and excretion of bile acids. The rhizomes of Alisma orientale is a well-known traditional Chinese medicine to treat edema, obesity, gonorrhea, leukorrhea, diarrhea, hyperlipidemia, and diabetes in China.
HYPOTHESIS/PURPOSE: We hypothesized Alisma orientale extract (AOE) to exert hepatoprotective effect against α-naphthylisothiocyanate (ANIT) induced cholestasis in rat. We aimed to investigate the mechanism of AOE.
STUDY DESIGN: Male Sprague Dawley rats with intrahepatic cholestasis induced by ANIT were treated with AOE (150, 300, or 600 mg/kg). Rats receiving vehicle (0.5% CMC-Na) served as control.
METHODS: 48 h after ANIT administration, rats were sacrificed. Blood was collected to obtain serum and livers were removed for histopathology and protein preparation. Biochemical indicators in serum were determined using commercial kits and triterpenoids were determined by liquid chromatography tandem Qtrap mass spectrometry. Proteomics was analyzed by liquid chromatography tandem ion-trap mass spectrometry. The differently expressed proteins were analyzed via the network database and verified by western blotting. The interaction between triterpenoids and FXR were evaluated by luciferase assay and molecular docking.
RESULTS: AOE treatment significantly decreased the serum AST, ALT, TBIL, and intrahepatic TBA and improved the liver pathologic change induced by ANIT. Proteomics analysis indicated that AOE regulated proteins related to bile acid homeostasis via activating farnesoid X receptor (FXR) signaling pathway. Luciferase assay and molecular docking results indicated that triterpenoids could activate FXR, which resulting in ameliorative accumulation of bile acids in the liver by increase of metabolism and transportation for bile acids, and decrease of synthesis for bile acids.
CONCLUSION: AOE protected against rat liver injury and cholestasis induced by ANIT by activation of farnesoid X receptor, suggesting that A. orientale could be regarded as a potential hepatoprotective drug.

Keywords:  Alisma orientale; Cholestasis; Farnesoid X receptor; Proteomics

DOI:  https://doi.org/10.1016/j.phymed.2018.03.017
Curr Opin Immunol. 2018 Apr 11. pii: S0952-7915(18)30029-3. [Epub ahead of print]51 187-196

Mass cytometry: a powerful tool for dissecting the immune landscape.

Yannick Simoni, Melissa Hui Yen Chng, Shamin Li, Michael Fehlings, Evan W Newell.

Advancement in methodologies for single cell analysis has historically been a major driver of progress in immunology. Currently, high dimensional flow cytometry, mass cytometry and various forms of single cell sequencing-based analysis methods are being widely adopted to expose the staggering heterogeneity of immune cells in many contexts. Here, we focus on mass cytometry, a form of flow cytometry that allows for simultaneous interrogation of more than 40 different marker molecules, including cytokines and transcription factors, without the need for spectral compensation. We argue that mass cytometry occupies an important niche within the landscape of single-cell analysis platforms that enables the efficient and in-depth study of diverse immune cell subsets with an ability to zoom-in on myeloid and lymphoid compartments in various tissues in health and disease. We further discuss the unique features of mass cytometry that are favorable for combining multiplex peptide-MHC multimer technology and phenotypic characterization of antigen specific T cells. By referring to recent studies revealing the complexities of tumor immune infiltrates, we highlight the particular importance of this technology for studying cancer in the context of cancer immunotherapy. Finally, we provide thoughts on current technical limitations and how we imagine these being overcome.

DOI: https://doi.org/10.1016/j.coi.2018.03.023
Biochim Biophys Acta. 2018 Apr 12. pii: S1570-9639(18)30047-5. [Epub ahead of print]1866(5-6): 712-721

Proteomic approach and expression analysis revealed the differential expression of predicted leptospiral proteases capable of ECM degradation.

Gunasekaran Dhandapani, Thoduvayil Sikha, Sneha M Pinto, M Kiran Kumar, Krishna Patel, Manish Kumar, Vikram Kumar, Jebasingh Tennyson, P K Satheeshkumar, Harsha Gowda, T S Keshava Prasad, M G Madanan.

  Leptospira, the causative agent of leptospirosis is known to have many proteases with potential to degrade extracellular matrix. However, a multipronged approach to identify, classify, characterize and elucidate their role has not been attempted. Our proteomic approach using high-resolution LC-MS/MS analysis of Triton X-114 fractions of Leptospira interrogans resulted in the identification of 104 proteases out of 130 proteases predicted by MEROPS. In Leptospira approximately 3.5% of the genome complements for proteases, which include catalytic types of metallo-, serine-, cysteine-, aspartic-, threonine- and asparagine- peptidases. Comparison of proteases from different serovars revealed that M04, M09B, M14A, M75, M28A, A01 and U73 protease families are exclusively present in pathogenic form. The M23 and S33 protease families are represented with >14 members in Leptospira. The differential expression under physiological temperature (37 °C) and osmolarity (300 mOsM) showed that proteases belonging to the catalytic type of Metallo-peptidases are upregulated significantly in pathogenic conditions. In silico prediction and characterization of the proteases revealed that several proteases are membrane anchored and secretory, classical as well as non-classical system. The study demonstrates the diversity and complexity of proteases, while maintaining conservation across the serovars in Leptospira and their differential expression under pathogenic conditions.

