CSIC UAB Proteomics Laboratory

Barcelona, Spain

CSIC UAB Proteomics Laboratory

Barcelona, Spain
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Quero C.,CSIC | Colome N.,CSIC UAB Proteomics Laboratory | Rodriguez C.,CSIC UAB Proteomics Laboratory | Eichhorn P.,CSIC UAB Proteomics Laboratory | And 4 more authors.
Chemico-Biological Interactions | Year: 2011

Objectives: Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. Epidemiological work traced the origin to the ingestion of aniline-adulterated rapeseed oil, fraudulently marketed and sold as edible oil. It affected more than 20,000 people with over 400 deaths in the first 2 years. In 2001 evidence was presented that genetic factors could play a role in the susceptibility of individuals to the disease. Thus, a prospective study on the differences in gene expression in sera between control versus TOS-affected populations, both originally exposed to the toxic oil, was undertaken in our laboratory. Methods: Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Problems related with serum analysis by 2-DE were addressed to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins. The use of nonionic reductants or the presence of thiourea in the gels, were also tested. Results: From the resulting optimized images, a group of 329 major gel spots was located, matched and compared with serum samples. Thirty-five of these protein spots were found to be under- or over-expressed in TOS patients (threefold increase or decrease). Proteins in these spots were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) peptide map fingerprinting and database search. Several haptoglobin (Hp) isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS. Resolution of the homologous α-1s and α-1f chains, with a mass difference of only 0.043 Da, was obtained after guanidation of the protein with O-methylisourea. We applied this procedure to the study of the distribution of the Hp alleles HP2, HP1s and HP1f in control versus TOS-affected populations. The MALDI-TOF proteotyping method was validated by a parallel analysis of the serum samples by 2-DE. Conclusions: Data obtained from 54 TOS cases and 48 controls indicate significant differences in the distribution of Hp phenotypes in the two populations. Haptoglobin phenotypes have been reported to have biological and clinical consequences and have been described as risk factors for several diseases. Consequently, it was concluded that haptoglobin polymorphism could play a role in TOS. © 2010 Elsevier Ireland Ltd. All Rights Reserved.


Casas V.,CSIC UAB Proteomics Laboratory | Casas V.,Autonomous University of Barcelona | Rodriguez-Asiain A.,CSIC UAB Proteomics Laboratory | Pinto-Llorente R.,CSIC UAB Proteomics Laboratory | And 4 more authors.
Frontiers in Microbiology | Year: 2017

The spirochetes Brachyspira hyodysenteriae and B. pilosicoli are pig intestinal pathogens that are the causative agents of swine dysentery (SD) and porcine intestinal spirochaetosis (PIS), respectively. Although some inactivated bacterin and recombinant vaccines have been explored as prophylactic treatments against these species, no effective vaccine is yet available. Immunoproteomics approaches hold the potential for the identification of new, suitable candidates for subunit vaccines against SD and PIS. These strategies take into account the gene products actually expressed and present in the cells, and thus susceptible of being targets of immune recognition. In this context, we have analyzed the immunogenic pattern of two B. pilosicoli porcine isolates (the Spanish farm isolate OLA9 and the commercial P43/6/78 strain) and one B. hyodysenteriae isolate (the Spanish farm V1). The proteins from the Brachyspira lysates were fractionated by preparative isoelectric focusing, and the fractions were analyzed by Western blot with hyperimmune sera from challenged pigs. Of the 28 challenge-specific immunoreactive bands detected, 21 were identified as single proteins by MS, while the other 7 were shown to contain several major proteins. None of these proteins were detected in the control immunoreactive bands. The proteins identified included 11 from B. hyodysenteriae and 28 from the two B. pilosicoli strains. Eight proteins were common to the B. pilosicoli strains (i.e., elongation factor G, aspartyl-tRNA synthase, biotin lipoyl, TmpB outer membrane protein, flagellar protein FlaA, enolase, PEPCK, and VspD), and enolase and PEPCK were common to both species. Many of the identified proteins were flagellar proteins or predicted to be located on the cell surface and some of them had been previously described as antigenic or as bacterial virulence factors. Here we report on the identification and semiquantitative data of these immunoreactive proteins which constitute a unique antigen collection from these bacteria. © 2017 Casas, Rodríguez-Asiain, Pinto-Llorente, Vadillo, Carrascal and Abian.


PubMed | Complutense University of Madrid, CSIC UAB Proteomics Laboratory and Institute for Systems Biology
Type: | Journal: Journal of proteomics | Year: 2015

To provide new and expanded proteome documentation of the opportunistically pathogen Candida albicans, we have developed new protein extraction and analysis routines to provide a new, extended and enhanced version of the C. albicans PeptideAtlas. Two new datasets, resulting from experiments consisting of exhaustive subcellular fractionations and different growing conditions, plus two additional datasets from previous experiments on the surface and the secreted proteomes, have been incorporated to increase the coverage of the proteome. High resolution precursor mass spectrometry (MS) and ion trap tandem MS spectra were analyzed with three different search engines using a database containing allele-specific sequences. This approach, novel for a large-scale C. albicans proteomics project, was combined with the post-processing and filtering implemented in the Trans Proteomic Pipeline consistently used in the PeptideAtlas project and resulted in 49,372 additional peptides (3-fold increase) and 1630 more proteins (1.6-fold increase) identified in the new C. albicans PeptideAtlas with respect to the previous build. A total of 71,310 peptides and 4174 canonical (minimal non-redundant set) proteins (4115 if one protein per pair of alleles is considered) were identified representing 66% of the 6218 proteins in the predicted proteome. This makes the new PeptideAtlas build the most comprehensive C. albicans proteomics resource available and the only large-scale one with detections of individual alleles.


Badia-Villanueva M.,University of Barcelona | Carulla P.,University of Barcelona | Carrascal M.,CSIC UAB Proteomics Laboratory | Abian J.,CSIC UAB Proteomics Laboratory | And 3 more authors.
Biochemical and Biophysical Research Communications | Year: 2014

Lipoprotein lipase (LPL) hydrolyzes circulating triacylglycerols (TAG) into free fatty acids and glycerol. It is present in almost all tissues and its tissue-specific regulation directs the flow of circulating TAG in the body. We demonstrated in a previous study that, in rat heart and post-heparin plasma (PHP), LPL consists of a pattern of more than 8 forms of the same apparent molecular weight, but different isoelectric point (pI). In the present study we describe, for the first time, the existence of at least nine LPL pI isoforms in human PHP, with apparent pI between 6.8 and 8.6. Separation and characterization of these forms was carried out by 2DE combined with Western blotting and mass spectrometry (MALDI-TOF/MS and LC-MS/MS). Further studies are needed to discover their molecular origin, the pattern of pI isoforms in human tissues, their possible physiological functions and possible modifications of their pattern in different pathologies. © 2014 Elsevier Inc. All rights reserved.


PubMed | University of Southern Denmark, University of Barcelona and CSIC UAB Proteomics Laboratory
Type: Journal Article | Journal: Biochemical and biophysical research communications | Year: 2014

Lipoprotein lipase (LPL) hydrolyzes circulating triacylglycerols (TAG) into free fatty acids and glycerol. It is present in almost all tissues and its tissue-specific regulation directs the flow of circulating TAG in the body. We demonstrated in a previous study that, in rat heart and post-heparin plasma (PHP), LPL consists of a pattern of more than 8 forms of the same apparent molecular weight, but different isoelectric point (pI). In the present study we describe, for the first time, the existence of at least nine LPL pI isoforms in human PHP, with apparent pI between 6.8 and 8.6. Separation and characterization of these forms was carried out by 2DE combined with Western blotting and mass spectrometry (MALDI-TOF/MS and LC-MS/MS). Further studies are needed to discover their molecular origin, the pattern of pI isoforms in human tissues, their possible physiological functions and possible modifications of their pattern in different pathologies.


Nguyen T.D.,CSIC UAB Proteomics Laboratory | Nguyen T.D.,Protein Biochemistry Laboratory | Carrascal M.,CSIC UAB Proteomics Laboratory | Vidal-Cortes O.,CSIC UAB Proteomics Laboratory | And 5 more authors.
Journal of Proteomics | Year: 2016

Phosphorylation is a reversible post-translational modification, playing a vital role in protein function. In T cells, protein phosphorylation is the key mechanism regulating T cell receptor-driven signaling pathways. In order to gain insights into the phosphoproteome evolution of T cell activation, we performed a large-scale quantitative phosphoproteomics study of Jurkat E6.1 (wild type) and Jurkat gamma1 (Phospholipase gamma1 null) cell clones upon costimulation with anti-CD3 and anti-CD28 antibodies at times ranging from 15. min to as long as 120. min. In total, we identified 5585 phosphopeptides belonging to 2008 phosphoproteins from both cell clones. We detected 130 and 114 novel phosphopeptides in Jurkat E6.1 and Jurkat gamma1 clones, respectively. A significantly lower number of proteins containing regulated phosphorylation sites were identified in Jurkat gamma1 in comparison to Jurkat E6.1, reflecting the vital role of Phospholipase gamma1 in T cell signaling. Several new phosphorylation sites from lymphocyte-specific protein tyrosine kinase (Lck) were identified. Of these, serine-121 showed significant changes in JE6.1 while only small changes in the Jgamma1 clone. Our data may contribute to the current human T cell phosphoproteome and provide a better understanding on T cell receptor signaling. Data are available via ProteomeXchange with identifier PXD002871. © 2015 Elsevier B.V.


