Proteome Analysis Of Tissues Research Articles

Integrative proteomics and immunochemistry analysis of the factors in the necrosis and repair in acetaminophen-induced acute liver injury in mice.

Acetaminophen (APAP) overdose-induced acute liver injury (AILI) is a significant clinical problem worldwide, the hepatotoxicity mechanisms are well elucidated, but the factors involved in the necrosis and repair still remain to be investigated. APAP was injected intraperitoneally in male Institute of Cancer Research (ICR) mice. Quantitative proteome analysis of liver tissues was performed by 2-nitrobenzenesulfenyl tagging, two-dimensional-nano high-performance liquid chromatography separation, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Diffrenetial proteins were verified by the immunochemistry method. 36 and 44 differentially expressed proteins were identified, respectively, at 24 hr after APAP (200 or 300 mg·kg -1 ) administration. The decrease in the mitochondrial protective proteins Prdx6, Prdx3, and Aldh2 accounted for the accumulation of excessive reactive oxygen species (ROS) and aldehydes, impairing mitochondria structure and function. The Gzmf combined with Bax and Apaf-1 jointly contributed to the necrosis. The blockage of Stat3 activation led to the overexpression of unphosphorylated Stat3 and the overproduction of Bax. The overexpression of unphosphorylated Stat3 represented necrosis; the alternation from Stat3 to p-Stat3 in necrotic regions represented hepatocytes from death to renewal. The high expressions of P4hα1, Ncam, α-SMA, and Cygb were involved in the liver repair, they were not only the markers of activated HSC but also represented an intermediate stage of hepatocytes from damage or necrosis to renewal. Our data provided a comprehensive report on the profile and dynamic changes of the liver proteins in AILI; the involvement of Gzmf and the role of Stat3 in necrosis were revealed; and the role of hepatocyte in liver self-repair was well clarified.

Proteomics Analysis of Myocardial Tissues in a Mouse Model of Coronary Microembolization.

Coronary microembolization (CME) is an important clinical problem, and it is related to poor outcome. The specific molecular mechanisms of CME are not fully understood. In the present study, we established a mice model of CME. Isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) technologies identified 249 differentially expressed proteins in the myocardial tissues of CME mice as compared with sham-operated mice. Bioinformatics analysis demonstrated that these differentially expressed proteins were enriched in several energy metabolism or cytoskeleton organization related processes or pathways. Quantitative PCR and Western blotting validation experiments revealed that succinate dehydrogenase (SDHA and SDHB) were upregulated, Rho GDP dissociation inhibitor α (RhoGDIα) and Filamin-A (FLNA) were downregulated significantly in CME mice. These findings indicated that the alternations of the cytoskeleton and energy metabolism pathways play important roles in the pathogenesis of CME, future studies are warranted to verify if targeting these molecules might be useful to alleviate CME injury or not.

Spatially Resolved Proteome Profiling of

Due to sensitivity limitations, global proteome measurements generally require large amounts of biological starting material, which masks heterogeneity within the samples and differential protein expression among constituent cell types. Methods for spatially resolved proteomics are being developed to resolve protein expression for distinct cell types among highly heterogeneous tissues, but have primarily been applied to mammalian systems. Here we evaluate the performance of cell-type-specific proteome analysis of tomato fruit pericarp tissues by a platform integrating laser-capture microdissection (LCM) and a recently developed automated sample preparation system (nanoPOTS, nanodroplet processing in one pot for trace samples). Tomato fruits were cryosectioned prior to LCM and tissues were dissected and captured directly into nanoPOTS chips for processing. Following processing, samples were analyzed by nanoLC-MS/MS. Approximately 1900 unique peptides and 422 proteins were identified on average from ∼0.04 mm2 tissues comprising ∼8-15 parenchyma cells. Spatially resolved proteome analyses were performed using cells of outer epidermis, collenchyma, and parenchyma. Using ≤200 cells, a total of 1,870 protein groups were identified and the various tissues were easily resolved. The results provide spatial and tissue-specific insights into key enzymes and pathways involved in carbohydrate transport and source-sink relationships in tomato fruit. Of note, at the time of fruit ripening studied here, we identified differentially abundant proteins throughout the pericarp related to chlorophyll biogenesis, photosynthesis, and especially transport.

