Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.     

Physical mapping by PFGE localizes the COL3A1 and COL5A2 genes to a 35-kb region on human chromosome 2

The genes encoding the alpha 1 chain of Type III collagen (COL3A1) and the alpha 2 chain of Type V (COL5A2) collagen have been mapped to the long arm of human chromosome 2. Linkage analysis in CEPH families indicated that these two genes are close to each other, with no recombination in 37 informative meioses. In the present study, DNA probes from the 3' ends of each gene have been physically mapped by pulsed-field gel electrophoresis. The probes recognized 11 macrorestriction fragments in common, ranging from greater than 1000 kb MluI and NotI fragments to a 35-kb SfiI fragment. Therefore, the COL3A1 and COL5A2 genes appear to exist as a gene cluster on chromosome 2. This is the third example of a collagen gene cluster. Other examples include the COL4A1-COL4A2 genes on chromosome 13q and the COL6A1-COL6A2 genes on chromosome 21q. The physical proximity of these genes may indicate common evolution and/or regulation.     

Assessment of the partitioning capacity of high abundant proteins in human cerebrospinal fluid using affinity and immunoaffinity subtraction spin columns

The performance of three different affinity and immunoaffinity subtraction spin columns was investigated for the removal of the most abundant proteins in human cerebrospinal fluid (CSF). A pool of human CSF was processed with the spin columns and both the bound and flow through fractions were compared with each other and with intact CSF using 1D gel electrophoresis and nanoLC–MALDI-TOF/TOF-MS analysis. MASCOT MS/MS ionscores were compared before and after processing with the columns. The non-specific co-removal of proteins bound to the high abundant proteins, so called “sponge effect” was also examined for each spin column. The reproducibility of one of the spin columns, ProteomeLab IgY-12 proteome partitioning spin column, was further investigated by isobaric tags for relative and absolute quantification (iTRAQ) labeling and MS/MS analysis. Overall, 173 unique proteins were identified on a 95% MudPIT confidence scoring level. For all three spin columns, the number of proteins identified and their MASCOT scores were increased up to 10 times. The largest degree of non-specific protein removal was observed for a purely affinity based albumin removal column, where 28 other proteins also were present. The ProteomeLab IgY-12 proteome partitioning spin column showed very high reproducibility when combined with iTRAQ labeling and MS/MS analysis. The combined relative standard deviation (R.S.D.) for the high abundant protein removal, iTRAQ labeling and nanoLC–MALDI-TOF/TOF-MS analysis was less than 17.5%.     

胃癌及癌旁组织定量比较蛋白质组学研究

为寻找胃癌特异的肿瘤标记物,用于胃癌临床诊断及药物治疗靶点的选择,本研究采用荧光差异显示凝胶电泳（DIGE）技术分离并筛选 Cy3、Cy5 及 Cy2 荧光素标记的胃癌及对应癌旁组织差异表达蛋白质,用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）或串联质谱技术进行鉴定并分析。结果共筛选出 33 个差异表达蛋白质点,其中 9 个蛋白质点在胃癌组织中上调,24 个蛋白质点下调。对 22 个蛋白质点采用质谱技术成功鉴定,突变结蛋白、锰超氧化物歧化酶、热休克蛋白 60等在胃癌中高表达,热休克蛋白 27、前列腺素 F 合酶、硒结合蛋白 1、锌指蛋白 160、微管蛋白 α6、真核生物翻译延伸因子 1 α1 等在胃癌组织中低表达,并筛选出 5 个未知蛋白。这些差异表达蛋白可望成为胃癌诊断的特异标记物,并与胃癌的发生、发展及预后等有关,为胃癌的诊断、发生机制的研究提供了新的思路。     

Identification of differential expressed proteins and characterization their mRNA expression in thermally stressed Apostichopus japonicus

In this study, we present a comparative proteomic analysis of the global protein expression changes in sea cucumber after 7 days exposure at 25 °C. Using two-dimensional electrophoresis followed by MALDI-TOF MS/MS, 27 protein spots with significant differences in abundance were identified and characterized. The identified proteins belonged primarily to the following four functional categories: cytoskeletal, material and energy metabolism, calcium homeostasis and extracellular matrix. The mRNA expression levels of 7 differentially expressed proteins were further assessed by qRT-PCR. The expression levels of 6 genes, including collagen, ATP synthase, major yolk protein, ferritin, nectin and protein disulfide isomerase showed significant differences under thermal stress, and among them, only two genes—ATP synthase and major yolk protein—showed consistent levels of protein and mRNA expression. Our results offer insight into the complex changes in protein turnover during higher temperature exposure in sea cucumber.     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

Determination of protein synthesis in vivo using labeling from deuterated water and analysis of MALDI-TOF spectrum.

