P404 IDENTIFICATION OF NEW BIOMARKERS IN HEPATITIS B RELATED HEPATOCELLULAR CARCINOMA PATIENTS USING THE MASS SPECTROMETRY AND WESTERN BLOT ANALYSIS

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

Download Full-text

Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches

BMC Veterinary Research ◽

10.1186/s12917-020-02591-1 ◽

2020 ◽

Vol 16 (1) ◽

Author(s):

Nathamon Yimpring ◽

Sittiruk Roytrakul ◽

Janthima Jaresitthikunchai ◽

Narumon Phaonakrop ◽

Sucheewin Krobthong ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Expression ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Expression Profiles ◽

Tumor Necrosis Factor Receptor ◽

Principal Component ◽

Peptide Mass ◽

Different Ages

Abstract Background Cryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1–2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1–2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography–tandem mass spectrometry. Results A PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1–2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age. Conclusions The present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

Download Full-text

Identification of cytoplasmic sialidase NEU2-associated proteins by LC-MS/MS

Turkish Journal of Biochemistry ◽

10.1515/tjb-2018-0089 ◽

2018 ◽

Vol 44 (4) ◽

pp. 462-472

Author(s):

Secil Akyildiz Demir ◽

Volkan Seyrantepe

Keyword(s):

Mass Spectrometry ◽

Western Blot ◽

Blot Analysis ◽

Actin Filaments ◽

Western Blot Analysis ◽

Mammalian Cells ◽

Protein S ◽

Mass Spectrometry Analysis ◽

Interacting Protein ◽

Spectrometry Analysis

Abstract Background Cytoplasmic sialidase (NEU2) plays an active role in removing sialic acids from oligosaccharides, glycopeptides, and gangliosides in mammalian cells. NEU2 is involved in various cellular events, including cancer metabolism, neuronal and myoblast differentiation, proliferation, and hypertrophy. However, NEU2-interacting protein(s) within the cell have not been identified yet. Objective The aim of this study is to investigate NEU2 interacting proteins using two-step affinity purification (TAP) strategy combined with mass spectrometry analysis. Methods In this study, NEU2 gene was cloned into the pCTAP expression vector and transiently transfected to COS-7 cells by using PEI. The most efficient expression time of NEU2- tag protein was determined by real-time PCR and Western blot analysis. NEU2-interacting protein(s) were investigated by using TAP strategy combined with two different mass spectrometry experiment; LC-MS/MS and MALDI TOF/TOF. Results Here, mass spectrometry analysis showed four proteins; α-actin, β-actin, calmodulin and histone H1.2 proteins are associated with NEU2. The interactions between NEU2 and actin filaments were verified by Western blot analysis and immunofluorescence analysis. Conclusions Our study suggests that association of NEU2 with actin filaments and other protein(s) could be important for understanding the biological role of NEU2 in mammalian cells.

Download Full-text

Identification of Immmunogenic Salivary Proteins of Anopheles vagus based on Mass Spectrometry Analysis

Jurnal ILMU DASAR ◽

10.19184/jid.v18i2.3106 ◽

2017 ◽

Vol 18 (2) ◽

pp. 73

Author(s):

Dwi Esti Febriyantiningsih ◽

Kartika Senjarini ◽

Rike Oktarianti

Keyword(s):

