Proteomics Laboratory

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Proteomics Laboratory

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Mato A.,University of Santiago de Compostela | Salgado F.J.,University of Santiago de Compostela | Lopez-Pedrouso M.,University of Santiago de Compostela | Carrera M.,CSIC - Institute of Marine Research | And 4 more authors.
Journal of Proteomics | Year: 2015

Pre-slaughter stress has adverse effects on meat quality that can lead to the occurrence of Dark Firm Dry (DFD) meat in cattle. This study explores the previously uncharacterized proteome changes linked to pre-slaughter stress in the longissimus thoracis (LT) bovine muscle. Differential proteome profiles of DFD and normal (non-DFD) LT meat samples from male calves of the Rubia Gallega breed were assessed by 2-DE coupled to MS analysis (LC-MS/MS and MALDI TOF/TOF MS). A total of seven structural-contractile proteins (three different myosin light chain isoforms, two fast skeletal myosin light chain 2 isoforms, troponin C type 2 and cofilin-2) and three metabolism enzymes (triosephosphate isomerase, ATP synthase and beta-galactoside alpha-2,6-sialyltransferase) were found to have statistically significant differential abundance in sample groups. In addition, 2-DE in combination with the phosphoprotein-specific fluorescent dye Pro-Q DPS revealed that highly phosphorylated fast skeletal myosin regulatory light chain 2 isoforms underwent the most intense relative change in muscle conversion to DFD meat. Therefore, they appear to be the most sensitive biomarkers of stress just prior to slaughter in Rubia Gallega. Overall, these findings will facilitate a more integrative understanding of the biochemical processes associated with stress in cattle muscle and their effects in meat quality. Biological significance: Pre-slaughter stress is a crucial factor in meat production. Animals destined for slaughter are stressed by a variety of endogenous and exogenous factors that negatively affect the complex post-mortem biochemical events underlying the conversion of muscle into meat. The study of the muscle proteome has a great relevance for understanding the molecular mechanisms associated with stress. However, there is no information available on the molecular changes linked to pre-slaughter stress in cattle on the proteome scale. Our study led to the identification of a number of candidate proteins associated with the response to pre-slaughter stress in the LT bovine muscle of the Rubia Gallega breed. The functions of those significantly changed proteins have a clear biological relationship with stress response. These findings contribute to a deeper insight into the molecular pathways that respond to stress in cattle. © 2015 Elsevier B.V.


PubMed | Proteomics Laboratory, University of Santiago de Compostela and CSIC - Institute of Marine Research
Type: | Journal: Data in brief | Year: 2015

Proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress were assessed on the basis of two-dimensional electrophoresis (2-DE) data. In this study, the bootstrap resampling statistical technique and a new measure of relative change of the volume of 2-DE protein spots are shown to be more efficient than commonly used statistics to reliably quantify changes in protein abundance in stress response. The data are supplied in this article and are related to Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress by Franco et al. [1].


PubMed | Proteomics Laboratory, University of Santiago de Compostela and CSIC - Institute of Marine Research
Type: | Journal: Journal of proteomics | Year: 2015