Keywords:  Leptospira; Metalloprotease; Pathogenesis; Protease; Proteomics; Triton X-114

DOI:  https://doi.org/10.1016/j.bbapap.2018.04.006
Methods. 2018 Apr 12. pii: S1046-2023(17)30368-7. [Epub ahead of print]

Tumor classification with MALDI-MSI data of tissue microarrays: a case study.

Nadine E Mascini, Jannis Teunissen, Rob Noorlag, Stefan M Willems, Ron M A Heeren.

  With mass spectrometry imaging (MSI) on tissue microarrays (TMAs) a large number of biomolecules can be studied for many patients at the same time, making it an attractive tool for biomarker discovery. Here we investigate whether lymph node metastasis can be predicted from MALDI-MSI data. Measurements are performed on TMAs and then filtered based on spectral intensity and the percentage of tumor cells, after which the resulting data for 122 patients is further preprocessed. We assume differences between patients with and without metastasis are expressed in a limited number of features. Two univariate feature selection methods are applied to reduce the dimensionality of the MALDI-MSI data. The selected features are then used in combination with three classifiers. The best classification scores are obtained with a decision tree classifier, which classifies about 72% of patients correctly. Almost all the predictive power comes from a single peak (m/z 718.4). The sensitivity of our classification approach, which can be generically used to search for biomarkers, is investigated using artificially modified data.

Keywords:  Data analytics; FFPE tissue; Head and neck cancer; MALDI; Mass spectrometry imaging; Tissue microarray

DOI:  https://doi.org/10.1016/j.ymeth.2018.04.004
Mol Metab. 2018 Mar 09. pii: S2212-8778(18)30071-1. [Epub ahead of print]

Epoxygenase inactivation exacerbates diet and aging-associated metabolic dysfunction resulting from impaired adipogenesis.

Antoni Olona, Ximena Terra, Jeong-Hun Ko, Carme Grau-Bové, Montserrat Pinent, Anna Ardevol, Ana Garcia Diaz, Aida Moreno-Moral, Matthew Edin, David Bishop-Bailey, Darryl C Zeldin, Timothy J Aitman, Enrico Petretto, Mayte Blay, Jacques Behmoaras.

   OBJECTIVE: When molecular drivers of healthy adipogenesis are perturbed, this can cause hepatic steatosis. The role of arachidonic acid (AA) and its downstream enzymatic cascades, such as cyclooxygenase, in adipogenesis is well established. The exact contribution of the P450 epoxygenase pathway, however, remains to be established. Enzymes belonging to this pathway are mainly encoded by the CYP2J locus which shows extensive allelic expansion in mice. Here we aimed to establish the role of endogenous epoxygenase during adipogenesis under homeostatic and metabolic stress conditions.
METHODS: We took advantage of the simpler genetic architecture of the Cyp2j locus in the rat and used a Cyp2j4 (orthologue of human CYP2J2) knockout rat in two models of metabolic dysfunction: physiological aging and cafeteria diet (CAF). The phenotyping of Cyp2j4-/- rats under CAF was integrated with proteomics (LC-MS/MS) and lipidomics (LC-MS) analyses in the liver and the adipose tissue.
RESULTS: We report that Cyp2j4 deletion causes adipocyte dysfunction under metabolic challenges. This is characterized by (i) down-regulation of white adipose tissue (WAT) PPARγ and C/EBPα, (ii) adipocyte hypertrophy, (iii) extracellular matrix remodeling, and (iv) alternative usage of AA pathway. Specifically, in Cyp2j4-/- rats treated with a cafeteria diet, the dysfunctional adipogenesis is accompanied by exacerbated weight gain, hepatic lipid accumulation, and dysregulated gluconeogenesis.
CONCLUSION: These results suggest that AA epoxygenases are essential regulators of healthy adipogenesis. Our results uncover their synergistic role in fine-tuning AA pathway in obesity-mediated hepatic steatosis.

Keywords:  Adipogenesis; Aging; Arachidonic acid; Cafeteria diet; Cytochrome P450 2j4; Steatosis

DOI:  https://doi.org/10.1016/j.molmet.2018.03.003
J Proteomics. 2018 Apr 12. pii: S1874-3919(18)30173-8. [Epub ahead of print]

Systematic analysis of the cerebrospinal fluid proteome of fibromyalgia patients.