Mass Spectrometry ◽

Salivary Gland ◽

Western Blot ◽

Salivary Glands ◽

Blot Analysis ◽

Western Blot Analysis ◽

Mass Spectrometry Analysis ◽

Salivary Proteins ◽

Immunogenic Proteins ◽

Anopheles Vagus

Malaria has been prevalent for a long time in tropical developing regions causing great morbidity and mortality. Among the malaria vectors, Anopheles vagus has been known as secondary malaria vector in East Java. Salivary glands of mosquitoes perform various functions for survival of the vectors and also conducive for blood feeding, harbouring of malaria parasites, and eventual parasite transmission. The salivary gland proteomes of An. vagus have not been carried out yet. The aim of our study was to identify and characterize the immunogenic proteins of salivary glands proteins of An. vagus. A proteomic approach combining one-dimensional electrophoresis (1DE) followed by western blot analysis using human sera from healthy people living in an endemic area (Kendal); liquid chromatography mass spectrometry (LC-MS/MS) and bioinformatic analysis was adopted to provide the first direct insight into identification and characterization of salivary proteins of An. vagus. Identification of immunogenic proteins using western blot analysis has revealed three immunogenic bands which had molecular weights of 69, 75 and 232 kDa. Among those proteins analysed by LC-MS/MS, there were alpha,1-4 glucan phosphorylase, putative myosin class I heavy chain which have the highest number of total spectrum count peptide. Other proteins like vitellogenin and heat shock protein 82 (Hsp82) were also identified. The majority of proteins were scrutinized marked for their role in metabolism, cytoskeleton protein and stress response. Keywords: Anopheles vagus, salivary gland, immunogenic, proteomics

Download Full-text

CRALBP is a Highly Prevalent Autoantigen for Human Autoimmune Uveitis

Clinical and Developmental Immunology ◽

10.1155/2007/39245 ◽

2007 ◽

Vol 2007 ◽

pp. 1-6 ◽

Cited By ~ 27

Author(s):

Cornelia A. Deeg ◽

Albert J. Raith ◽

Barbara Amann ◽

John W. Crabb ◽

Stephan R. Thurau ◽

...

Keyword(s):

Mass Spectrometry ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Two Dimensional ◽

Autoimmune Uveitis ◽

Equine Recurrent Uveitis ◽

And Control ◽

Normal Controls ◽

Subsequent Identification

Cellular retinaldehyde binding protein (CRALBP) is an autoantigen in spontaneous equine recurrent uveitis. In order to test whether CRALBP contributes to human autoimmune uveitis, the specificity of antibodies from human uveitis patient's sera was first evaluated in two-dimensional (2D) Western blot analysis. Subsequent identification of the immunoreactive proteins by mass spectrometry resulted in the identification of CRALBP as a putative autoantigen. Additionally, sera from human uveitis and control patients were by Western blot using purified human recombinant CRALBP. Anti-CRALBP autoantibodies occur more frequently (P<.01) in human uveitis patients than in normal controls. Thirty out of 56 tested uveitis patient's sera contained autoantibodies reactive against CRALBP, compared to only four out of 23 normal control subjects. The presence of CRALBP autoantibodies in 54% of tested uveitis patients supports CRALBP as a possible autoantigen in human autoimmune uveitis.

Download Full-text

Widely used commercial ELISA does not detect preHP-2, but recognizes properdin as a potential second member of the zonulin family

10.1101/157578 ◽

2017 ◽

Cited By ~ 1

Author(s):

Lucas Scheffler ◽

Alyce Crane ◽

Henrike Heyne ◽

Anke Tönjes ◽

Dorit Schleinitz ◽

...

Keyword(s):

Mass Spectrometry ◽

Western Blot ◽

Intestinal Permeability ◽

Blot Analysis ◽

Western Blot Analysis ◽

Metabolic Diseases ◽

Polyclonal Antibodies ◽

Complement C3 ◽

Elisa Kit ◽

Haptoglobin Genotype

AbstractBACKGROUNDThere is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating zonulin and metabolic traits related to obesity.METHODSSerum zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects.RESULTSAs zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the zonulin ELISA assay using antibody capture experiments, mass spectrometry and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin 2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the zonulin family.CONCLUSIONSOur study suggests that the zonulin ELISA does not recognize pre-haptoglobin 2, rather structural (and possibly functional) analogue proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.

Download Full-text

Ribonucleic acid-binding protein CPSF6 promotes glycolysis and suppresses apoptosis in hepatocellular carcinoma cells by inhibiting the BTG2 expression

BioMedical Engineering OnLine ◽

10.1186/s12938-021-00903-6 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Yang Liu ◽

