Nanoscale Liquid Chromatography-tandem Mass Research Articles

Proteomic Analysis of Dysfunctional Liver Sinusoidal Endothelial Cells Reveals Substantial Differences in Most Common Experimental Models of Chronic Liver Diseases.

Molecular markers of dedifferentiation of dysfunctional liver sinusoidal endothelial cells (LSEC) have not been fully elucidated. We aimed at deciphering the molecular profile of dysfunctional LSEC in different pathological scenarios. Flow cytometry was used to sort CD11b-/CD32b+ and CD11b-/CD32b- LSEC from three rat models of liver disease (bile duct ligation-BDL; inhaled carbon tetrachloride-CCl4; and high fat glucose/fructose diet-HFGFD). A full proteomic profile was performed applying nano-scale liquid chromatography tandem mass spectrometry (nLC-MS) and analyzed with PEAKS software. The percentage of CD32b- LSEC varied across groups, suggesting different capillarization processes. Both CD32+ and CD32b- LSEC from models are different from control LSEC, but differently expressed proteins in CD32b- LSEC are significantly higher. Heatmaps evidenced specific protein expression patterns for each model. Analysis of biological significance comparing dysfunctional CD32b- LSEC with specialized CD32b+ LSEC from controls showed central similarities represented by 45 common down-regulated proteins involved in the suppression of the endocytic machinery and 63 common up-regulated proteins associated with the actin-dependent cytoskeleton reorganization. In summary; substantial differences but also similarities in dysfunctional LSEC from the three most common models of liver disease were found, supporting the idea that LSEC may harbor different protein expression profiles according to the etiology or disease stage.

Proteomic analysis of the mouse sperm acrosome - towards an understanding of an organelle with diverse functionality

The acrosome located within the mammalian sperm head is essential for successful fertilization, as it enables the sperm to penetrate the extracellular layers of the oocyte and fuse with oolemma. However, the mammalian acrosomal vesicle is no longer considered to contain only hydrolytic enzymes. Using label-free nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) proteomics, we identified a total of 885 proteins in the acrosome isolated from spermatozoa obtained from cauda epididymis of free-living house mice Mus musculus musculus contains a total of 885 proteins. Among these, 334 proteins were significantly enriched in the acrosome thus representing 27.3% of the whole proteome of the intact sperm. Importantly, we have detected a total of nine calycins while eight of them belong to the lipocalin protein family. In mice, lipocalins are involved in multi-level chemical communication between individuals including pheromone transport and odor perception. Using an indirect immunofluorescence assay, we demonstrated that lipocalin 5 (LCN5) is expressed in the mouse germ cells, and after completing spermatogenesis, it remains localized in the sperm acrosome until the last step of the extratesticular maturation, the acrosome reaction. The presence of lipocalins in the acrosome and acrosome-reacted sperm suggests their original role as chelators of organic and potentially toxic compounds resulting from ongoing spermiogenesis. Along with this evidence, detected mitochondrial (e.g., a subunit of the cytochrome c oxidase MTCO1) and proteasomal proteins (subunits of both 20 S core proteasome [PSMA2, PSMBs] and 19 S regulatory particle [PSMDs]) in acrosomes provide further evidence that acrosomes could also function as `waste baskets` after testicular sperm maturation.

Screening for Lipid-Metabolism-Related Genes and Identifying the Diagnostic Potential of ANGPTL6 for HBV-Related Early-Stage Hepatocellular Carcinoma.