Payam Emami Khoonsari, Sravani Musunri, Stephanie Herman, Camilla I Svensson, Lars Tanum, Torsten Gordh, Kim Kultima.

Fibromyalgia (FM) is a syndrome characterized by widespread muscular pain, fatigue and functional symptoms, which is known to be difficult to diagnose as the various symptoms overlap with many other conditions. Currently, there are no biomarkers for FM, and the diagnosis is made subjectively by the clinicians. We have performed shotgun proteomics on cerebrospinal fluid (CSF) from FM patients and non-pain controls to find potential biomarker candidates for this syndrome. Based on our multivariate and univariate analyses, we found that the relative differences in the CSF proteome between FM patients and controls were moderate. Four proteins, important to discriminate FM patients from non-pain controls, were found: Apolipoprotein C-III, Galectin-3-binding protein, Malate dehydrogenase cytoplasmic and the neuropeptide precursor protein ProSAAS. These proteins are involved in lipoprotein lipase (LPL) activity, inflammatory signaling, energy metabolism and neuropeptide signaling.
SIGNIFICANCE: Fibromyalgia is present in as much as 2% of the population, causing pain, stiffness, and tenderness of the muscles. Upon accurate diagnostic, nonpharmacological and pharmacological therapies can be used to alleviate pain and manage other symptoms. However, lack of objective, universal applicable diagnostic criteria as well as vague and diffused symptoms, have made diagnosis difficult. In this context, our findings can shed light on potential value of CSF proteome for objectively diagnosing FM.

DOI: https://doi.org/10.1016/j.jprot.2018.04.014
Int J Biochem Cell Biol. 2018 Apr 11. pii: S1357-2725(18)30084-0. [Epub ahead of print]

Urinary Extracellular Vesicle Biomarkers in Urological Cancers: From Discovery Towards Clinical Implementation.

Bert Dhondt, Jan Van Deun, Silke Vermaerke, Ario de Marco, Nicolaas Lumen, Olivier De Wever, An Hendrix.

  Urine contains cellular elements, biochemicals, and proteins derived from glomerular filtration of plasma, renal tubule excretion, and urogenital tract secretions that reflect an individual's metabolic and pathophysiologic state. Despite intensive research into the discovery of urinary biomarkers to facilitate early diagnosis, accurate prognosis and prediction of therapy response in urological cancers, none of these markers has reached widespread use. Their implementation into daily clinical practice is hampered by a substantial degree of heterogeneity in performance characteristics and uncertainty about reliability, clinical utility and cost-effectiveness, in addition to several technical limitations. Extracellular vesicles (EV) have raised interest as a potential source of biomarker discovery because of their role in intercellular communication and the resemblance of their molecular content to that of the releasing cells. We review currently used urinary biomarkers in the clinic and attempts that have been made to identify EV-derived biomarkers for urological cancers. In addition, we discuss technical and methodological considerations towards their clinical implementation.

Keywords:  Biomarkers; Cancer; Exosomes; Extracellular Vesicles; Omics; Urine

DOI:  https://doi.org/10.1016/j.biocel.2018.04.009
Cell Syst. 2018 Apr 10. pii: S2405-4712(18)30109-1. [Epub ahead of print]

A Library of Phosphoproteomic and Chromatin Signatures for Characterizing Cellular Responses to Drug Perturbations.

Lev Litichevskiy, Ryan Peckner, Jennifer G Abelin, Jacob K Asiedu, Amanda L Creech, John F Davis, Desiree Davison, Caitlin M Dunning, Jarrett D Egertson, Shawn Egri, Joshua Gould, Tak Ko, Sarah A Johnson, David L Lahr, Daniel Lam, Zihan Liu, Nicholas J Lyons, Xiaodong Lu, Brendan X MacLean, Alison E Mungenast, Adam Officer, Ted E Natoli, Malvina Papanastasiou, Jinal Patel, Vagisha Sharma, Courtney Toder, Andrew A Tubelli, Jennie Z Young, Steven A Carr, Todd R Golub, Aravind Subramanian, Michael J MacCoss, Li-Huei Tsai, Jacob D Jaffe.

  Although the value of proteomics has been demonstrated, cost and scale are typically prohibitive, and gene expression profiling remains dominant for characterizing cellular responses to perturbations. However, high-throughput sentinel assays provide an opportunity for proteomics to contribute at a meaningful scale. We present a systematic library resource (90 drugs × 6 cell lines) of proteomic signatures that measure changes in the reduced-representation phosphoproteome (P100) and changes in epigenetic marks on histones (GCP). A majority of these drugs elicited reproducible signatures, but notable cell line- and assay-specific differences were observed. Using the "connectivity" framework, we compared signatures across cell types and integrated data across assays, including a transcriptional assay (L1000). Consistent connectivity among cell types revealed cellular responses that transcended lineage, and consistent connectivity among assays revealed unexpected associations between drugs. We further leveraged the resource against public data to formulate hypotheses for treatment of multiple myeloma and acute lymphocytic leukemia. This resource is publicly available at https://clue.io/proteomics.

