Prediction of a Missing Protein Expression Map in the Context of the Human Proteome Project

Experimental evidence for the entire human proteome has been defined in the Human Proteome Project, and it is publicly available in the neXtProt database. However, there are still human proteins for which reliable experimental evidence does not exist, and the identification of such information has become one of the overriding objectives in the chromosome-centric study of the human proteome. With this aim and considering the complexity of protein detection using shotgun and targeted proteomics, the research community has addressed the integration of transcriptomics and proteomics landscapes. Here, we describe an analytical pipeline that predicts the probability of a missing protein being expressed in a biological sample based on (1) gene sequence characteristics, (2) the probability of an expressed gene being a coding gene of a missing protein in a certain sample, and (3) the probability of a gene being expressed in a transcriptomic experiment. More than 3400 microarray experiments were analyzed corresponding to three biological sources: cell lines, normal tissues, and cancer samples. A gene classification based on gene expression profiles distinguished among ubiquitous, nonubiquitous, nonexpressed, and coding genes of missing proteins. In addition, a different tissue-specific expression pattern for the coding genes of missing proteins is reported. Our results underline the relevance of selecting an appropriate sample for the detection of missing proteins and provide a comprehensive method to score their expression probability. Testis, brain, and skeletal muscle are the most promising normal tissues

Proteome of the Early Embryo–Maternal Dialogue in the Cattle Uterus

We analyzed embryo–maternal interactions in the bovine uterus on day 8 of development. Proteomic profiles were obtained by two-dimensional difference gel electrophoresis from 8 paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus. Results were contrasted with UF obtained after artificial insemination. We detected 50 differential protein spots (<i>t</i> test, <i>p</i> < 0.05). Subsequent protein characterization by nano-LC–ESI–MS/MS enabled us to identify 38 proteins, obtaining for first time the earliest evidence of involvement of the down-regulated NFkB system in cattle as a pregnancy signature pathway. Embryos enhanced the embryotrophic ability of UF and decreased uterine protein, while blood progesterone was unaltered. Twinfilin, hepatoma-derived growth factor, and synaptotagmin-binding cytoplasmic RNA interacting protein have not previously been identified in the mammalian uterus. TNFα and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNFα, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNFα were up-regulated. The embryonic signaling agents involved could be TNFα and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development

Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach

The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations

Embryonic Sex Induces Differential Expression of Proteins in Bovine Uterine Fluid

The bovine endometrium recognizes early embryos and reacts differently depending on the developmental potential of the embryo. However, it is unknown whether the endometrium can distinguish embryonic sex. Our objective was to analyze sexual dimorphism in the uterus in response to male and female embryos. Differentially expressed (DE) proteins, different levels of hexoses, and other embryotrophic differences were analyzed in uterine fluid (UF). Proteomic analysis of day-8 UF recovered from heifers after the transfer of day-5 male or female embryos identified 23 DE proteins. Regulated proteasome/immunoproteasome protein subunits indicated differences in antigen processing between UF carrying male embryos (male-UF) or female embryos (female-UF). Several enzymes involved in glycolysis/gluconeogenesis and antioxidative/antistress responses were up-regulated in female-UF. Fructose concentration was increased in female-UF versus male-UF, while glucose levels were similar. <i>In vitro</i> cultures with molecules isolated from male-UF were found to improve male embryo development compared to female embryos cultured with molecules isolated from female-UF. We postulated that, <i>in vivo</i>, male embryos induce changes in the endometrium to help ensure their survival. In contrast, female embryos do not appear to induce these changes

Detection of Missing Proteins Using the PRIDE Database as a Source of Mass Spectrometry Evidence