PubMed | Protein Biochemistry Laboratory and CSIC UAB Proteomics Laboratory
Type: | Journal: Journal of proteomics | Year: 2015

Phosphorylation is a reversible post-translational modification, playing a vital role in protein function. In T cells, protein phosphorylation is the key mechanism regulating T cell receptor-driven signaling pathways. In order to gain insights into the phosphoproteome evolution of T cell activation, we performed a large-scale quantitative phosphoproteomics study of Jurkat E6.1 (wild type) and Jurkat gamma1 (Phospholipase gamma1 null) cell clones upon costimulation with anti-CD3 and anti-CD28 antibodies at times ranging from 15min to as long as 120min. In total, we identified 5585 phosphopeptides belonging to 2008 phosphoproteins from both cell clones. We detected 130 and 114 novel phosphopeptides in Jurkat E6.1 and Jurkat gamma1 clones, respectively. A significantly lower number of proteins containing regulated phosphorylation sites were identified in Jurkat gamma1 in comparison to Jurkat E6.1, reflecting the vital role of Phospholipase gamma1 in T cell signaling. Several new phosphorylation sites from lymphocyte-specific protein tyrosine kinase (Lck) were identified. Of these, serine-121 showed significant changes in JE6.1 while only small changes in the Jgamma1 clone. Our data may contribute to the current human T cell phosphoproteome and provide a better understanding on T cell receptor signaling. Data are available via ProteomeXchange with identifier PXD002871.


Ponce J.,Fundacio Institute Dinvestigacio En Ciencies Of La Salut Germans Trias I Pujol | Brea D.,University of Santiago de Compostela | Carrascal M.,CSIC UAB Proteomics Laboratory | Guirao V.,Fundacio Institute Dinvestigacio En Ciencies Of La Salut Germans Trias I Pujol | And 5 more authors.
Proteomics | Year: 2010

Cell death induced by over-activation of glutamate receptors occurs in different neuropathologies. Cholesterol depletors protect from neurotoxic over-activation of glutamate receptors, and we have recently reported that this neuroprotection is associated with a reduction of the N-methyl-D-aspartate subtype of glutamate receptors in detergent-resistant membrane domains (DRM). In the present study we used comparative proteomics to further identify which proteins, besides the N-methyl-D-aspartate receptor, change its percentage of association to DRM after treatment of neurons with simvastatin. We detected 338 spots in neuronal DRM subjected to 2-DE; eleven of these spots changed its intensity after treatment with simvastatin. All 11 differential spots showed reduced intensity in simvastatin-treated samples and were identified as adipocyte plasma membrane associated protein, enolase, calretinin, coronin 1a, f-actin capping protein α1, f-actin capping protein α2, heat shock cognate protein 71, malate dehydrogenase, n-myc downregulated gene 1, prohibitin 2, Rab GDP dissociation inhibitor, translationally controlled tumor protein and voltage dependent anion selective channel protein 1. The proteins tested colocalized with the lipid raft marker caveolin-1. Interestingly, the proteins we have identified in the present study had been previously reported to play a role in cell fate and, thus, they might represent novel targets for neuroprotection. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.


Villanueva J.,CSIC UAB Proteomics Laboratory | Carrascal M.,CSIC UAB Proteomics Laboratory | Abian J.,CSIC UAB Proteomics Laboratory
Journal of Proteomics | Year: 2014

Isotope dilution mass spectrometry is a reference technique for quantitative analysis, given that it combines the sensitivity and selectivity of MS instruments with the precision and accuracy associated with the use of internal standards. Isotope-labeled proteins are the optimal internal standards for quantitative proteomics as they closely mimic the behavior of their natural counterparts during the analytical process. A major complication of isotope dilution mass spectrometry proteomics is the technical difficulty of obtaining these internal standards, especially in studies where a high number of proteins have to be quantified simultaneously. In this paper, we review some of the characteristics of the isotope dilution mass spectrometry approach, its benefits in terms of reliability and quality control in targeted proteomic analysis and the different strategies developed for its application in proteomics.© 2013 Elsevier B.V.


PubMed | CSIC UAB Proteomics Laboratory
Type: | Journal: Journal of proteomics | Year: 2014

Isotope dilution mass spectrometry is a reference technique for quantitative analysis, given that it combines the sensitivity and selectivity of MS instruments with the precision and accuracy associated with the use of internal standards. Isotope-labeled proteins are the optimal internal standards for quantitative proteomics as they closely mimic the behavior of their natural counterparts during the analytical process. A major complication of isotope dilution mass spectrometry proteomics is the technical difficulty of obtaining these internal standards, especially in studies where a high number of proteins have to be quantified simultaneously. In this paper, we review some of the characteristics of the isotope dilution mass spectrometry approach, its benefits in terms of reliability and quality control in targeted proteomic analysis and the different strategies developed for its application in proteomics.

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