Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. Biological significance: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups. (C) 2016 Elsevier B.V. All rights reserved.

The aim of this study was to develop a novel method to detect circulating histones H3 and H2B in plasma based on multiple reaction monitoring targeted mass spectrometry and a multiple reaction monitoring approach (MRM-MS) for its clinical application in critical bacteriaemic septic shock patients. Plasma samples from 17 septic shock patients with confirmed bacteraemia and 10 healthy controls were analysed by an MRM-MS method, which specifically detects presence of histones H3 and H2B. By an internal standard, it was possible to quantify the concentration of circulating histones in plasma, which were significantly higher in patients, and thus confirmed their potential as biomarkers for diagnosing septic shock. After comparing surviving patients and non-survivors, a correlation was found between higher levels of circulating histones and unfavourable outcome. Indeed, histone H3 proved a more efficient and sensitive biomarker for septic shock prognosis. In conclusion, these findings suggest the accuracy of the MRM-MS technique and stable isotope labelled peptides to detect and quantify circulating plasma histones H2B and H3. This method may be used for early septic shock diagnoses and for the prognosis of fatal outcomes.

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Primary liver cancer (HCC) is recognized as the fifth most common neoplasm and the second leading cause of cancer death worldwide. Most risk factors are known, and the molecular pathogenesis has been widely studied in the past decade; however, the underlying molecular mechanisms remain to be unveiled, as they will facilitate the definition of novel biomarkers and clinical targets for more effective patient management. We utilize the B/D-HPP popular protein strategy. We report a list of popular proteins that have been highly cocited with the expression "liver cancer". Several enzymes highlight the known metabolic remodeling of liver cancer cells, four of which participate in one-carbon metabolism. This pathway is central to the maintenance of differentiated hepatocytes, as it is considered the connection between intermediate metabolism and epigenetic regulation. We designed a targeted selective reaction monitoring (SRM) method to follow up one-carbon metabolism adaptation in mouse HCC and in regenerating liver following exposure to CCl4. This method allows systematic monitoring of one-carbon metabolism and could prove useful in the follow-up of HCC and of chronically liver-diseased patients (cirrhosis) at risk of HCC. The SRM data are available via ProteomeXchange in PASSEL (PASS01060)

Acute intermittent porphyria (AIP) is an autosomal dominant metabolic disorder caused by a deficiency of hepatic porphobilinogen deaminase (PBGD). The disease is characterized by life threatening acute neurovisceral attacks. The aim of this study was to identify metabolites secreted by the hepatocytes that reflect differential metabolic status in the liver and that may predict response to the acute attack treatment. Plasma vitamin D binding protein (VDBP) from a mouse model of AIP displayed an abnormal migration in 2D-electrophoresis that is efficiently recovered upon gene therapy leading to liver specific over-expression of the PBGD protein. The change in VDBP mobility results from a differential isoelectric point suggesting a post-translational modification that takes place preferably in the liver. Liquid chromatography-mass spectrometry (LC-MS) analysis of human samples before and after glycosidase treatment revealed glycosylated plasma VDBP specifically in patients with recurrent attacks of ALP. Glycosylated VDBP recovered normal values in three severely afflicted AIP patients submitted to therapeutic liver transplantation. Our findings suggest that post-translational modification of VDBP might be considered as a promising biomarker to study and monitor the liver metabolic status in patients with AIP. Significance: We describe an increased glycosylation of VDBP in porphyric livers. Normal glycosylation was recovered upon liver gene therapy in a mouse model of porphyria or after liver transplantation in severely afflicted patients with AIP. Moreover, quantification of glycosylated VDBP by our ELISA immunoassay or LC-MS protocol in patients undergoing PBGD-gene therapy (www.aipgene.org) may be used as a marker indicating improvement or normalization of the patient's hepatic metabolism.

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. In vitro 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, in vivo, male embryos induce changes in the endometrium to help ensure their survival. In contrast, female embryos do not appear to induce these changes.

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 (t test, p < 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. TNF¿ and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNF¿, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNF¿ were up-regulated. The embryonic signaling agents involved could be TNF¿ and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development.

Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal+ infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal+ induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27Kip1 protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16¿24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator.