Exploration of Potential Tumor Markers for Lung Adenocarcinomas by Two‐Dimensional Gel Electrophoresis Coupled with Nano‐LC/MS/MS

To explore the potential tumor markers for lung adenocarcinoma, two‐dimensional gel electrophoresis (2DE) coupled with nano‐LC/MS/MS was used to analyze the differentially expressed proteins in 10 surgical resected lung adenocarcinoma tissues. 16 proteins were significantly different between the cancer tissue and adjacent normal tissue. Galectin‐1, peroxiredoxin II (Prx II), proapolipoprotein, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), aldolase A, enolase 1, neuropolypeptide h3, Prx V, cyclophilin A, vimentin, protein disulfide isomerase (PDI), tropomyosin 3 (TPM 3), glutathione S‐transferase Pi (GST‐Pi), manganese superoxide dismutase (MnSOD), and cofilin 1 were up‐regulated in the cancer tissue. On the other hand, profilin was down‐regulated in the cancer tissue. Among these proteins, six proteins were validated by Western blot analysis. The identified proteins contributing to the spectrum of cancer progression may be used as potential diagnostic biomarkers for lung adenocarcinomas.     

Proteomics Analysis of Up-regulated NPM1 Protein in Colorectal Cancer

In order to identify potential protein targets involved in colorectal cancer(CRC), we used a liquid chromatography coupled with mass spectrometry(LC-MS)/MS-based proteomics approach to characterize global protein expression patterns in malignant tissues and their adjacent healthy tissues from Dukes C stage CRC patients. A total number of 34 differentially expressed proteins were detected and identified by LC-MS/MS and database searching, which are supposed to be relevant to progression of colorectal tumor. Among these proteins, nucleophosmin 1(NPM1) was found to be remarkably up-regulated in colorectal carcinoma tissues, as compared with that in their normal counterparts. The results presented here could provide clues to elucidate the pathological significance of NPM1 in regulation of carcinogenesis of Dukes C stage colorectal tumors.     

5‐Methoxytryptophan‐dependent inhibition of oral squamous cell carcinoma metastasis

The metastatic status of oral cancer is highly associated with the overall survival rate of patients. Previous studies have revealed that the endogenous tryptophan metabolite 5‐methoxytryptophan (5‐MTP) can downregulate cyclooxygenase‐2 expression; suppress tumor proliferation, migration, and invasion; and reduce the tumor size. To improve the understanding of the molecular mechanisms involved in the regulation of 5‐MTP in the tumorigenesis of oral cancer, we conducted a comparative wound healing and transwell invasion assays. Our results revealed that 5‐MTP reduce oral cancer cell migration and invasion ability. In addition, the results of an in vivo assay demonstrated that the growth of primary tumors was significantly inhibited by 5‐MTP in OC3 oral cancer cells and in invasive OC3‐I5 oral cancer cells. Moreover, enlarged spleens were observed in OC3‐I5‐implanted severe combined immunodeficiency mice although 5‐MTP can inhibit spleen enlargement. Through comparative proteomics, we identified 32 differentially regulated protein spots by using 2D‐DIGE/MALDI‐TOF MS analyses. Some of the differentially regulated proteins such as amadillo‐repeat‐containing X‐linked protein 1, phosphoglycerate kinase 1, tropomyosin alpha‐1, and tropomyosin alpha‐4 may be associated with the 5‐MTP‐dependent inhibition of oral cancer growth and metastasis. We conclude that 5‐MTP plays a crucial role in inhibiting in vitro and in vivo cancer invasion and metastasis.     

Down-regulation of Vinculin in Human Colorectal Carcinoma Identified by Proteomics Analysis

In the present study, we aimed to globally profile the proteins involved in colorectal carcinoma(CRC), in order to find clues to the pathological process of CRC. Pairs of malignant tissues and their adjacent healthy tissues from patients with colorectal cancer were subject to differential proteomics analysis. Two dimensional electrophoresis coupled with mass spectrometry(2-DE/MS) was used to identify differentially expressed proteins between pairs of tissue samples. A list of proteins relevant to the progression of colorectal tumor was identified by two dimensional gel electrophoresis(2-DE)-based proteomics approach. Among the identified proteins, vinculin was found to be remarkably down-regulated in colorectal carcinoma tissues. In addition, three phosphorylation modifications within the isolated vinculin were identified by in-depth liquid chromatography-tandem mass spectrometry(LC-MS/MS) analysis. Our results provide a basis for further understanding the pathological significance of vinculin in the regulation of carcinogenesis, invasion and metastasis of colorectal tumors.     

