2D map of proteins from human renal stone matrix and evaluation of their effect on oxalate induced renal tubular epithelial cell injury

Purpose Proteins constitute a major portion of the organic matrix of human calcium oxalate (CaOx) renal stones and the matrix is considered to be important in stone formation and growth. The present study evaluates the effect of these proteins on oxalate injured renal epithelial cells accompanied by a 2D map of these proteins. Materials and Methods Proteins were isolated from the matrix of kidney stones containing CaOx as the major constituent using EGTA as a demineralizing agent. The effect of more than 3kDa proteins from matrix of human renal (calcium oxalate) CaOx stones was investigated on oxalate induced cell injury of MDCK renal tubular epithelial cells. A 2D map of >3kDa proteins was also generated followed by protein identification using MALDI-TOF MS. Results The >3kDa proteins enhanced the injury caused by oxalate on MDCK cells. Also, the 2D map of proteins having MW more than 3kDa suggested the abundance of proteins in the matrix of renal stone. Conclusion Studies indicate that the mixture of >3kDa proteins in the matrix of human renal stones acts as promoter of calcium oxalate crystal nucleation and growth as it augments the renal epithelial cell injury induced by oxalate. The effect of promoters masks the inhibitors in the protein mixture thereby leading to enhanced renal cell injury. 2D map throws light on the nature of proteins present in the kidney stones. .

Screening and identifying oxaliplatin-resistance-associated proteins in colorectal cancer cell lines / 肿瘤

Objective: To screen and identify oxaliplatin-resistance-associated proteins in CRC (colorectal cancer) cell lines using proteomics technologies in order to find new biomarkers for individual therapy of CRC. Methods: Oxaliplatin-resistant human CRC cell line HT-29/L-OHP (oxaliplatin) was established. The total proteins in HT-29 and HT-29/L-OHP cells were extracted. The differentially expressed proteins between HT-29 and HT-29/L-OHP cells were screened and identified using 2-DE (two-dimensional gel electrophoresis) and MALDI-TOF-MS (matrix assisted laser desorption-ionization time-of-flight tandem mass spectrometry). Some proteins obtained were validated by Western blotting. Results: The 2-DE maps of total proteins in HT-29 and HT-29/L-OHP cells were established. Of the 38 protein spots identified as differentially expressed proteins (over two-fold, P < 0.05) between HT-29 and HT-29/L-OHP cells, 37 protein spots were positively identified by MALDI-TOF-MS (17 proteins were up-regulated and 20 proteins were down-regulated as compared with the parental HT-29 cells). The result of Western blotting showed that the PCBP1 [poly (C)-binding protein-1] and TUBB2A (tubulin beta 2A ) proteins were up-regulated while ANXA3 (annexin A3) and STIP1 (stress-induced-phosphoprotein 1) proteins were down-regulated in HT-29/L-OHP cells. The result of Western blotting was consistent with that of proteomics. Conclusion: There were 37 oxaliplatin-resistance-associated proteins in CRC identified in this study which may provide useful evidence in further research on mechanism of oxaliplatin-resistance in CRC. Copyright © 2013 by TUMOR.

Establishment of a "Four-step method" for extraction and separation of Candida albicans total proteins / 第二军医大学学报

Objective: To introduce a "Four-step method" for extraction and separation of Candida albicans total proteins. Methods: Proteins of C. albicans were extracted step-by-step with 4 kinds of solutions with different solubilities. After quantification, the protein samples were separated by isoelectric focusing electrophoresis and then by SDS-PAGE. Results: Proteins with different solubilities were successfully extracted step-by-step from C. albicans and were separated by two-dimensional gel electrophoresis. Conclusion: The "Four-step method" for extraction of C. albicans proteins is an effective approach to study C. albicans membrane proteome and lays a foundation for further investigation of the mechanisms of antifungal agents and drug resistance in C. albicans.