This paper describes a method of determining protein synthesis and turnover using in vivo labeling of protein with deuterated water and analysis of matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF) spectrum. Protein synthesis is calculated using mass isotopomer distribution analysis instead of precursor to product amino acid enrichment ratio. During protein synthesis, the incorporation of deuterium from water changes the mass isotopomer distribution (isotope envelop) according to the number of deuterium atoms (0, 1, 2, 3, etc.) incorporated, and the distribution of the protein with 0, 1, 2, 3,... atoms of deuterium follows a binomial distribution. A mathematical algorithm by which the distribution of deuterium isotopomers can be extracted from the observed MALDI-TOF spectrum is presented. Since deuterium isotopomers are unique to newly synthesized proteins, the quantitation of their distribution provides a method for the quantitation of newly synthesized proteins. The combined use of postsource decay sequence identification and mass isotopomer distribution analysis makes the use of in vivo labeling with deuterated water a precise method to determine specific protein synthesis.     

Turnover Rates of Hepatic Collagen and Circulating Collagen-Associated Proteins in Humans with Chronic Liver Disease

Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of ²H₂O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2–0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates.     

Proteomic identification of collagens and related proteins in human fibroblasts

Summary. Fibroblasts are used for diagnosis of a series of metabolic diseases and are particularly suitable for the diagnosis of collagen disorders. We aimed to generate a skin fibroblast map that would be suitable for the concomitant determination of collagen and collagen-related proteins.A human skin fibroblast cell line was cultivated, homogenised, proteins extracted and subject to two-dimensional gel electrophoresis with subsequent in-gel-digestion of protein spots and mass spectrometrical identification (MALDI-TOF).Collagen alpha1 (I) chain precursor, collagen alpha1 (III) chain precursor, collagen alpha2 (VI) precursor and collagen modifying enzymes prolyl 4-hydroxylase alpha-2-subunit precursor, procollagen-lysine 2-oxoglutarate 5-dioxygenase 1 and 2, protein disulfide isomerase ER-60 precursor and peptidyl-prolyl cis-trans isomerase were among the abundant proteins.The finding of collagen and collagen-related structures as well as the identification of other metabolic enzyme systems on one 2D gel may propose the use of this proteomic method for further characterization of collagen and collagen-related proteins or for preliminary screening of metabolic disorders.     

Difference in mass analysis using labeled lysines (DIMAL-K): a new, efficient proteomic quantification method applied to the analysis of astrocytic secretomes

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.     

Proteomic Analysis of Osteoblasts Exposed to Fluoride In Vitro

Proteomical analysis is defined as the characterization of the entire set of protein encoded a genome. Two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) are main techniques used in proteomic analysis to achieve information about protein expression profiles. Knowledge about the mechanism of skeletal fluorosis can be gained by recognizing changes in protein expression. To better understand the skeletal fluorosis process, osteoblasts isolated from calvarial of neonatal mouse were cultured and treated with 2 ppm fluoride for 72 h, and proteins of the osteoblast were profiled by 2-DE. With the analysis of Image-Master 2D analysis software, we detected a total number of 493 matching spots on 2-DE images. Among them, 28 protein spots showed twofold significant alteration (P < 0.05) in fluoride-exposed groups. Moreover, 12 proteins were identified by MALDI-TOF MS. These identified proteins in fluoride-exposed group were associated with cell proliferation, metabolism, and oxidative folding. Thus, our study provides useful information on fluoride-related changes of proteome and shows that proteomical analysis is a powerful methodology for the better understanding of skeletal fluorosis.     

Comparative Proteome Analysis of Human Temporal Cortex Lobes by Two-Dimensional Electrophoresis and Identification of Selected Common Proteins

Human brain proteins were isolated from left and right temporal cortex lobes at the age of 73, 23, 84 years and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient strip in the first dimension and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over 800 polypeptide spots were resolved with a silver-staining protocol by computerized 2-D gel analsis. Seven of the polypeptide spots were evidently distinguishable between human left and right temporal lobes. Four of the polypeptide spots were larger and three were smaller in human right temporal lobe. One of these three protein spots that have descendent expression in human right temporal lobe was identified as carbonyl reductase (NADPH) 1 by MALDI-TOF MS. Thirty-three common spots were identified by ESI-MS/MALDI-TOF MS/Edman sequencing and a protein database search. These identified proteins include some important enzymes and regulating proteins.     

Technical evaluation of MALDI-TOF mass spectrometry for quantitative proteomic profiling matrix formulation and application

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been recently used to identify disease markers by directly profiling and quantifying the peptide/proteins in biological samples under different physiological or experimental conditions. The information of reproducibility of such quantitative profiling method has not been available. It is important to evaluate and reduce error from technical variation. In this study, an unbiased signal acquisition strategy was used to evaluate the effects of three sample-matrix spotting methods and two matrix chemicals, α-cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid, on the reproducibility of the peptide/protein signal intensities. The sandwich spotting method using 0.1% nitrocellulose coating film and CHCA gave the best quantitative results for the standard peptides and proteins with mass<66.5 kDa. The normalized signal intensities of the standard peptides and proteins were directly proportional to their concentrations with intra-assay (within-day) coefficient of variations (CVs) ranging from 6.5% to 17%. When analyzing serum peptides <6000 m/z, the interassay (between-days) CVs of all the evaluated peptide peaks were <15%. These data indicate that with the right MS analysis conditions, MALDI-TOF MS appears to be a feasible tool for directly profiling and quantifying the peptide/ proteins in biological samples.     