Hongbo Zou ◽

Qichao Xie ◽

Lan Zou ◽

Rui Kong ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Rna Binding ◽

Binding Protein ◽

Terminal Deoxynucleotidyl Transferase ◽

Glucose Assay ◽

Normal Hepatocyte ◽

Hcc Cells

AbstractHepatocellular carcinoma (HCC) is currently the sixth most common malignancy and the second major cause of tumor-related deaths in the world. This study aimed to investigate the role of cleavage and polyadenylation factor-6 (CPSF6) and B-cell translocation gene 2 (BTG2) in regulating the glycolysis and apoptosis in HCC cells. The RNA and protein expression of CPSF6 and BTG2 in normal hepatocyte and HCC were, respectively, detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis and Western blot analysis. The viability and apoptosis of transfected Huh-7 cells were, respectively, analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. The expression of apoptosis-related proteins and HK-2 in transfected Huh-7 cells was also detected by Western blot analysis. The levels of glucose and lactate in the culture supernatant of transfected Huh-7 cells were, respectively, detected with the glucose assay kit and lactate assay kit. The interaction of CPSF6 and BTG2 was confirmed by RNA binding protein immunoprecipitation (RIP) assay. As a result, CPSF6 expression was increased while BTG2 expression was decreased in Huh-7 cells. Interference with CPSF6 suppressed the viability and glycolysis, and promoted the apoptosis of Huh-7 cells. Furthermore, CPSF6 interacted with BTG2 and interference with CPSF6 upregulated the BTG2 expression and inhibited the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK)/nuclear factor (NF)-κB pathway. Interference with BTG2 could partially reverse the above cell changes caused by interference with CPSF6. In conclusion, CPSF6 inhibited the BTG2 expression to promote glycolysis and suppress apoptosis in HCC cells by activating AKT/ERK/NF-κB pathway.

Download Full-text

Overproduction and Purification of Soluble Recombinant Human Granulocyte Colony Stimulating Factor in Escherichia coli Using Thioredoxin as Fusion

ANNALES BOGORIENSES ◽

10.14203/ann.bogor.2017.v21.n1.1-8 ◽

2017 ◽

Vol 21 (1) ◽

pp. 1

Author(s):

Dian Fitria Agustiyanti ◽

Debbie Sofie Retnoningrum ◽

Heni Rachmawati ◽

Asrul Muhamad Fuad

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Fusion Protein ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Soluble Form ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Stimulating Factor

Recombinant human Granulocyte Colony Stimulating Factor (G-CSF) has been produced in a soluble form in Escherichia coli BL21 (DE3) as a fusion protein. The open reading frame of G-CSF was synthetically constructed in previous work and was codon optimized for best expression in E. coli. In this research, the gene was fused to thioredoxin (Trx) at the N-terminal in pET32 vector. The purpose of this research was to optimize the overproduction and purification processes to obtain high yield recombinant protein in soluble form, and to characterize the Trx-G-CSF fusion protein. Overproduction was performed using IPTG induction method for 3 and 6 hours. The protein was purified by Ni-NTA affinity chromatography and separated using gradient concentration of imidazole. The purified protein was then characterized by SDS-PAGE and Western Blot analysis. Further, enterokinase was used to separate G-CSF from the fusion protein. The purified form of G-CSF was subsequently characterized using Western Blot and mass spectrometry using MALDI-TOF. The results showed that the fusion protein was successfully produced in soluble part as much as 48.25% were obtained after 3 hours of induction. The yield of fusion protein was 67.37% from total protein (229.65 mg protein/L culture). The Western Blot analysis showed the G-CSF band at around 18.6 kDa. Mass spectrometry with MALDI-TOF/ TOF revealed that 25.86% of amino acid residue was recognized as part of human G-CSF sequence.

Download Full-text

Detection of carbonyl-modified proteins in interfibrillar rat mitochondria usingN′-aminooxymethylcarbonylhydrazino-D-biotin as an aldehyde/keto-reactive probe in combination with Western blot analysis and tandem mass spectrometry

Electrophoresis ◽

10.1002/elps.200700606 ◽

2008 ◽

Vol 29 (6) ◽

pp. 1317-1324 ◽

Cited By ~ 33

Author(s):

Woon-Gye Chung ◽

Cristobal L. Miranda ◽

Claudia S. Maier

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Tandem Mass ◽

Modified Proteins

Download Full-text

PTBP1 Interacts with PKM2 in an NPM-ALK-Dependent Manner and Contributes to the Pathogenesis of Anaplastic Large Cell Lymphoma (ALCL)

Blood ◽

10.1182/blood.v124.21.3010.3010 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3010-3010

Author(s):

Johnvesly Basappa ◽

Steven R Hwang ◽

Scott RP McDonnell ◽

Venkatesh Basrur ◽

Carlos Murga-Zamalloa ◽

...