Pre-slaughter stress has adverse effects on meat quality that can lead to the occurrence of Dark Firm Dry (DFD) meat in cattle. This study explores the previously uncharacterized proteome changes linked to pre-slaughter stress in the longissimus thoracis (LT) bovine muscle. Differential proteome profiles of DFD and normal (non-DFD) LT meat samples from male calves of the Rubia Gallega breed were assessed by 2-DE coupled to MS analysis (LC-MS/MS and MALDI TOF/TOF MS). A total of seven structural-contractile proteins (three different myosin light chain isoforms, two fast skeletal myosin light chain 2 isoforms, troponin C type 2 and cofilin-2) and three metabolism enzymes (triosephosphate isomerase, ATP synthase and beta-galactoside alpha-2,6-sialyltransferase) were found to have statistically significant differential abundance in sample groups. In addition, 2-DE in combination with the phosphoprotein-specific fluorescent dye Pro-Q DPS revealed that highly phosphorylated fast skeletal myosin regulatory light chain 2 isoforms underwent the most intense relative change in muscle conversion to DFD meat. Therefore, they appear to be the most sensitive biomarkers of stress just prior to slaughter in Rubia Gallega. Overall, these findings will facilitate a more integrative understanding of the biochemical processes associated with stress in cattle muscle and their effects in meat quality.Pre-slaughter stress is a crucial factor in meat production. Animals destined for slaughter are stressed by a variety of endogenous and exogenous factors that negatively affect the complex post-mortem biochemical events underlying the conversion of muscle into meat. The study of the muscle proteome has a great relevance for understanding the molecular mechanisms associated with stress. However, there is no information available on the molecular changes linked to pre-slaughter stress in cattle on the proteome scale. Our study led to the identification of a number of candidate proteins associated with the response to pre-slaughter stress in the LT bovine muscle of the Rubia Gallega breed. The functions of those significantly changed proteins have a clear biological relationship with stress response. These findings contribute to a deeper insight into the molecular pathways that respond to stress in cattle.


News Article | November 9, 2015
Site: cen.acs.org

If you’ve used gas chromatography-mass spectrometry (GC-MS) to analyze unknown compounds from cells and biological tissue, a new study suggests you may want to throw away most—if not all—of your data. But some researchers believe it might be better to keep the data and throw away the new study instead. Gary Siuzdak of Scripps Research Institute California and coworkers heated standard samples of known small molecules and samples of unknown metabolites from cells to mimic the GC stage of a GC-MS instrument. This initial stage uses heating to volatilize and separate samples. The researchers then used liquid chromatography-MS (LC-MS), which does not use heat in its initial LC separation stage, to analyze the heated samples as well as unheated, but identical, samples (Anal. Chem. 2015, DOI: 10.1021/acs.analchem.5b03003). The result: Up to 40% of the heated compounds were modified or destroyed, compared with the unheated ones—even in samples whose components were derivatized by trimethylsilylation, a method widely used to protect compounds from heating. “We found that even relatively low temperatures used in GC-MS can have a detrimental effect on small-molecule analysis,” he continues. GC and GC-MS have been used to identify and measure small molecules for more than 50 years. So the new results might trigger an “uh-oh moment” for analytical chemists. Using standard laboratory procedures, Siuzdak and coworkers used LC-MS to analyze small-molecule standards and human plasma metabolites stored at room temperature (25 °C). Then they compared the results with LC-MS analyses of the same samples heated to 60 °C, 100 °C, or 250 °C, to mimic a variety of common heating conditions used in GC-MS. The heating changed the identities of or destroyed roughly 5%, 15%, and 30% of compounds in both types of samples, respectively. Siuzdak suggests that work-arounds would involve using GC-MS primarily to measure sample concentrations by using reference standards rather than trying to identify unknowns, or switching to techniques such as LC-electrospray ionization MS, which does not use heating. “In fact, that is the direction things are going,” he says. “However, tens