Polymorphisms of COTL1 gene identified by proteomic approach and their association with autoimmune disorders

To select candidate genes, we attempted to comparative analysis of protein levels between rheumatoid arthritis (RA) patients and healthy controls by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). We identified 17 proteins that showed up- or down-regulated spots in RA patients. We found that coactosin-like1 (COTL1) were highly expressed in RA patients compared with healthy controls. We performed a case-control study to determine whether the COTL1 gene polymorphisms were associated with RA and systemic lupus erythematosus (SLE). The genotype frequency of c.-1124G>T and the allelic frequency of c.484G>A in RA patients, and the genotype frequency of c.484G>A in SLE patients were significantly different from healthy controls (P = 0.009, 0.027, and 0.025, respectively). We also investigated the correlation with the levels of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibody in RA patients, and anti-nuclear antibodies (ANA) in SLE patients. The c.484G>A polymorphism in RA patients has significant association with the levels of anti-CCP antibody (P = 0.03). Our findings demonstrated that c.-1124G>T and c.484G>A polymorphisms of the COTL1 gene might be associated with the genetic susceptibility of autoimmune disorders.

Polymorphisms of COTL1 gene identified by proteomic approach and their association with autoimmune disorders

To select candidate genes, we attempted to comparative analysis of protein levels between rheumatoid arthritis (RA) patients and healthy controls by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). We identified 17 proteins that showed up- or down-regulated spots in RA patients. We found that coactosin-like1 (COTL1) were highly expressed in RA patients compared with healthy controls. We performed a case-control study to determine whether the COTL1 gene polymorphisms were associated with RA and systemic lupus erythematosus (SLE). The genotype frequency of c.-1124G>T and the allelic frequency of c.484G>A in RA patients, and the genotype frequency of c.484G>A in SLE patients were significantly different from healthy controls (P = 0.009, 0.027, and 0.025, respectively). We also investigated the correlation with the levels of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibody in RA patients, and anti-nuclear antibodies (ANA) in SLE patients. The c.484G>A polymorphism in RA patients has significant association with the levels of anti-CCP antibody (P = 0.03). Our findings demonstrated that c.-1124G>T and c.484G>A polymorphisms of the COTL1 gene might be associated with the genetic susceptibility of autoimmune disorders.

Comparative proteomic study at the different stages during initiation and evolution of experimental colorectal carcinoma / 肿瘤

Objective: To research the proteins differentially expressed during evolution of experimental colorectal carcinoma (normal mucosa→adenoma→carcinoma→liver metastasis) so as to find the early diagnostic biomarker of colorectal cancer as well as to understand its pathogenesis mechanism. Methods: Ninety male rats were injected with 1, 2-dimethylhydrazine intraperitoneally and sacrificed at different weeks to establish the experimental colorectal tumor models (from normal mucosa to liver metastasis). These samples at different stages were collected and divided into four groups (normal mucosa group, adenoma group, carcinoma group, and liver metastasis group). The proteins of these 4 groups were extracted to conduct 2-dimensional gel electrophoresis. The differential protein spots were examined by mass spectrometry and analyzed by bioinformatics. Results: Ten differentially expressed proteins were identified by 2-dimensional gel electrophoresis and mass spectrometry including α-enolase, cardiac α-actin (CA), transgelin protein, myosin regulatory light chain smooth muscle isoform (MRLC), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), haptoglobin, disulfideisomerase (DI), creatine kinase mitochondrial (CKm), heat shock protein-8 (HSP-8) and Keratin complex-2 (KC-2). Conclusions: There exist differentially expressed proteins at various stages during the evolution of colorectal carcinoma. These proteins may be the candidate biomarkers for the early diagnosis of colorectal carcinoma. Proteomic technology is an effective way for preliminary identification of the tumor biomarkers.