Keyword(s):

Mass Spectrometry ◽

Western Blot ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cancer Metabolism ◽

Dependent Manner ◽

Polypyrimidine Tract Binding Protein ◽

Stat3 Phosphorylation

Abstract Introduction: Pyruvate kinase (PKM2) is a glycolytic enzyme that plays an important role in cancer metabolism. Our previous work demonstrated that NPM-ALK phosphorylates PKM2 at Y105 and that this regulates altered metabolism that promoted ALK-mediated lymphomagenesis. There is emerging evidence that PKM2 may contribute to oncogenesis independent of its role in cell metabolism. We hypothesized that identification of novel PKM2 interactors via mass spectrometry could provide additional insights on its expanding functional role in cancer. Methods: In order to identify novel proteins that interact with PKM2, we generated an ALCL-derived cell line (DEL) stably transduced to express FLAG-tagged WT PKM2. We isolated the FLAG-tagged PKM2-immunocomplex and subjected it to proteomic analysis by using liquid-chromatography tandem mass spectrometry (LC-MS/MS). A subset of the proteins unique to the PKM2 bait as compared to control was selected for subsequent studies including reciprocal immunoprecipitations (IP), western blot analysis and functional experiments. Results: Mass spectrometry identified 63 proteins that interacted with WT PKM2 including -catenin and FBP-6, both known interactors of PKM2. Among these we also noted the polypyrimidine tract-binding protein (PTBP1), a protein with a role in RNA stability as a candidate interactor of PKM2. Reciprocal immunoprecipitations confirmed the interaction between endogenous PKM2 and PTBP1 in two ALCL-derived cell lines. In order to determine if phosphorylation of PKM2 at Y105 was necessary for its interaction with PTBP1, FLAG-WT-PKM2 and FLAG-Y105F-PKM2 were used for IP and western blot analysis which confirmed that the PTBP1 interaction occurred with WT-PKM2 and not Y105F-PKM2. Similarly, the interaction of PTBP1 with PKM2 was significantly decreased in the presence of a selective ALK inhibitor. To determine whether the interaction of PTBP1 with pY105-PKM2 occurred in distinct subcellular compartments, we carried out subcellular fractionation of ALCL-derived cell lines and evaluated the interaction of PKM2 with PTBP1. Forward and reciprocal immunoprecipitations demonstrated that the interaction of PTBP1 and PKM2 and occurs primarily in the nucleus and selective to pY105-PKM2. Analysis of subcellular fractions after selective inhibition of ALK by crizotinib also showed that the expression of nuclear pY105 PKM2 was abolished. Based on the previous observation that PKM2 acts as a protein kinase and phosphorylates STAT3, we assessed the role of pY105-PKM2 on STAT3 phosphorylation in DEL cells stably expressing either FLAG-WT PKM2 WT or Y105F-PKM2. Western blot analysis demonstrated that nuclear expression of active (pY705)-STAT3 was decreased in cells expressing Y105F-PKM2 relative to WT-PKM2. Stable knockdown by lenti-viral transduction of PTBP1 shRNA in DEL cells demonstrated a marked decrease in expression of pY105-PKM2 and Y705-STAT3 without affecting total STAT3 protein. Moreover, knockdown of PTBP1 led to decreased ALCL cell proliferation and colony formation in soft agar relative to control. Conclusion: These data demonstrate that PTBP1 is a novel PKM2 interacting protein and phosphorylation of PKM2 at Y105 by NPM-ALK regulates the interaction. The interaction of PTBP1 occurs preferentially with pY105-PKM2 within the nucleus and regulates the phosphorylation of Y705-STAT3. PTBP1 promotes oncogenesis in ALCL by regulating the nuclear localization of Y105-PKM2 and phosphorylation of Y705-STAT3. Our studies provide evidence for a novel role of PTBP1 in mediating oncogenesis in NPM-ALK expressing ALCLs. Disclosures No relevant conflicts of interest to declare.

Download Full-text