of thousands of GC-MS instruments are still being used on a daily basis” to analyze unknowns in nutrition, forensics, clinical and environmental analysis, and similar fields. The findings have sparked some strong opinions, both heated and unheated. For example, “I am unclear why a scientific journal would publish work that is such clear nonsense,” says metabolomics expert Oliver Fiehn of the University of California, Davis. “The publication of this paper is, in my opinion, a major embarrassment for Analytical Chemistry. There was a peer review process involved, but perhaps not all peer reviewers understood the study design the Siuzdak group used.” When analytical chemists “develop, validate, and implement an analytical method for GC-MS, they carefully control for important method parameters,” such as the temperatures used and the conditions used to derivatize compounds to protect them from heating, he says, noting that the Siuzdak group sidestepped such controls. In some experiments, Siuzdak’s team “used underivatized metabolites and heated them intensely,” Fiehn says. “That is called cooking—like in a kitchen. Primary metabolites have lots and lots of hydroxyl and amino groups, and blood plasma has a lot of sugars,” groups that should be protected to prevent breakdown before heating them for analysis, he adds. Fiehn believes the Scripps researchers made other methodological mistakes, such as analyzing trimethylsilyl-derivatized samples in a water-containing LC-MS solvent, because water cleaves trimethylsilyl groups. “You cannot inject trimethylsilylated compounds in an aqueous solvent into an LC-MS system and expect that peaks survive,” he says. “That’s why their MS spectra could not identify compounds: The authors destroyed them.” And metabolomics specialist Warwick Dunn of the University of Birmingham, in England, pointed out that “the published study heated dry samples, whereas GC heats samples in a liquid solution for a few seconds during injection and then in the gas phase. Heat transfer in a solid can be expected to be higher and could therefore provide a higher level of degradation than in the gas phase.” Hence, “further validation of the degradation of many metabolites is required before we should worry about the terabytes of data already collected.” Other scientists who spoke with C&EN were less critical. For example, Stephen Barnes, director of the Targeted Metabolomics & Proteomics Laboratory at the University of Alabama, Birmingham, says that whole-metabolome analysis typically identifies fewer than 20% of cellular metabolites, and the new study could help explain why. At this year’s International Conference of the Metabolomics Society, Barnes says, “a group reported incubating a series of pure metabolites at 70 °C and analyzing them to see if new compounds appeared without added enzymes. The answer was yes.” He notes that compounds in samples can react with each other, albeit slowly, and that any elevated temperatures to which they are exposed can also alter their composition. “I don’t think the problem is confined to GC-MS,” Barnes adds. “Those doing LC-MS analyses need to think hard about this too.” For example, samples can get modified during the extraction process used to prepare them, in which heated solvents are sometimes used. Or they can even get modified at the source from which they are obtained—for example, the human body is about 37 °C. GC-MS and LC-MS analyses are used as evidence in criminal cases, Barnes says, and “it seems like one could defend guilty people on the basis that prosecutors have no way of knowing whether the data are valid. The forensic science community needs to develop a more rigorous understanding of the pitfalls of analysis.” Liang Li of the University of Alberta, whose group develops MS methods for proteomics and metabolomics applications, says the new study is important because it reminds people, particularly new practitioners in the metabolomics field, not to use high temperatures or other conditions that are more extreme than necessary for processing complex metabolomic samples. Siuzdak says his team’s study wasn’t intended to disparage “thousands of papers’ worth of research.” But molecular transformation from sample heating “has been a fundamental yet unrecognized problem with GC-MS technology since its inception,” he says. “I remember asking someone about it many years ago during the question period after his talk, and he simply didn’t know how to respond. For me, it has always been the elephant in the room.”