Proteomic analysis reveals changes in the hippocampus protein pattern of rats exposed to dietary zinc deficiency

An experiment was performed to observe protein changes in the hippocampus of zinc‐deficient (ZD) rats. Twenty‐four male weanling Wistar rats were randomly assigned to ZD (n=12) and control groups (n=12). After 4‐wk treatment, we used 2‐DE and MALDI‐TOF‐MS to analyze the proteomes of hippocampus in the two groups. One of the important differential proteins, ubiquitin C‐terminal hydrolase L1 (Uch‐L1), was confirmed by Western blot assays. The results demonstrated that compared with the controls, ZD rats had significantly reduced plasma zinc concentration and alkaline phosphatase activity. The latency period in passive avoidance performance was also significantly shorter for the ZD rats. Nine proteins were differentially expressed between the two groups. Eight of them were identified. Tubulin β chain and voltage‐dependent anion channel 1 were upregulated, while mitochondrial aldehyde dehydrogenase, α‐enolase, dimethylarginine dimethylaminohydrolase 1, F‐actin capping protein α‐2 subunit, pyruvate dehydrogenase β and Uch‐L1 were downregulated, respectively. Importantly, some of the identified proteins (e.g. Uch‐L1) are known to be involved in cognitive impairment. Western blot analysis of hippocampus Uch‐L1 expression confirmed the proteomic findings. The data indicated that there may be common mechanisms or pathways in cognitive dysfunction between neurodegenerative diseases and zinc deficiency.     

The use of matrix coating assisted by an electric field (MCAEF) to enhance mass spectrometric imaging of human prostate cancer biomarkers

In this work, we combined a newly developed matrix coating technique – matrix coating assisted by an electric field (MCAEF) and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) to enhance the imaging of peptides and proteins in tissue specimens of human prostate cancer. MCAEF increased the signal‐to‐noise ratios of the detected proteins by a factor of 2 to 5, and 232 signals were detected within the m/z 3500–37500 mass range on a time‐of‐flight mass spectrometer and with the sinapinic acid MALDI matrix. Among these species, three proteins (S100‐A9, S100‐A10, and S100‐A12) were only observed in the cancerous cell region and 14 proteins, including a fragment of mitogen‐activated protein kinase/extracellular signal‐regulated kinase kinase kinase 2, a fragment of cAMP‐regulated phosphoprotein 19, 3 apolipoproteins (C‐I, A‐I, and A‐II), 2 S100 proteins (A6 and A8), β‐microseminoprotein, tumor protein D52, α‐1‐acid glycoprotein 1, heat shock protein β‐1, prostate‐specific antigen, and 2 unidentified large peptides at m/z 5002.2 and 6704.2, showed significantly differential distributions at the p < 0.05 (t‐test) level between the cancerous and the noncancerous regions of the tissue. Among these 17 species, the distributions of apolipoprotein C‐I, S100‐A6, and S100‐A8 were verified by immunohistological staining. In summary, this study resulted in the imaging of the largest group of proteins in prostate cancer tissues by MALDI‐MS reported thus far, and is the first to show a correlation between S100 proteins and prostate cancer in a MS imaging study. The successful imaging of the three proteins only found in the cancerous tissues, as well as those showing differential expressions demonstrated the potential of MCAEF‐MALDI/MS for the in situ detection of potential cancer biomarkers. Copyright © 2015 John Wiley & Sons, Ltd.     