Abstract A058: Integrative prostate cancer tissue proteomics dissects clear and distinct proteomes for human prostate cancer and benign prostatic hyperplasia

Abstract Current approaches in proteomics techniques and MS systems have revived the quest for novel biomarkers in prostate cancer (PCa) boosting the molecular characterization of the disease. To reveal fundamental differences between benign and malignant growth of prostate cells, we combined a new protein extraction procedure that disrupts the crosslinked proteins from the previously formaldehyde-fixed, paraffin-embedded tissue samples with in-depth proteomic analysis (ESI-MS/MS). Human PCa and benign prostatic hyperplasia (BPH) tissue samples were obtained using phase-transfer surfactant-aided extraction/tryptic digestion of formalin-fixed and paraffin-embedded sections mounted on microscope slides. Data analysis was based on label-free spectral counting, identifying with a minimum of two peptides, 1331 and 1239 proteins in PCa and BPH tissue proteomes, respectively. 71 proteins were exclusively present in PCa samples, while 122 proteins where exclusively present in BPH samples. In order to prioritize candidate markers for PCa, we compared protein expression based on normalized spectral counts between tissue samples. We set as cut-offs proteins that were found with a minimum of three peptides within the PCa and BPH proteomes. This filter resulted in the selection of two clusters of 11 and 16 proteins, respectively. The data sets highlighted distinct proteins that were previously studied in the context of prostate cancer progression, including SSBP1, GDF15, NDRG1, C4A, and APOE for PCa and DUSP3, MME, SRI, and DSG1 for BPH, thus providing further confirmation for the robustness of our quantification method. We next subjected our candidate list to bioinformatics analysis (Oncomine). Accordingly, the 5 proteins mentioned for PCa were significantly upregulated (fold change >1.5, P≤0.05) in prostate adenocarcinoma vs. normal prostate gland. Whole-exome analysis (cBioportal) revealed amplification as the most frequent genetic alteration and RNASeq data also confirmed a significant upregulation for these proteins (P≤0.05). Strikingly, proteins associated with BPH were significantly downregulated (fold change >1.5, P≤0.05) across the same comparison and RNA-seq data also confirmed a significant downregulation for these proteins (P≤0.05). This report showcases significant and extensive differences in protein expression patterns between BPH and prostate carcinoma. Proteome analysis of prostate tissues should help to disclose the molecular mechanisms underlying prostate malignant growth, resulting in new sets of biomarkers for diagnostic, prognostic, and therapeutic use. Citation Format: Sofia Lage Vickers, Juan Antonio Bizzotto, Alejandra Paez, Javier Cotignola, Carlos Scorticati, Osvaldo Mazza, Pia Valacco, Geraldine Gueron, Elba Vazquez. Integrative prostate cancer tissue proteomics dissects clear and distinct proteomes for human prostate cancer and benign prostatic hyperplasia [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A058.

Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry.

Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)-10) for multiplexed analysis and relative quantification, and provides 3× higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra- and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two distinct breast cancer subtypes from patient-derived xenograft models to enable assessment of proteome and phosphoproteome depth and quantitative reproducibility. Each plex consisted of ten samples, each being 300 μg of peptide derived from <50 mg of wet-weight tissue. Of the 10,000 proteins quantified per sample, we could distinguish 7,700 human proteins derived from tumor cells and 3100 mouse proteins derived from the surrounding stroma and blood. The maximum deviation across replicates and laboratories was <7%, and the inter-laboratory correlation for TMT ratio-based comparison of the two breast cancer subtypes was r > 0.88. The maximum deviation for the phosphoproteome coverage was <24% across laboratories, with an average of >37,000 quantified phosphosites per sample and differential quantification correlations of r > 0.72. The full procedure, including sample processing and data generation, can be completed within 10 d for ten tissue samples, and 100 samples can be analyzed in ~4 months using a single LC-MS/MS instrument. The high quality, depth, and reproducibility of the data obtained both within and across laboratories should enable new biological insights to be obtained from mass spectrometry-based proteomics analyses of cells and tissues together with proteogenomic data integration.