News Article | February 23, 2017
Site: www.chromatographytechniques.com

If a new, personalized medical treatment saves you from a stroke or kidney failure in the future, you may have to thank Flipper and his friends. NIST research chemist Ben Neely and his team have just finished creating a detailed, searchable index of all the proteins found in the bottlenose dolphin genome. While the dolphin genome was first compiled in 2008, recent technology allowed Neely to develop a more exhaustive map of all the proteins produced by the marine animal’s DNA. Since dolphins and humans are such similar mammals, this proteomics approach has wide applications. “Once you can identify all of the proteins and know their amounts as expressed by the genome, you can figure out what’s going on in the bottlenose dolphin’s biological systems in this really detailed manner,” Neely explained. In addition to improving care for bottlenose dolphins in captivity as well as assessment of wild populations, dolphin proteomics can yield useful data on the safety and health of the world’s oceanic food web. The generated proteomic data can also be compared to human’s proteins, providing researchers with new information about how the body works, leading to new medical treatment options. Specifically, researchers like Neely are exploring the dolphins vanin-1 protein, which as a human counterpart, but in much smaller amounts. Scientists believe the vanin-1 protein is the key to how dolphins quickly descend in water without damaging their organs. When marine mammals swim toward the ocean floor, they actually shut off blood flow to their organs. During dives that can last as long as 90 minutes, marine mammals restrict blood floor to their kidneys, liver, heart and lungs to shunt more oxygen to the brain. In contrast, if blood stops flowing to the organs of a human’s body for even a few minutes, the result is often catastrophic, including stroke, kidney failure and even death. So, could elevated levels of the vanin-1 protein in humans actually protect our kidneys? “There’s this gap in the knowledge about genes and the proteins [dolphins] make. We are missing a huge piece of the puzzle in how these animals do what they do,” said Mike Janech, director of the Nephrology Proteomics Laboratory at the Medical University of South Carolina. Janech, a kidney researcher and expert in proteomics, draws from the field of biomimicry, where researchers look to nature for creative solutions to human problems. In Janech’s case, his nature inspiration comes from dolphins who seem to have protective proteins that may contain clues to treatments for aging-associated diseases in humans. During a study to determine why captive dolphins were living longer in the wild and developing an insulin resistance, Janech discovered that excessively high vanin-1 levels were correlated with decreased liver function in wild dolphins, which suggests they provide a protective effect in avoiding metabolic syndrome. But the researchers weren’t done just yet. Janech and his colleagues also noticed another potential use for the special protein. The function of vanin-1 is to make vitamin B5 and in doing so it releases an antioxidant that has been shown to protect tissues from injury, like that which occurs after the hypoxia and re-oxygenation of diving and resurfacing in marine mammals. So, again, the question becomes, could this be the key to helping humans resist the hypoxia that causes acute kidney injury? While Janech has applied for a grant to study that very question, Neely and his team will continue to apply newer, high-tech analytical techniques to the study of marine animals. The protein map Neely developed was done so using an ultra-high resolution tribrid mass spectrometer, which is currently the most powerful tool available to identify and quantify proteins.


PubMed | National Research Council Italy and Proteomics Laboratory
Type: | Journal: BMC cancer | Year: 2016

We have previously demonstrated that the hydroxylated biphenyl compound D6 (3E,3E)-4,4-(5,5,6,6-tetramethoxy-[1,1-biphenyl]-3,3-diyl)bis(but-3-en-2-one), a structural analogue of curcumin, exerts a strong antitumor activity on melanoma cells both in vitro and in vivo. Although the mechanism of action of D6 is yet to be clarified, this compound is thought to inhibit cancer cell growth by arresting the cell cycle in G2/M phase, and to induce apoptosis through the mitochondrial intrinsic pathway. To investigate the changes in protein expression induced by exposure of melanoma cells to D6, a differential proteomic study was carried out on D6-treated and untreated primary melanoma LB24Dagi cells.Proteins were fractionated by SDS-PAGE and subjected to in gel digestion. The peptide mixtures were analyzed by liquid chromatography coupled with tandem mass spectrometry. Proteins were identified and quantified using database search and spectral counting. Proteomic data were finally uploaded into the Ingenuity Pathway Analysis software to find significantly modulated networks and pathways.Analysis of the differentially expressed protein profiles revealed the activation of a strong cellular stress response, with overexpression of several HSPs and stimulation of ubiquitin-proteasome pathways. These were accompanied by a decrease of protein synthesis, evidenced by downregulation of proteins involved in mRNA processing and translation. These findings are consistent with our previous results on gene expression profiling in melanoma cells treated with D6.Our findings confirm that the curcumin analogue D6 triggers a strong stress response in melanoma cells, turning down majority of cell functions and finally driving cells to apoptosis.


Gharesi-Fard B.,Shiraz University of Medical Sciences | Gharesi-Fard B.,Proteomics Laboratory | Zolghadri J.,Shiraz University of Medical Sciences | Kamali-Sarvestani E.,Shiraz University of Medical Sciences | Kamali-Sarvestani E.,Proteomics Laboratory
Reproductive Sciences | Year: 2015