凝胶电泳-毛细管液相色谱-质谱联用技术分析大肠癌肿瘤与癌旁组织的差异蛋白质组

通过凝胶电泳将大肠癌患者的手术标本组织(肿瘤与癌旁)中的总蛋白按照分子量分成不同的组分,经胶内水解后,利用毛细管液相色谱-质谱联用技术对各个组分的多肽混合物进行蛋白质组学分析,共鉴定出29种差异表达的蛋白质,其中肿瘤组织中表达上调的蛋白质19种,如Fibrinogen beta chain,Vimentin,Fi-brinogen gamma chain,Histone H2A type 1-B/E,Isoform 1 of Periostin,Fibrinogen alpha chain,Histone H3.1,Al-pha-enolase,Protein disulfide-isomerase A3等;在肿瘤组织中表达下调的有10种蛋白质,如Sarcomeric tropomy-osin kappa,Transgelin,Heat shock protein beta-1,Talin-1,Isoform 2 of Vinculin,Isoform 1 of Filamin-C等.通过蛋白质印迹实验对其中的踝蛋白1(Talin-1)和烯醇化酶(Alpha-enolase)的蛋白质组学鉴定结果进行了验证.生物信息学分析表明在大肠癌组织中表达上调的蛋白多具有胞外分泌的属性,提示这些蛋白很有可能会渗透到机体内的循环系统中,从而在患者血液或者腹水中能够被检测到;而在大肠癌组织中表达下调的蛋白则多为细胞骨架、胞外基质纤维等的组成成分,这些蛋白的下调常与肿瘤细胞向周围组织的侵袭和转移有密切关系.本研究为大肠癌的早期诊断、大肠癌初筛等方面的研究等提供了基础数据,对进一步深入研究大肠癌发病机制具有重要意义.     

Using Single‐Cell Amperometry To Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis

The pretreatment of cultured pheochromocytoma (PC12) cells with cis‐diamminedichloroplatinum (cisplatin), an anti‐cancer drug, influences the exocytotic ability of the cells in a dose‐dependent manner. Low concentrations of cisplatin stimulate catecholamine release whereas high concentrations inhibit it. Single‐cell amperometry reflects that 2 μm cisplatin treatment increases the frequency of exocytotic events and reduces their duration, whereas 100 μm cisplatin treatment decreases the frequency of exocytotic events and increases their duration. Furthermore, the stability of the initial fusion pore that is formed in the lipid membrane during exocytosis is also regulated differentially by different cisplatin concentrations. This study thus suggests that cisplatin influences exocytosis by multiple mechanisms.     

lncRNA-miRNA-mRNA interaction network for colorectal cancer; An in silico analysis

BackgroundColorectal cancer (CRC) is one of the most frequent and diagnosed diseases. Accumulating evidences showed that mRNAs and noncoding RNAs play important regulatory roles in tumorigenesis. Identification and determining the relationship between them can help diagnosis and treatment of cancer.MethodsHere we analyzed three microarray datasets; GSE110715, GSE32323 and GSE21510, to identify differentially expressed lncRNAs and mRNAs in CRC. The adjusted p-value ≤0.05 was considered statistically significant. Gene set enrichment analysis was carried out using DAVID tool. The miRCancer database was searched to obtain differentially expressed miRNAs in colorectal cancer, and the miRDB database was used to attain the targets of the obtained miRNAs. To predict the lncRNA-miRNA interactions we used DIANA-LncBase v2 and RegRNA 2.0. Finally the lncRNA-miRNA-mRNA-signaling pathway network was constructed using Cytoscape v3.1.ResultsBy analyzing the three datasets, a total of 21 mRNAs (15 up- and 6 down-regulated) and 24 lncRNAs (18 up- and 6 down-regulated) were identified as common differentially expressed genes between CRC tumor and marginal tissues. Nevertheless, the constructed lncRNA-miRNA-mRNA-signaling pathway network revealed a convergence on 6 lncRNAs (3 up- and 3 downregulated), 7 mRNAs (2 up- and 5 downregulated) and 6 miRNAs (3 up- and 3 downregulated). We found that dysregulation of lncRNAs such as PCBP1-AS1, UCA1 and SNHG16 could sequester several miRNAs such as hsa-miR-582-5p and hsa-miR-198 and promote the proliferation, invasion and drug resistance of colorectal cancer cells.ConclusionsWe introduced a set of lncRNAs, mRNAs and miRNAs differentially expressed in CRC which might be considered for further experimental research as potential biomarkers of CRC development.     