Lipid metabolic reprogramming is one of the hallmarks of hepatocarcinogenesis and development. Therefore, lipid-metabolism-related genes may be used as potential biomarkers for hepatocellular carcinoma (HCC). This study aimed to screen for genes with dysregulated expression related to lipid metabolism in HCC and explored the clinical value of these genes. We screened differentially expressed proteins between tumorous and adjacent nontumorous tissues of hepatitis B virus (HBV)-related HCC patients using a Nanoscale Liquid Chromatography-Tandem Mass Spectrometry platform and combined it with transcriptomic data of lipid-metabolism-related genes from the GEO and HPA databases to identify dysregulated genes that may be involved in lipid metabolic processes. The potential clinical values of these genes were explored by bioinformatics online analysis tools (GEPIA, cBioPortal, SurvivalMeth, and TIMER). The expression levels of the secreted protein (angiopoietin-like protein 6, ANGPTL6) in serum were analyzed by ELISA. The ability of serum ANGPTL6 to diagnose early HCC was assessed by ROC curves. The results showed that serum ANGPTL6 could effectively differentiate between HBV-related early HCC patients with normal serum alpha-fetoprotein (AFP) levels and the noncancer group (healthy participants and chronic hepatitis B patients) (AUC = 0.717, 95% CI: from 0.614 to 0.805). Serum ANGPTL6 can be used as a potential second-line biomarker to supplement serum AFP in the early diagnosis of HBV-related HCC.

From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8.

With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography—tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and –activating properties, occurs in different NH2-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.

Proteomic analysis of a hom-disrupted, cephamycin C overproducing Streptomyces clavuligerus

Streptomyces clavuligerus is prolific producer of cephamycin C, a medically important antibiotic. In our former study, cephamycin C titer was 2-fold improved by disrupting homoserine dehydrogenase (hom) gene of aspartate pahway in Streptomyces clavuligerus NRRL 3585. In this article, we aimed to provide a comprehensive understanding at the proteome level on potential complex metabolic changes as a consequence of hom disruption in Streptomyces clavuligerus AK39. A comparative proteomics study was carried out between the wild type and its hom disrupted AK39 strain by 2 Dimensional Electrophoresis-Matrix Assisted Laser Desorption and Ionization Time-Of-Flight Mass Spectrometry (2DE MALDI-TOF/MS) and Nanoscale Liquid Chromatography- Tandem Mass Spectrometry (nanoLC-MS/MS) analyses. Clusters of Orthologous Groups (COG) database was used to determine the functional categories of the proteins. The theoretical pI and Mw values of the proteins were calculated using Expasy pI/Mw tool. "Hypothetical/Unknown" and "Secondary Metabolism" were the most prominent categories of the differentially expressed proteins. Upto 8.7-fold increased level of the positive regulator CcaR was a key finding since CcaR was shown to bind to cefF promoter thereby direcly controlling its expression. Consistently, CeaS2, the first enzyme of CA biosynthetic pathway, was 3.3- fold elevated. There were also many underrepresented proteins associated with the biosynthesis of several Non-Ribosomal Peptide Synthases (NRPSs), clavams, hybrid NRPS/Polyketide synthases (PKSs) and tunicamycin. The most conspicuously underrepresented protein of amino acid metabolism was 4-Hydroxyphenylpyruvate dioxygenase (HppD) acting in tyrosine catabolism. The levels of a Two Component System (TCS) response regulator containing a CheY-like receiver domain and an HTH DNA-binding domain as well as DNA-binding protein HU were elevated while a TetR-family transcriptional regulator was underexpressed. The results obtained herein will aid in finding out new targets for further improvement of cephamycin C production in Streptomyces clavuligerus.

Identification of Isopeptides Between Human Tissue Transglutaminase and Wheat, Rye, and Barley Gluten Peptides

Celiac disease (CD) is a chronic immune-mediated enteropathy of the small intestine, which is triggered by the ingestion of storage proteins (gluten) from wheat, rye, and barley in genetically predisposed individuals. Human tissue transglutaminase (TG2) plays a central role in the pathogenesis of CD, because it is responsible for specific gluten peptide deamidation and covalent crosslinking, resulting in the formation of Nε-(γ-glutamyl)-lysine isopeptide bonds. The resulting TG2-gluten peptide complexes are assumed to cause the secretion of anti-TG2 autoantibodies, but the underlying mechanisms are only partly known. To gain more insight into the structures of these complexes, the aim of our study was to identify TG2-gluten isopeptides. With the use of discovery-driven as well as targeted nanoscale liquid chromatography tandem mass spectrometry, we detected 29 TG2-gluten isopeptides in total, involving seven selected TG2 lysine residues (K205, K265, K429, K468, K590, K600, K677). Several gluten peptides carried known B-cell epitopes and/or T-cell epitopes, either intact 9-mer core regions or partial sequences, as well as sequences bearing striking similarities to already known epitopes. These novel insights into the molecular structures of TG2-gluten peptide complexes may help clarify their physiological relevance in the initiation of CD autoimmunity and the role of anti-TG2 autoantibodies.

Urine proteomics of primary membranous nephropathy using nanoscale liquid chromatography tandem mass spectrometry analysis

BackgroundPrimary membranous nephropathy (PMN) is an important cause of nephrotic syndrome in adults. Urine proteome may provide important clues of pathophysiological mechanisms in PMN. In the current study, we analyzed and compared the proteome of urine from patients with PMN and normal controls.MethodsWe performed two technical replicates (TMT1 and TMT2) to analyze and compare the urine proteome from patients with PMN and normal controls by tandem mass tag (TMT) technology coupled with nanoscale liquid chromatography tandem mass spectrometry analysis (LC–MS/MS). Gene ontology (GO) enrichment analysis was performed to analyse general characterization of the proteins. The proteins were also matched against the database of Kyoto Encyclopedia of Genes and Genomes (KEGG). For validation, Western blot was used to analyze the selected proteins.ResultsA total of 509 proteins and 411 proteins were identified in TMT1 and TMT2, respectively. 249 proteins were both identified in two technical replicates. GO analysis and KEGG analysis revealed immunization and coagulation were predominantly involved. Among the differential protein, the overexcretion of alpha-1-antitrypsin (A1AT) and afamin (AFM) were validated by Western blot analysis.ConclusionsOur data showed the important role of immunologic mechanism in the development of PMN, and the value of urinary A1AT and AFM in biomarker discovery of patients with PMN. The discovery of the overexcretion of A1AT and AFM in the urine can help to further elucidate pathogenetic mechanisms involved in PMN.

Detection of Carbofuran-Protein Adducts in Serum of Occupationally Exposed Pesticide Factory Workers in Pakistan.

This study was conducted to investigate the protein adducts with pesticides in a cohort of 172 factory workers that were exposed to a mixture of pesticides. The 35 samples showing considerable variation in biochemical parameters, i.e., butyrylcholinestrase (BChE), serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), gamma-glutamyl transferase (GGT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP/ALKP), lactate dehydrogenase (LDH), creatine phosphokinase (CPK) enzymes, and controls were analyzed by reversed-phase nanoscale liquid chromatography tandem mass spectrometry (nLC-MS/MS) on an Orbitrap mass spectrometer employing a shotgun proteomics approach. Only protein adducts with carbofuran were found on serum proteins of these workers. These adducts were of carbofuran labeled lysine (Lys-142, Lys-183, Lys-287, and Lys-467), arginine (Arg-210, Arg-242, and Arg-256) from serum albumin, and serine (Ser-07, Ser-54, and Ser-150) from immunoglobulin proteins. The arginine residues (Arg-210, Arg-242, Arg-246, and Arg-434) from albumin were also found to be glycated in serum of workers showing a high level of glucose who also had glycated arginine (Arg-1120) modified with carbofuran in their tankyrase-1-binding protein. The number of tandem mass spectra of modified peptides increased with increasing time of exposure. This is the first report to demonstrate the presence of carbofuran-labeled albumin, immunoglobulin, and glycated arginine, which shows that lysine and arginine of human albumin and serine of immunoglobulin are covalently modified in the serum of workers that were occupationally exposed to carbofuran, and the modification is detectable by tandem mass spectrometry. These peptides modified with carbofuran can potentially be used as a biomarker of carbofuran exposure.

CCN1 (Cyr61) Is Overexpressed in Human Osteoarthritic Cartilage and Inhibits ADAMTS-4 (Aggrecanase 1) Activity.