Infrared laser ablation sampling coupled with data independent high resolution UPLC-IM-MS/MS for tissue analysis

Infrared laser ablation microsampling was used with data-dependent acquisition (DDA) and ion mobility-enhanced data-independent acquisition (HDMSE) for mass spectrometry based bottom-up proteomics analysis of rat brain tissue. Results from HDMSE and DDA analyses of the 12 laser ablation sampled tissue sections showed that HDMSE consistently identified approximately seven times more peptides and four times more proteins than DDA. To evaluate the impact of ultra-performance liquid chromatography (UPLC) peak congestion on HDMSE and DDA analysis, whole tissue digests from rat brain were analyzed at six different UPLC separation times. Analogous to results from laser ablated samples, HDMSE analyses of whole tissue digests yielded about four times more proteins identified than DDA for all six UPLC separation times.

Label-Free Quantitative Proteomics Unravels Carboxypeptidases as the Novel Biomarker in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with a high mortality rate and poor prognosis. However, little is known concerning the molecular mechanism of PDAC at the proteomics level. Here we report a proteomics analysis of PDAC tumor and adjacent tissues by shotgun proteomics followed by label-free quantification, and in total, 3031 and 3306 proteins were identified in three pairs of PDAC tumor and adjacent tissues, respectively; 40 of them were differentially expressed for at least three-fold in PDAC tumor tissues. Ontological and interaction network analysis highlighted the dysregulation of a set of four proteins in the carboxypeptidase family: carboxypeptidase A1 (CPA1), A2 (CPA2), B1 (CPB1), and chymotrypsin C (CTRC). Western blotting confirmed the downregulation of the carboxypeptidase network in PDAC. Immunohistochemistry of tissue microarray from 90 PDAC patients demonstrated that CPB1 was downregulated 7.07-fold (P<.0001, n=81) in tumor comparing with the peritumor tissue. Further 208 pancreatic tissues from PDAC tumor, peritumor, and pancreatis confirmed the downregulation of CPB1 in the PDAC patients. In summary, our results displayed that the expression of carboxypeptidase is significantly downregulated in PDAC tumor tissues and may be novel biomarker in the patient with PDAC.

Abstract A14: Downregulation of desmin involves gene promoter hypermethylation in gallbladder cancer

Abstract Gallbladder cancer (GBC) is a fatal malignancy with widespread geographical variations in incidence worldwide. There are limited studies on systematic molecular profiling of gallbladder cancer in literature. Previously, we carried out differential proteomic and methylome-based analysis in gallbladder tumors and adjacent non-tumors to identify differentially expressed proteins and global methylation status of genes involved in gallbladder cancer. Quantitative tissue proteomics analysis using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) based Liquid Chromatography-Mass Spectrometry (LC-MS/MS) showed that desmin protein was significantly downregulated (~3 folds) in tumors. Global methylation profiling using Illumina's Infinium Human Methylation 450 BeadChip array revealed hypermethylation in desmin in gallbladder tumors compared to non-tumor tissue. These findings lead us to further investigate the epigenetic regulation of desmin gene expression in gallbladder cancer. Real-time PCR analysis revealed significant downregulation of desmin (p=0.001) expression in tumors (n=12) as compared to the non-tumor tissue (n=12). Immunohistochemical analysis also showed significantly (p&amp;lt;0.0001) decreased level of desmin in 95% (26/27) of tumor cells in comparison to non-tumors 35.71% (5/14). This was again validated by Western blotting, showing downregulation of desmin (p=0.0081) at protein level in tumor tissues compared to the non-tumors. Methylation specific polymerase chain reaction analysis showed 86.67% methylation of desmin in corresponding gallbladder tumors compared to non-tumor tissue (p=0.004). Taken together, our observations showing decreased expression and hypermethylation of desmin gene suggest desmin as a potential epigenetically controlled biomarker for GBC. However, further studies are warranted to explore its role in tumorogenesis of gallbladder cancer. Citation Format: Shushruta Bhunia, Mustafa Ahmed Barbhuiya, Sanjiv Gupta, Braj Raj Shrivastava, Pramod Kumar Tiwari. Downregulation of desmin involves gene promoter hypermethylation in gallbladder cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A14.

Blood Pressure Control by a Secreted FGFBP1 (Fibroblast Growth Factor-Binding Protein).