Proteomic identification of exosomal LRG1: a potential urinary biomarker for detecting NSCLC

In the present research, we aimed to screen for non-small cell lung cancer (NSCLC)-related proteins in urinary exosomes by comparing urinary exosomes proteome of normal controls and NSCLC patients. Urinary exosomes were isolated by ultracentrifugation and identified by electron microscopy. Exosomal proteins were separated by 1-D SDS-PAGE and the differentially expressed bands between healthy controls and NSCLC patients ranging in size from 35 to 45?kD were cut from the gel. After tryptic digestion, 18 proteins were identified by nano-HPLC-chip-MS/MS. The differential expression of leucine-rich α-2-glycoprotein (LRG1) was further validated in urinary exosomes by Western blot and in lung tissue by immunohistochemistry. The LRG1 was found to be expressed at higher levels in urinary exosomes and lung tissue of NSCLC patients. These results suggested that LRG1 may be a candidate biomarker for non-invasive diagnosis of NSCLC in urine.     

Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE

Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method.     

Screening of exon methylation biomarkers for colorectal cancer via LC‐MS/MS strategy

The identification of biomarkers would be of benefit for the diagnosis and treatment of colorectal cancer. DNA methylation in specific genomic regions, which had shown strongly association with disease genotypes, was an effective indicator to reveal the occurrence and development of cancers. To screen out methylation biomarkers for colorectal cancer (CRC), genomic DNA was isolated from colorectal cancerous and corresponding cancer‐adjacent tissues collected from 30 CRC patients and then bisulfite‐converted. The exon regions of 5 targeted genes (CNRIP1 , HIC1 , RUNX3 , p15 , and SFRP2 ) were amplified by using nested polymerase chain reaction with specific primers, and the amplicon was purified and hydrolyzed. The methylation levels of these specific regions were detected by liquid chromatography tandem mass spectrometry (LC‐MS/MS). The results showed that 5 targeted exon regions were successfully amplified and confirmed by sequencing. The methodological validations indicated that LC‐MS/MS was highly sensitive and accurate. The methylation levels of CNRIP1 and RUNX3 were remarkably high in CRC tissues with statistical difference when compared with corresponding cancer‐adjacent individuals, while that of HIC1 , p15 , and SFRP2 had no difference between 2 subjects. These findings supported CNRIP1 and RUNX3 as potential DNA methylation biomarkers for CRC diagnosis and treatment, and our LC‐MS/MS approach exhibited great advantages in the identification of regional DNA methylation biomarkers.     

Low C24‐OH and C22‐OH sulfatides in human renal cell carcinoma

Histopathologic diagnosis of renal cell carcinoma (RCC) may sometimes be difficult with small biopsy samples. We applied histology‐directed matrix‐assisted laser desorption/ionization mass spectrometry to RCC samples to evaluate whether and how lipid profiles are different between RCC and normal tissue. We evaluated 59 RCC samples and 24 adjacent normal tissue samples collected from patients who underwent surgery. Five peaks were significantly differently expressed (p < 10^?7) between RCCs and adjacent normal tissue samples. C24‐OH sulfatide (ST‐OH {18:1/24:0}[M‐H]^?; m/z 906.7 in the negative ion mode) and C22‐OH sulfatide (ST‐OH {18:1/22:0}[M‐H]^?; m/z 878.6 in the negative ion mode) were most significantly underexpressed in RCC samples, compared with adjacent normal tissue samples. With 100 random training‐to‐test partitions within these samples, the median prediction accuracy (RCC vs. normal) ranged from 96.3% to 100% at p cutoff values for feature selection ranging from 0.001 to 10^?7. Two oncocytoma samples were predicted as normal tissue by five lipids that were differentially expressed between RCC and normal tissue at p < 10^?7. Clear‐cell, papillary, and chromophobe RCCs were different in lipid profiles. Permutation p‐ values for 0.632+ bootstrap cross‐validated misclassification rates were less than 0.05 for all the classifiers. Thus, lipid profiles differentiate RCC from normal tissue and may possibly classify the histology of RCC. © 2014 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons, Ltd.     

Rapid identification of glycerophospholipids from RAW264.7 cells by UPLC/ESI ‐QTOF‐MS

The study aims to develop a rapid, sensitive ultra‐performance liquid chromatography coupled with an electrospray ionization quadruple time‐of‐flight tandem mass spectrometry (UPLC/Q‐TOF‐MS) analytical method for identifying glycerophospholipids (GPLs) from RAW264.7 cells. A total of 78 GPLs including 22 phosphatidylethanolamines (PEs), 49 phosphatidylcholines (PCs), four phosphatidylglycerols, one phosphatidylinositol and two unknown GPLs were identified. PC (14:0/16:1), PC (14:0/16:0), PE (0:0/20:3), PE (22:5/0:0) and PE (22:3/0:0) were identified for the first time. The UPLC/Q‐TOF‐MS method is suitable for targeting analysis of GPLs from RAW264.7 cells, which allows us to find out new GPLs compositions related to inflammatory diseases and to explain their pharmacological roles in inflammatory process. Copyright © 2014 John Wiley & Sons, Ltd.     

Studies of keratins in tongue coating samples of hepatitis B patients by mass spectrometry

Pooled tongue coating samples from 64 hepatitis B patients and 24 healthy adults were studied and a major band of differential proteins was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). The differential proteins in this band were identified and proved to be keratins by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and Western blot analysis. Furthermore, relative quantification of the identified keratins was performed via using stable isotopic labeling and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS), showing the higher expression level of these keratins in tongue coating samples of hepatitis B patients than healthy adults. These results provided additional information to understand the medical diagnosis depending on the tongue coating. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparative proteomic analysis of different Toxoplasma gondii genotypes by two‐dimensional fluorescence difference gel electrophoresis combined with mass spectrometry

Toxoplasma gondii is a protozoan parasite infecting almost all warm‐blooded animals and humans. There are three infective stages of T. gondii: the tachyzoites, the bradyzoites, and the oocysts. The tachyzoite is a rapidly multiplying stage and the main pathogenic factor. In North America and Europe, T. gondii is consisted of four major clonal lineages (namely Types I, II, III, and Type 12). In this study, we explored the proteomic profiles of different genotypes (Type I‐RH strain, Type II‐PRU strain, Type II‐TgQHO strain, and ToxoDB 9‐TgC7 strain) of T. gondii tachyzoites by using 2D DIGE combined with MALDI‐TOF MS. Totally, 110 differentially abundant protein spots were selected. Of these, 98 spots corresponding to 56 proteins from T. gondii were successfully identified. These included surface antigen (SAG1), heat shock protein 70 (Hsp 70), disulfide isomerase, coronin, heat shock protein 60 (Hsp 60), pyruvate kinase, receptor for activated C kinase 1, and peroxiredoxin. Gene ontology enrichment analysis revealed that most of the differentially abundant proteins were involved in biological regulation, metabolic process, response to stress, binding, antioxidant activity, and transporter activity. According to the KEGG metabolic pathway maps of T. gondii, some identified proteins were involved in the glycolytic/gluconeogenesis pathway. The present study identified differentially abundant proteins among different genotypes of T. gondii and these findings have implications for the better understanding of the phenotypic differences among the examined T. gondii genotypes, which in turn may contribute to the better control of toxoplasmosis.     

Unveiling the rat urinary proteome with three complementary proteomics approaches

Urine is a suitable biological fluid to look for markers of physiological and pathological processes, including renal and nonrenal diseases. In addition, it is an optimal body sample for diagnosis, because it is easily obtained without invasive procedures and can be sampled in large quantities at almost any time. Rats are frequently used as a model to study human diseases, and rat urine has been analyzed to search for disease biomarkers. The normal human urinary proteome has been studied extensively, but the normal rat urinary proteome has not been studied in such depth. In light of this, we were prompted to analyze the normal rat urinary proteome using three complementary proteomics platforms: SDS‐PAGE separation, followed by LC‐ESI‐MS/MS; 2DE, followed by MALDI‐TOF‐TOF and 2D‐liquid chromatography‐chromatofocusing, followed by LC‐ESI‐Q‐TOF. A total of 366 unique proteins were identified, of which only 5.2% of unique proteins were identified jointly by the three proteomics platforms used. This suggests that simultaneous proteomics techniques provide complementary and nonredundant information. Our analysis affords the most extensive rat urinary protein database currently available and this may be useful in the study of renal physiology and in the search for biomarkers related to renal and nonrenal diseases.