ADAMTS-4, also called aggrecanase 1, is considered to play a key role in aggrecan degradation in human osteoarthritic (OA) cartilage, but information about regulators of ADAMTS-4 aggrecanase activity remains limited. We undertook this study to search for molecules that modulate ADAMTS-4 activity. Molecules copurified with ADAMTS-4 from ADAMTS-4-transfected chondrocytic cells were sequenced by nanoscale liquid chromatography tandem mass spectrometry. Binding activity was determined by immunoprecipitation and solid-phase binding assay. Effects on ADAMTS-4 activity were examined by aggrecan digestion assay. Expression of the binding molecule in OA cartilage and chondrocytes was examined by immunohistochemistry and reverse transcription-polymerase chain reaction. We identified CCN1 (Cyr61) as an ADAMTS-4-binding protein and showed specific binding to the ADAMTS-4 cysteine-rich domain. Aggrecanase activity of ADAMTS-4 was inhibited by interaction with CCN1. Expression of messenger RNA for CCN1 was significantly higher in human OA cartilage than in normal cartilage. CCN1 was immunolocalized to chondrocytes in OA cartilage, showing direct correlations of immunoreactivity with the Mankin score of cartilage lesions and chondrocyte cloning. CCN1 and ADAMTS-4 were commonly coexpressed in clustered chondrocytes. CCN1 expression in OA chondrocytes was down-regulated by interleukin-1α (IL-1α) and up-regulated by transforming growth factor β (TGFβ). ADAMTS-4 expression was induced by treatment with IL-1α or TGFβ, but aggrecanase activity was detected only under stimulation with IL-1α. TGFβ-treated chondrocytes exhibited aggrecanase activity when CCN1 expression was knocked down. Our findings provide the first evidence that CCN1 suppresses ADAMTS-4 activity and that CCN1 overexpression is directly correlated with chondrocyte cloning in OA cartilage. Our results suggest that the TGFβ/CCN1 axis plays a role in chondrocyte cluster formation through inhibition of ADAMTS-4.

Site‐specific proteomic approach for study protein S‐nitrosylation

Protein S‐nitrosylation, the covalent attachment of nitric oxide (NO) to cysteine residues, is an emerging post‐translational modification (PTM) which regulates a large variety of cellular functions and signalling events. Characterization of S‐nitrosylated proteins and their modification sites is essential to understand the action of NO in biological systems. Here we present a novel and robust method for the identification of protein S‐nitrosylation sites in complex protein mixtures. The approach utilizes the Cysteinyl affinity resin to selectively enrich S‐nitrosylated peptides. The enriched samples were analyzed by nanoscale liquid chromatography tandem mass spectrometry on LTQ‐Orbitrap mass spectrometer. We applied this approach to MDA‐MB‐231 cells treated with Angeli's salt. Angeli's salt has been shown to inhibit breast cancer tumor growth and angiogenesis. A total of 177 S‐nitrosylation sites were identified and an S‐nitrosylation motif was revealed in our study. The 177 sites are significantly more than the number reported by previous methods, demonstrating the efficiency of our approach.

Site-Specific Proteomics Approach for Study Protein S-Nitrosylation

Here we present a novel and robust method for the identification of protein S-nitrosylation sites in complex protein mixtures. The approach utilizes the cysteinyl affinity resin to selectively enrich S-nitrosylated peptides reduced by ascorbate followed by nanoscale liquid chromatography tandem mass spectrometry. Two alkylation agents with different added masses were employed to differentiate the S-nitrosylation sites from the non-S-nitrosylation sites. We applied this approach to MDA-MB-231 cells treated with Angeli's salt, a nitric oxide donor that has been shown to inhibit breast tumor growth and angiogenesis. A total of 162 S-nitrosylation sites were identified and an S-nitrosylation motif was revealed in our study. The 162 sites are significantly more than the number reported by previous methods, demonstrating the efficiency of our approach. Our approach will further facilitate the functional study of protein S-nitrosylation in cellular processes and may reveal new therapeutic targets.