Effects of hSSTR2 gene in vitro transfection on differential proteins expression in pancreatic cancer cell line Panc-1 / 中华胰腺病杂志

Objective To study the effects of hSSTR2 gene in vitro transfection on differential proteins expression in pancreatic cancer cell line Panc-1 and search new sensitive therapeutic targets of pancreatic cancer. Methods The full length hSSTR2 cDNA was introduced into pancreatic cancer cell line Panc-1 by adenovirus vector ( Ad. CMV. hSSTR2. GFP) mediated transfection. The differential expressed proteins between the hSSTR2 transfection group, vector control and mock control were isolated and screened by 2D-DIGE analysis. Protein identification was performed by peptide mass finger printing with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF). Results The hSSTR2 gene was transfected into Panc-1 pancreatic cancer cells in vitro successfully, and fluorescence difference protein expression patterns were established between hSSTR2 negative and positive expression of Panc-1 cell. Analysis by DeCyder v6.5 software showed a total of 18 protein spots ( > 1.3-fold) and these protein spots were identified by mass spectrometry as 13 proteins. Proteins with lower abundance levels included GMP synthase, stress induced phosphoprotein 1, glutamate dehydrogenase 1, Septin-11, vimentin, Isocitrate dehydrogenase [NAD] subunit alpha, Import inner membrane translocase subunit TIM50. Proteins with high abundance levels included Elongation factor 1-alpha-1, Isoform M2 of Pyruvate kinase isozymes M1/M2, Enoyl-CoA hydratase,tripartite motif-containing 28 protein, Mitofilin, HSP105. Conclusions The proteins expression changed after hSSTR2 gene in vitro transfection in Panc-1 cells, and the function of difference proteins involved the process of metabolism of sugar, fat and nucleic acid, and the regulation of cell growth. The present study paved the way for searching new sensitive therapeutic targets of pancreatic cancer.

Comparative study on the proteomic analysis in cervical carcinoma tissues with different radiosensitivities / 肿瘤

Objective: To compare the proteomic differences in early cervical carcinoma tissues with various radiosensitivities and provide the basis for determining of radiosensitivity-associated proteins in cervical carcinoma. Methods: The fresh carcinoma tissues were collected from 10 untreated cervical carcinoma patients and preserved at - 80C. According to WHO Criterion of Therapeutical Effect of Solid Carcinoma, the tissues were classified into two groups: high sensitive group (HS) and low sensitive group (LS). The total protein was extracted and separated by using two-dimensional gel electrophoresis (2-DE). The PD-quest 7.0 software was used to perform image matching and difference analysis to identify the differentially expressed protein-spots between the two groups. The identified protein spot was cut in electrophoretic gel in situ, digested with trypsin, and analyzed by Matrix-assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS). The peptide mass fingerprintings (PMF) was obtained. The proteins were identified by data searching in the Mascot database. Some differentially expressed proteins were confirmed by Western blotting. Results: The constructed two-dimensional gel electrophoresis profiles had high resolution and good reproducibility for both HS and IS groups. There were (754 + 64) differentially expressed protein spots (n = 5) in HS group and (777 ± 48) in LS group (n = 5). Some protein spots with differential ratio more than 2-fold were selected and analyzed by mass spectrometry. The average matching rate was 87.6% between the two groups. Eight differentially expressed proteins were successfully identified among which five proteins had overexpression and three proteins had weak expression in HS group. The results of Western blotting were consistant with proteomic screening. Conclusion: There was significant difference in protein profilings between HS and LS early cervical carcinoma after radiotherapy. The differentially expressed proteins may be radiosensitivity-associated proteins.

Mass spectrometry based proteomic analysis of human stem cells: a brief review

Stem cells can give rise to various cell types and are capable of regenerating themselves over multiple cell divisions. Pluripotency and self-renewal potential of stem cells have drawn vast interest from different disciplines, with studies on the molecular properties of stem cells being one example. Current investigations on the molecular basis of stem cells pluripotency and self-renewal entail traditional techniques from chemistry and molecular biology. In this mini review, we discuss progress in stem cell research that employs proteomics approaches. Specifically, we focus on studies on human stem cells from proteomics perspective. To our best knowledge, only the following types of human stem cells have been examined via proteomics analysis: human neuronal stem cells, human mesenchymal stem cells, and human embryonic stem cells. Protein expression serves as biomarkers of stem cells and identification and expression level of such biomarkers are usually determined using two-dimensional electrophoresis coupled mass spectrometry or non-gel based mass spectrometry.

Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues and its potential post-translational modification

Gastric cancer is one of the most common cancers worldwide. The purpose of this study was to find out potential markers for gastric cancer. Tumor and normal tissues from 152 gastric cancer cases were analyzed by two-dimensional gel electrophoresis (2-DE). The images of silver stained gels were analyzed and statistical analysis of spot intensities revealed that spot 4262 showed higher expression (5.7-fold increase) in cancer tissues than in normal tissues (P< 0.001). It was identified by peptide mass fingerprinting as nicotinamide N-methyltransferase (NNMT). A monoclonal antibody with a detection limit down to 10 ng was produced against NNMT in mouse. Using the prepared monoclonal antibody, western blot analysis of NNMT was performed for gastric tissues from 15 gastric cancer patients and two gastric ulcer patients. The results corroborated those of 2-DE experiments. A single spot was detected in gastric ulcer tissues while four to five spots were detected in gastric cancer tissues. In cancer tissues, two additional spots of acidic and basic form were mainly detected on 2-DE gels. This suggests that NNMT receives a post-translational modification in cancer- specific manner.

Identification of Proteins Differentially Expressed in the Conventional Renal Cell Carcinoma by Proteomic Analysis

Renal cell carcinoma (RCC) is one of the most malignant tumors in urology, and due to its insidious onset patients frequently have advanced disease at the time of clinical presentation. Thus, early detection is crucial in management of RCC. To identify tumor specific proteins of RCC, we employed proteomic analysis. We prepared proteins from conventional RCC and the corresponding normal kidney tissues from seven patients with conventional RCC. The expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The overall protein expression patterns in the RCC and the normal kidney tissues were quite similar except some areas. Of 66 differentially expressed protein spots (p<0.05 by Student t-test), 8 different proteins from 11 spots were identified by MALDI-TOF-MS. The expression of the following proteins was repressed (p<0.05); aminoacylase-1, enoyl-CoA hydratase, aldehyde reductase, tropomyosin alpha-4 chain, agmatinase and ketohexokinase. Two proteins, vimentin and alpha-1 antitrypsin precursor, were dominantly expressed in RCC (p<0.05).

Identification of New Proteins in Follicular Fluid from Mature Human Follicles by Direct Sample Rehydration Method of Two-Dimensional Polyacrylamide Gel Electrophoresis

Human follicular fluid (HFF) includes various biologically active proteins which can affect follicle growth and oocyte fertilization. Thus far, these proteins from mature follicles in human follicular fluid have been poorly characterized. Here, two-dimensional polyacrylamide gel electrophoresis (2-DE) with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to identify new proteins in HFF. Mature follicular fluids were obtained from five females after oocyte collection during in vitro fertilization (IVF). We directly rehydrated HFF samples, obtained high-resolution 2-DE maps, and processed them for 2-DE and MALDI-MS. One hundred eighty spots were detected and 10 of these spots were identified. By the 2-DE database, six of them had been reported, as proteins already existing in HFF. Hormone sensitive lipase (HSL), Unnamed protein product 1 (UPP1), Unnamed protein product 2 (UPP2), and apolipoprotein A-IV precursor were newly detected. HSL and apolipoprotein A-IV participate in lipid metabolism. UPP1 has a homology with selenocysteine lyase. We found by RT-PCR that these genes are expressed from human primary granulosa cells. The proteins identified here may emerge as potential candidates for specific functions during folliculogenesis, hormone secretion regulation, or oocyte maturation. Further functional analysis of these proteins is necessitated to determine their biological implications.