Fibroblast growth factors (FGFs) participate in organ development and tissue maintenance, as well as the control of vascular function. The paracrine-acting FGFs are stored in the extracellular matrix, and their release is controlled by a secreted FGF-binding protein (FGF-BP, FGFBP1, and BP1) that modulates FGF receptor signaling. A genetic polymorphism in the human FGFBP1 gene was associated with higher gene expression and an increased risk of familial hypertension. Here, we report on the effects of inducible BP1 expression in a transgenic mouse model. Induction of BP1 expression in adult animals leads to a sustained rise in mean arterial pressure by >30 mm Hg. The hypertensive effect of BP1 expression is prevented by candesartan, an angiotensin II (AngII) receptor antagonist, or by tempol, an inhibitor of reactive oxygen species. In vivo, BP1 expression sensitizes peripheral resistance vessels to AngII constriction by 20-fold but does not alter adrenergic vasoconstriction. FGF receptor kinase inhibition reverses the sensitization to AngII. Also, constriction of isolated renal afferent arterioles by AngII is enhanced after BP1 expression and blocked by FGF receptor kinase inhibition. Furthermore, AngII-mediated constriction of renal afferent arterioles is abolished in FGF2-/- mice but can be restored by add-back of FGF2 plus BP1 proteins. In contrast to AngII, adrenergic constriction is not affected in the FGF2-/- model. Proteomics and gene expression analysis of kidney tissues after BP1 induction show that MAPK (mitogen-activated protein kinase) signaling via MKK4 (MAPK kinase 4), p38, and JNK (c-Jun N-terminal kinase) integrates the crosstalk of the FGF receptor and AngII pathways and thus impact vascular tone and blood pressure.

Abstract 2244: Pharmacological TLR4 antagonism using topical resatorvid blocks solar UV-induced skin tumorigenesis in SKH-1 mice

Abstract An urgent need exists for the development of more efficacious molecular strategies targeting non-melanoma skin cancer (NMSC), the most common malignancy worldwide. Inflammatory signaling downstream of Toll-like receptor 4 (TLR4) is a causative factor in several forms of tumorigenesis, yet its role in solar UV-induced skin carcinogenesis remains undefined. In our recently published work based upon immunohistochemical (IHC) analysis of NMSC tissue microarrays and proteomic analysis of reverse-phase protein microarrays (RPPA) from banked human tissue, we observed increased TLR4 expression in association with tumorigenic progression from normal skin to actinic keratosis and cutaneous squamous cell carcinoma. In immortalized keratinocytes expressing luciferase reporter constructs, resatorvid potently inhibits UV-induced AP-1 and NF-κB signaling, associated with downregulation of inflammatory mediators (IL-6, IL-8, IL-10) and MAP Kinase phosphorylation. In a subsequent acute exposure model in SKH-1 mice, topical resatorvid efficiently antagonized UV-induced stress signaling while potentiating UV-induced epidermal apoptosis. In the current report, we show for the first time that pharmacological inhibition of TLR4 using the specific antagonist resatorvid (TAK-242) blocks UV-induced tumorigenesis in SKH-1 mice. After assessing photostability, efficient cutaneous delivery, and skin residence time of topical resatorvid, we then tested the feasibility of TLR4-directed inhibition of UV-induced tumorigenesis. To this end, SKH-1 mice were split into three groups (n = 20). Each group was exposed to solar-simulated UV (three times a week; 15 weeks duration), followed by another 10 weeks in the absence of UV exposure (sacrifice at week 25). The control group received topical vehicle (acetone) on their backs 1 hour prior to UV exposure (three times a week until sacrifice). The “Prevention” group received topical resatorvid following an analogous dosing regimen. The “Intervention” group received vehicle 1hr prior to each UV treatment but switched to topical resatorvid three times a week only after UV was terminated. In the Prevention model, topical treatment with resatorvid significantly inhibited both tumor area (66% reduction, p = 0.0052) and tumor multiplicity (32% reduction, p = 0.0329; cross sectional Kruskal-Wallis test). In the Intervention model, a marked inhibition of both measures of tumor yield was observed, yet they did not reach statistical significance. Likewise, suppression of UV-induced TLR4-driven signaling was also confirmed in murine skin harvested at week 14 (prior to tumor onset), as assessed by IHC and RPPA analysis. Taken together, these data generated using resatorvid in a murine photocarcinogenesis model suggest that pharmacological TLR4 antagonism may represent a novel molecular strategy for topical prevention of solar UV-induced NMSC. Citation Format: Sally E. Dickinson, Karen Blohm-Mangone, Nichole B. Burkett, Shekha Tahsin, Paul B. Myrdal, Kelly L. Karlage, Jaroslav Janda, Kathylynn Saboda, Denise J. Roe, Zigang Dong, Ann M. Bode, Emanuel F. Petricoin, Valerie S. Calvert, Clara Curiel-Lewandrowski, David S. Alberts, Georg T. Wondrak. Pharmacological TLR4 antagonism using topical resatorvid blocks solar UV-induced skin tumorigenesis in SKH-1 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2244. doi:10.1158/1538-7445.AM2017-2244