Proteomic Analysis of Human Dermal Fibroblast Conditioned Medium (DFCM)

Growth factors and extracellular matrix (ECM) proteins are involved in wound healing. Human dermal fibroblasts secrete wound-healing mediators in culture medium known as dermal fibroblast conditioned medium (DFCM). However, the composition and concentration of the secreted proteins differ with culture conditions and environmental factors. We cultured human skin fibroblasts in vitro using serum-free keratinocyte-specific media (EpiLife? Medium [KM1] and defined keratinocyte serum-free medium [KM2]) and serum-free fibroblast-specific medium (FM) to obtain DFCM-KM1, DFCM-KM2 and DFCM-FM, respectively. We identified and compared their proteomic profiles using bicinchoninic acid assay (BCA), 1-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography MS (LC-MS/MS). DFCM-KM1 and DFCM-KM2 had higher protein concentrations than DFCM-FM but not statistically significant. MALDI-TOF/TOF MS identified the presence of fibronectin, serotransferrin, serpin and serum albumin. LC-MS/MS and bioinformatics analysis identified 59, 46 and 58 secreted proteins in DFCM-KM1, DFCM-KM2 and DFCM-FM, respectively. The most significant biological processes identified in gene ontology were cellular process, metabolic process, growth and biological regulation. STRING® analysis showed that most secretory proteins in the DFCMs were associated with biological processes (e.g. wound healing and ECM organisation), molecular function (e.g. ECM binding) and cellular component (e.g. extracellular space). ELISA confirmed the presence of fibronectin and collagen in the DFCMs. In conclusion, DFCM secretory proteins are involved in cell adhesion, attachment, proliferation and migration, which were demonstrated to have potential wound-healing effects by in vitro and in vivo studies.     

Measurement of protein turnover rates by heavy water labeling of nonessential amino acids

In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (2H2O) labeling can safely maintain stable body water 2H enrichments for weeks or months. 2H is metabolically incorporated into C-H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so 2H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of 2H2O-labeled rodent tissue proteins that metabolic 2H flux into C-H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By 2H2O labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl4 was detected. Kinetics of several human serum proteins were also measured, reproducing published t1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, 2H2O labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover.     

Intact protein profiling in breast cancer biomarker discovery: Protein identification issue and the solutions based on 3D protein separation,bottom‐up and top‐down mass spectrometry

Proteomics profiling of intact proteins based on MALDI‐TOF MS and derived platforms has been used in cancer biomarker discovery studies. This approach suffers from a number of limitations such as low resolution, low sensitivity, and that no knowledge is available on the identity of the respective proteins in the discovery mode. Nevertheless, it remains the most high‐throughput, untargeted mode of clinical proteomics studies to date. Here we compare key protein separation and MS techniques available for protein biomarker identification in this type of studies and define reasons of uncertainty in protein peak identity. As a result of critical data analysis, we consider 3D protein separation and identification workflows as optimal procedures. Subsequently, we present a new protocol based on 3D LC‐MS/MS with top‐down at high resolution that enabled the identification of HNRNP A2/B1 intact peptide as correlating with the estrogen receptor expression in breast cancer tissues. Additional development of this general concept toward next generation, top‐down based protein profiling at high resolution is discussed.     

Double standards in quantitative proteomics: direct comparative assessment of difference in gel electrophoresis and metabolic stable isotope labeling

Quantitative protein expression profiling is a crucial part of proteomics and requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. In this report we evaluate in a direct comparative assessment two state-of-the-art quantitative proteomic approaches, namely difference in gel electrophoresis (DiGE) and metabolic stable isotope labeling. Therefore, Saccharomyces cerevisiae was grown under well defined experimental conditions in chemostats under two single nutrient-limited growth conditions using (14)N- or (15)N-labeled ammonium sulfate as the single nitrogen source. Following lysis and protein extraction from the two yeast samples, the proteins were fluorescently labeled using different fluorescent CyDyes. Subsequently, the yeast samples were mixed, and the proteins were separated by two-dimensional gel electrophoresis. Following in-gel digestion, the resulting peptides were analyzed by mass spectrometry using a MALDI-TOF mass spectrometer. Relative ratios in protein expression between these two yeast samples were determined using both DiGE and metabolic stable isotope labeling. Focusing on a small, albeit representative, set of proteins covering the whole gel range, including some protein isoforms and ranging from low to high abundance, we observe that the correlation between these two methods of quantification is good with the differential ratios determined following the equation R(Met.Lab.) = 0.98R(DiGE) with r(2) = 0.89. Although the correlation between DiGE and metabolic stable isotope labeling is exceptionally good, we do observe and discuss (dis)advantages of both methods as well as in relation to other (quantitative) approaches.