Keywords:  GCP; L1000; LINCS project; P100; drug discovery; epigenetics; mass spectrometry; mechanism of action; proteomics; signaling

DOI:  https://doi.org/10.1016/j.cels.2018.03.012
Andrologia. 2018 Apr 15. e13015

Proteomic analysis of sperm proteins in infertile men with high levels of reactive oxygen species.

A Ayaz, A Agarwal, R Sharma, N Kothandaraman, Z Cakar, S Sikka.

  Oxidative stress is a significant risk factor for male infertility. A pro-oxidant testicular environment may alter the expression profile of functional sperm proteins and result in poor sperm quality. Patients and donors were divided into ROS (-) and ROS (+) groups. Using computational studies, and data mining of available literature on spermatozoa, oxidative stress and proteomics, we identified three core regulatory proteins angiotensin-converting enzyme (ACE), heat-shock protein (Hsp70) family A member 2 (HSPA2) and ribosomal protein subunit 27A (RPS27A) and seven interlink proteins NOS2, SUMO2, UBL4A, FBXO25, MAP3K3, APP and UBC. HSPA2 was validated by Western Blot, while the localisation of ACE, RPS27A, MAP3K3 and APP was identified by immunocytochemistry. The obtained results showed that HSPA2 was 1.2 (ROS+) and 2.1 (ROS-) fold downregulated in spermatozoa from patients with high levels of reactive oxygen species (ROS). ACE and APP were localised in the post-acrosomal region of spermatozoa, whereas RPS27A and MAP3K3 were localised either in the tail or sperm neck area. Our data show that these proteins may play a role in ROS-induced male infertility.

Keywords:  male infertility; network; oxidative stress; reactive oxygen species; spermatozoa

DOI:  https://doi.org/10.1111/and.13015
J Ethnopharmacol. 2018 Apr 11. pii: S0378-8741(17)33305-6. [Epub ahead of print]

Investigation of the therapy targets of Yi-Qi-Yang-Yin-Hua-Tan-Qu-Yu recipe on type 2 diabetes by serum proteome labeled with iTRAQ.

Jing Zhao, Ming Xie, Jin-Na Liu, Bang-Zhong Wang.

  Ethnopharmacology relevance Based on basic theories of Chinese medicine, Yi-Qi-Yang-Yin-Hua-Tan-Qu-Yu (YQYYHTQY) recipe was constituted by eleven kinds of Chinese herbs and effective in treatment of type 2 diabetes (T2DM). But the therapy target was unclear.
OBJECTIVE: In this study, we used the serum proteome labeled by iTRAQ to find therapy target of YQYYHTQY recipe on T2DM.
MATERIALS AND METHODS: The rat model was induced by high-fat diet (HFD) and streptozotocin (STZ, 30mg/kg). Drugs were administered to rats once daily for 14 days. Related laboratory parameters were observed. Serum proteome were compared between T2DM and YQYYHTQY group using the iTRAQ labeling quantitative proteomics technique. Functional differential proteins were analysis by STRING software. Target proteins were confirmed by ELISA kits.
RESULTS: Hyperglycemia, hyperinsulinemia, insulin resistance, decrease of glucose transporter, depilation, less activity, flock together, depression, ecchymosis of tongue and tail appearance, the typical diabetic patients "a little more than three" symptoms, as well as the decrease of grip strength, serum cyclic adenosine monophosphate (cAMP)/ cyclic guanosine monophosphate (cGMP) ratio, serum high density lipoprotein-cholesterol (HDL-C) and the increase of serum triglyceride (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), thromboxane B2 (TXB2)/ 6-keto prostaglandin F1α (6-keto PGF1α) ratio, endothelin-1 (ET-1) levels were found in T2DM group. After drugs treatment, all the above indexes almost were improved in different degrees and effect of YQYYHTQY recipe was superior to pioglitazone hydrochloride. In addition, there were 23 differential proteins, 5 up-regulated and 18 down-regulated proteins. Of them, there were 4 proteins related with diabetes, blood and behavior. Cell division control protein 42 homolog (CDC42) and Ras homolog gene family member A (RhoA) were the therapy targets of YQYYHTQY recipe on T2DM.
CONCLUSIONS: YQYYHTQY recipe showed therapy effect on T2DM. CDC42 and RhoA proteins were the therapy targets of YQYYHTQY recipe.

Keywords:  CDC42; Chinese herb formula; RhoA; serum proteome; type 2 diabetes

DOI:  https://doi.org/10.1016/j.jep.2018.03.027