New Apex in Proteome Analysis

Improved sample processing workflows enable rapid, deep analysis of human cellular and tissue proteomes.

Hippocampal Proteomic Analysis Reveals Distinct Pathway Deregulation Profiles at Early and Late Stages in a Rat Model of Alzheimer's-Like Amyloid Pathology.

The cerebral accumulation and cytotoxicity of amyloid beta (Aβ) is central to Alzheimer's pathogenesis. However, little is known about how the amyloid pathology affects the global expression of brain proteins at different disease stages. In order to identify genotype and time-dependent significant changes in protein expression, we employed quantitative proteomics analysis of hippocampal tissue from the McGill-R-Thy1-APP rat model of Alzheimer-like amyloid pathology. McGill transgenic rats were compared to wild-type rats at early and late pathology stages, i.e., when intraneuronal Aβ amyloid burden is conspicuous and when extracellular amyloid plaques are abundant with more pronounced cognitive deficits. After correction for multiple testing, the expression levels of 64 proteins were found to be considerably different in transgenic versus wild-type rats at the pre-plaque stage (3months), and 86 proteins in the post-plaque group (12months), with only 9 differentially regulated proteins common to the 2 time-points. This minimal overlap supports the hypothesis that different molecular pathways are affected in the hippocampus at early and late stages of the amyloid pathology throughout its continuum. At early stages, disturbances in pathways related to cellular responses to stress, protein homeostasis, and neuronal structure are predominant, while disturbances in metabolic energy generation dominate at later stages. These results shed new light on the molecular pathways affected by the early accumulation of Aβ and how the evolving amyloid pathology impacts other complex metabolic pathways.

Subventricular zone involvement in Glioblastoma \u2013 A proteomic evaluation and clinicoradiological correlation

Glioblastoma multiforme (GBM), the most malignant of all gliomas is characterized by a high degree of heterogeneity and poor response to treatment. The sub-ventricular zone (SVZ) is the major site of neurogenesis in the brain and is rich in neural stem cells. Based on the proximity of the GBM tumors to the SVZ, the tumors can be further classified into SVZ+ and SVZ−. The tumors located in close contact with the SVZ are classified as SVZ+, while the tumors located distantly from the SVZ are classified as SVZ−. To gain an insight into the increased aggressiveness of SVZ+ over SVZ− tumors, we have used proteomics techniques like 2D-DIGE and LC-MS/MS to investigate any possible proteomic differences between the two subtypes. Serum proteomic analysis revealed significant alterations of various acute phase proteins and lipid carrying proteins, while tissue proteomic analysis revealed significant alterations in cytoskeletal, lipid binding, chaperone and cell cycle regulating proteins, which are already known to be associated with disease pathobiology. These findings provide cues to molecular basis behind increased aggressiveness of SVZ+ GBM tumors over SVZ− GBM tumors and plausible therapeutic targets to improve treatment modalities for these highly invasive tumors.

Tissue Proteome Analysis of Different Grades of Human Gliomas Provides Major Cues for Glioma Pathogenesis.