The current catalogue of the human proteome is not yet complete, as experimental proteomics evidence is still elusive for a group of proteins known as the missing proteins. The Human Proteome Project (HPP) has been successfully using technology and bioinformatic resources to improve the characterization of such challenging proteins. In this manuscript, we propose a pipeline starting with the mining of the PRIDE database to select a group of data sets potentially enriched in missing proteins that are subsequently analyzed for protein identification with a method based on the statistical analysis of proteotypic peptides. Spermatozoa and the HEK293 cell line were found to be a promising source of missing proteins and clearly merit further attention in future studies. After the analysis of the selected samples, we found 342 PSMs, suggesting the presence of 97 missing proteins in human spermatozoa or the HEK293 cell line, while only 36 missing proteins were potentially detected in the retina, frontal cortex, aorta thoracica, or placenta. The functional analysis of the missing proteins detected confirmed their tissue specificity, and the validation of a selected set of peptides using targeted proteomics (SRM/MRM assays) further supports the utility of the proposed pipeline. As illustrative examples, DNAH3 and TEPP in spermatozoa, and UNCX and ATAD3C in HEK293 cells were some of the more robust and remarkable identifications in this study. We provide evidence indicating the relevance to carefully analyze the ever-increasing MS/MS data available from PRIDE and other repositories as sources for missing proteins detection in specific biological matrices as revealed for HEK293 cells

Oral administration of MTA to <i>Mdr2^−/−</i> mice reduces hepatomegaly and serum parameters of liver injury.

<p><i>Mdr2^+/+</i> and <i>Mdr2^−/−</i> mice were treated with MTA during three weeks. Increased liver-to-body weight ratio (%) in <i>Mdr2^−/−</i> mice was reduced by MTA (A). Serum transaminases (B), alkaline phosphatase (C) and bilirubin (D), levels that are increased in <i>Mdr2^−/−</i> mice were attenuated by MTA. *<i>P</i><0.05 <i>vs</i> untreated <i>Mdr2^+/+</i> mice, #<i>P</i><0.05 <i>vs</i> untreated <i>Mdr2^−/−</i> mice.</p

Potential mechanisms of action of MTA in <i>Mdr2^−/−</i> mice.

<p>Our <i>in vivo</i> and <i>in vitro</i> observations indicate that MTA displays different actions that may underlie its beneficial effects on the course of liver injury and fibrosis in <i>Mdr2^−/−</i> mice. MTA may exert a direct cytoprotective effect on hepatocytes by reducing JNK activity, preventing hepatocellular death and further inflammation. MTA also inhibits the production of cytokines by inflammatory cells, likely through interference with NFκB activity as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015690#pone.0015690-Hevia1" target="_blank">[16]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015690#pone.0015690-IglesiasAra1" target="_blank">[18]</a>. In addition, enhanced expression of adenosine A2B receptors may contribute to the anti-inflammatory pharmacological profile of MTA. This compound can also exert direct effects on extracellular matrix (ECM) producing cells (myofibroblasts), reducing their activation, proliferation and the production of pro-fibrogenic factors. Inhibition of JunD expression may be an important event in the antifibrogenic action of MTA.</p

Expression of fibrogenic activation and cellular proliferation-related genes in myofibroblasts isolated from the liver of control and MTA-treated <i>Mdr2−/−</i> mice.

<p><i>Mdr2−/−</i> mice were treated for three weeks with MTA or vehicle as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015690#s2" target="_blank">Methods</a> section. At the end of the treatments hepatic myofibroblasts were isolated and the mRNA levels of the indicated genes were measured. Data are means ±SEM of three independent cell preparations per condition. *<i>P</i><0.05 <i>vs</i> vehicle-treated <i>Mdr2−/−</i> mice.</p

MTA modulates intracellular signalling pathways activated in <i>Mdr2^−/−</i> mouse liver.

<p>Phosphorylation levels of Smad1/5/8 (A), Smad2 (B), JNK and c-Jun (C) and Erk1/2 (D) were analyzed by western blotting in liver extracts from <i>Mdr2^+/+</i>, control <i>Mdr2^−/−</i>, and MTA-treated <i>Mdr2^−/−</i> mice. *<i>P</i><0.05 <i>vs Mdr2^+/+</i> mice, #<i>P</i><0.05 <i>vs</i> untreated <i>Mdr2^−/−</i> mice.</p

Effect of MTA on liver myofibroblast intracellular signalling pathways.

<p>Liver myofibroblasts were pre-treated with MTA for 30 min in the absence of serum and then stimulated with 10% FCS for 30 min or PDGF (20 ng/ml) for 60 min. Phosphorylation levels of Erk1/2 and c-Jun upon FCS stimulation (A), and S6 ribosomal protein after PDGF treatment (B) were analyzed by western blotting. Representative blots are shown.</p