Placenta is a transient and unique pregnancy tissue that supports the fetus nutritionally and metabolically. Expression of the unique placental proteins in different stages may influence the development of the fetus as well as the pregnancy outcome. The present study aimed to compare the total placental proteome differences between the normal first-and third-trimester human placentas. In the current study, placental proteome was compared between normal first-and third-trimester placentas using 2-dimensional polyacrylamide gel electrophoresis method for separation and matrix-assisted laser desorption/ionization time-of flight mass spectrometry technique for identification of the proteins. Despite the overall similarities, comparison of the mean intensity of the protein spots between the first-and third-trimester placental proteomes revealed that 22 spots were differentially expressed (P <.05) among which 11 distinct spots were successfully identified. Of the 11 differentially expressed proteins, 4 were increased (protein disulfide isomerase, tropomyosin 4 isoform 2, enolase 1, and 78-kDa glucose-regulated protein), while the remaining 7 (actin γ1 propeptide, heat shock protein gp96, α1-antitrypsin, EF-hand domain family member D1, tubulin α1, glutathione S-transferase, and vitamin D binding protein) showed decreased expression in the placentas from the first-trimester compared to the full-term ones. In summary, the results of the present study as the first research on the comparison of the first-and third-trimester human placental proteomes introduced a group of 11 proteins with altered expression. Interestingly, some of these proteins are reported to be altered in pregnancy-related disorders. © The Author(s) 2014.


De La Fuente M.,Mision Biologica de Galicia CSIC | Borrajo A.,University of Santiago de Compostela | Bermudez J.,University of Santiago de Compostela | Lores M.,University of Santiago de Compostela | And 6 more authors.
Journal of Proteomics | Year: 2011

Common bean (Phaseolus vulgaris L.) is the most important grain legume for direct human consumption. Proteomic studies in legumes have increased significantly in the last years but few studies have been performed to date in P. vulgaris. We report here a proteomic analysis of bean seeds by two-dimensional electrophoresis (2-DE). Three different protein extraction methods (TCA-acetone, phenol and the commercial clean-up kit) were used taking into account that the extractome can have a determinant impact on the level of quality of downstream protein separation and identification. To demonstrate the quality of the 2-DE analysis, a selection of 50 gel spots was used in protein identification by mass spectrometry (MALDI-TOF MS and MALDI-TOF/TOF). The results showed that a considerable proportion of spots (70%) were identified in spite of incomplete genome/protein databases for bean and other legume species. Most identified proteins corresponded to storage protein, carbohydrate metabolism, defense and stress response, including proteins highly abundant in the seed of P. vulgaris such as the phaseolin, the phytohemagglutinin and the lectin-related α-amylase inhibitor. © 2010 Elsevier B.V.


Lopez-Pedrouso M.,University of Santiago de Compostela | Alonso J.,Proteomics Laboratory | Zapata C.,University of Santiago de Compostela
Plant Molecular Biology | Year: 2014

Phaseolin is the major seed storage protein of common bean, Phaseolus vulgaris L., accounting for up to 50 % of the total seed proteome. The regulatory mechanisms responsible for the synthesis, accumulation and degradation of phaseolin in the common bean seed are not yet sufficiently known. Here, we report on a systematic study in dormant and 4-day germinating bean seeds from cultivars Sanilac (S) and Tendergreen (T) to explore the presence and dynamics of phosphorylated phaseolin isoforms. High-resolution two-dimensional electrophoresis in combination with the phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescent stain and chemical dephosphorylation by hydrogen fluoride-pyridine enabled us to identify differentially phosphorylated phaseolin polypeptides in dormant and germinating seeds from cultivars S and T. Phosphorylated forms of the two subunits of type α and β that compose the phaseolin were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MALDI-TOF/TOF tandem MS. In addition, we found that the levels of phosphorylation of the phaseolin changed remarkably in the seed transition from dormancy to early germination stage. Temporal changes in the extent of phosphorylation in response to physiological and metabolic variations suggest that phosphorylated phaseolin isoforms have functional significance. In particular, this prospective study supports the hypothesis that mobilization of the phaseolin in germinating seeds occurs through the degradation of highly phosphorylated isoforms. Taken together, our results indicate that post-translational phaseolin modifications through phosphorylations need to be taken into consideration for a better understanding of the molecular mechanisms underlying its regulation. © 2013 Springer Science+Business Media Dordrecht.

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