Gliomas are heterogeneous and most commonly occurring brain tumors. Blood-brain barrier restricts the entry of brain tumor proteins into blood stream thus limiting the usage of serum or plasma for proteomic analysis. Our study aimed at understanding the molecular basis of aggressiveness of various grades of brain tumors using isobaric tagging for relative and absolute quantification (iTRAQ) based mass spectrometry. Tissue proteomic analysis of various grades of gliomas was performed using four-plex iTRAQ. We labeled five sets (each set consists of control, grade-II, III, and IV tumor samples) of individual glioma patients using iTRAQ reagents. Significantly altered proteins were subjected to bioinformatics analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID). Various metabolic pathways like glycolysis, TCA-cycle, electron transport chain, lactate metabolism, and blood coagulation pathways were majorly observed to be perturbed in gliomas. Most of the identified proteins involved in redox reactions, protein folding, pre-messenger RNA (mRNA) processing, antiapoptosis, and blood coagulation were found to be upregulated in gliomas. Transcriptomics data of glioblastoma multiforme (GBM), low-grade gliomas (LGGs), and controls were downloaded from The Cancer Genome Atlas (TCGA) data portal and further analyzed using BRB-Array tools. Expression levels of a few significantly altered proteins like lactate dehydrogenase, alpha-1 antitrypsin, fibrinogen alpha chain, nucleophosmin, annexin A5, thioredoxin, ferritin light chain, thymosin beta-4-like protein 3, superoxide dismutase-2, and peroxiredoxin-1 and 6 showed a positive correlation with increasing grade of gliomas thereby offering an insight into molecular basis behind their aggressive nature. Several proteins identified in different grades of gliomas are potential grade-specific markers, and perturbed pathways provide comprehensive overview of molecular cues involved in glioma pathogenesis.

Comparative Proteomics and Bioinformatics Analysis of Tissue from Non-Small Cell Lung Cancer Patients

Comparative Proteomics and Bioinformatics Analysis of Tissue from Non-Small Cell Lung Cancer Patients

Using proteomics to discover novel biomarkers for fatty liver development and response to CB1R antagonist treatment in an obese mouse model.

Over activity of cannabinoid receptor type 1 (CB1R) plays a key role in increasing the incidence of obesity-induced non-alcoholic fatty liver disease. Tissue proteome analysis has been applied to investigate the bioinformatics regarding the mode of action and therapeutic mechanism. The aim of this study was to explore the potential pathways altered with CB1R in obesity-induced fatty liver. Male C57BL/6 mice were fed either a standard chow diet (STD) or a high-fat diet (HFD) with or without 1-week treatment of CB1R inverse agonist AM251 at 5 mg/kg. Then, liver tissues were harvested for 2DE analysis and protein profiles were identified by using MALDI-MS. Results showed that eight of significantly altered protein spots at the level of changes > twofold were overlapped among the three groups, naming major urinary protein 1, ATP synthase subunit β, glucosamine-fructose-6-phosphate aminotransferase 1, zine finger protein 2, s-adenosylmethionine synthase isoform type-1, isocitrate dehydrogenase subunit α, epoxide hydrolase 2 and 60S acidic ribosomal protein P0. These identified proteins were involved in glucose/lipid metabolic process, xenobiotic metabolic system, and ATP synthesized process in mitochondria. Based on the findings, we speculated that CB1R blockade might exert its anti-metabolic disorder effect via improvement of mitochondrial function in hepatic steatosis in HFD condition.

Proteomics Analysis of Ovarian Cancer Cell Lines and Tissues Reveals Drug Resistance-associated Proteins.

Carboplatin and paclitaxel form the cornerstone of chemotherapy for epithelial ovarian cancer, however, drug resistance to these agents continues to present challenges. Despite extensive research, the mechanisms underlying this resistance remain unclear. A 2D-gel proteomics method was used to analyze protein expression levels of three human ovarian cancer cell lines and five biopsy samples. Representative proteins identified were validated via western immunoblotting. Ingenuity pathway analysis revealed metabolomic pathway changes. A total of 189 proteins were identified with restricted criteria. Combined treatment targeting the proteasome-ubiquitin pathway resulted in re-sensitisation of drug-resistant cells. In addition, examination of five surgical biopsies of ovarian tissues revealed α-enolase (ENOA), elongation factor Tu, mitochondrial (EFTU), glyceraldehyde-3-phosphate dehydrogenase (G3P), stress-70 protein, mitochondrial (GRP75), apolipoprotein A-1 (APOA1), peroxiredoxin (PRDX2) and annexin A (ANXA) as candidate biomarkers of drug-resistant disease. Proteomics combined with pathway analysis provided information for an effective combined treatment approach overcoming drug resistance. Analysis of cell lines and tissues revealed potential prognostic biomarkers for ovarian cancer.

Hydrophilic Strong Anion Exchange (hSAX) Chromatography Enables Deep Fractionation of Tissue Proteomes.

The bottom-up proteomic analysis of cell line and tissue samples to a depth > 10,000 proteins still represents a considerable challenge because of the sheer number of peptides generated by proteolytic digestions and the high dynamic range of protein expression. As a result, comprehensive protein coverage requires multidimensional peptide separation. Recently, off-line hydrophilic strong cation exchange (hSAX) chromatography has proven its merits for high resolution separation of peptides due to its high degree of orthogonality to reversed-phase liquid chromatography. Here we describe the use of hSAX for the deep analysis of tissue proteomes. The protocol includes optimized sample preparation steps (lysis with the aid of mechanical disruption, one-step disulfide bridge reduction and alkylation), setup and operation of hSAX columns and gradients, desalting of hSAX fractions prior to LC-MS/MS analysis, and suggestions for the choice of data acquisition parameters and data analysis using MaxQuant. Application of the protocol to the fractionation of 300 μg human brain tissue digest led to the identification of more than 100,000 unique peptide sequences representing over 10,195 proteins and 9,500 genes in 3 days of measurement time on a Q Exactive Plus mass spectrometer.

GeLC-MS: A Sample Preparation Method for Proteomics Analysis of Minimal Amount of Tissue.

Application of various proteomics methodologies have been implemented for the global and targeted proteome analysis of many different types of biological samples such as tissue, urine, plasma, serum, blood, and cell lines. Among the aforementioned biological samples, tissue has an exceptional role into clinical research and practice. Disease initiation and progression is usually located at the tissue level of different organs, making the analysis of this material very important for the understanding of the disease pathophysiology. Despite the significant advances in the mass spectrometry instrumentation, tissue proteomics still faces several challenges mainly due to increased sample complexity and heterogeneity. However, the most prominent challenge is attributed to the invasive procedure of tissue sampling which restricts the availability of fresh frozen tissue to minimal amounts and limited number of samples. Application of GeLC-MS sample preparation protocol for tissue proteomics analysis can greatly facilitate making up for these difficulties. In this chapter, a step by step guide for the proteomics analysis of minute amounts of tissue samples using the GeLC-MS sample preparation protocol, as applied by our group in the analysis of multiple different types of tissues (vessels, kidney, bladder, prostate, heart) is provided.

Comparative proteome analysis of abdominal adipose tissues between fat and lean broilers.

BackgroundThe molecular mechanism underlying broiler fat deposition is still poorly understood.MethodCurrently, we used two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in abdominal adipose tissues of birds at 4 week of age derived from Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF).ResultsThirteen differentially expressed protein spots were screened out and identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The protein spots were matched to thirteen proteins by searching against the NCBInr database. These identified proteins were apolipoprotein A-I (Apo A-I), cytokeratin otokeratin, ATP synthase subunit alpha, peptidyl-prolyl cis-trans isomerase FKBP4 (PPIase FKBP4), aspartate aminotransferase, carbonic anhydrase II (CA-II), prostaglandin-H2 D-isomerase precursor, fibrinogen alpha chain, lamin-A (LMNA), superoxide dismutase [Mn] (MnSOD), heat shock protein beta-1 (HSPβ1) and two predicted proteins. These differentially expressed proteins are involved mainly in lipid metabolism, amino acid metabolism, signal transduction, energy conversion, antioxidant, and cytoskeleton. Differential expression of Apo A-I, PPIase FKBP4, and cytokeratin otokeratin proteins were further confirmed by Western blot analysis. Quantitative real-time RT-PCR analyses showed that, of these 13 differentially expressed proteins, only PPIase FKBP4 and cytokeratin otokeratin were differentially expressed at mRNA level between the two lines.ConclusionsOur results have provided further information for understanding the basic genetics control of growth and development of broiler adipose tissue.