Manuel Lombardía

Measurement of wheat gluten and barley hordeins in contaminated oats from Europe, the United States and Canada by Sandwich R5 Elisa

Objectives We have investigated the extent of contamination with wheat, barley, rye or a mixture of these cereals in a large number of grains and commercial oats. We have also attempted to identify the type of cereal contaminant. Methods Sandwich R5 ELISA (using either gliadins or hordeins as standards), western blot, matrix-assisted laser desorption/ionization time-of-flight mass spectrometric and quantitative real-time PCR (Q-PCR) techniques have been used to analyze a total of 134 oats, comprising grains and commercial oat products collected from Europe, the United States and Canada. Results Twenty-five of the 134 pure, uncontaminated oat varieties were found to have undetectable levels of gluten, whereas most of the 109 grains and commercial oat products were mainly contaminated with mixtures of wheat, barley and rye, barley being the predominant contaminant. The percentages of these cereals in the oat samples have been calculated by specific wheat, barley and rye Q-PCR systems. The oat samples were grouped according to the avenin spectra determined by the mass spectrometric technique. The data confirmed that contaminated oat foods, based on the same variety, could have different levels of wheat, barley and rye contamination. Conclusion This study has verified that contamination with wheat gliadins or barley hordeins in oat samples can be measured by the Sandwich R5 ELISA, using either gliadins or hordeins as standards, and also the importance of using confirmatory techniques (such as western blot, Q-PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) to confirm that most oats are contaminated with mixtures of wheat, barley and rye.

Comprehensive analysis of gluten in processed foods using a new extraction method and a competitive ELISA based on the R5 antibody.

The only treatment for coeliac disease is to follow a strict, life-long gluten-free diet. It is therefore essential to use a highly sensitive, specific technique for gluten analysis in foods. Nowadays, the usual method for determining gluten content in gluten-free foods, internationally accepted by the Codex Alimentarius Commission, is the R5 antibody-based sandwich ELISA, combined with the cocktail-extraction solution. This technique requires at least two epitopes in the protein, but in hydrolysed foods, proteins are fragmented during food processing and converted into peptides in which only one toxic epitope may appear. Consequently, it was necessary to develop a new competitive immunoassay that, together with a reliable, compatible extraction solution, would provide a complete gluten analysis in any kind of food. We analysed commercial foods and home-made maize breads spiked with a known amount of gliadins using the sandwich R5 ELISA and the new competitive R5 ELISA that has been developed. These foods had previously been extracted with 60% ethanol/water, the cocktail solution or the new extracting solution called UPEX (universal prolamin and glutelin extractant solution). The complementary SDS-PAGE and western blot techniques were also used to confirm the gluten content. The limits of detection and quantification of the competitive R5 ELISA were 0.36 and 1.22 ng/ml of gliadins, respectively. The intra- and inter-assay precisions based on two samples were, respectively, 7.3% and 5.4% for the first sample and 9.9% and 6.3% for the second. This new assay was a better technique than the sandwich R5 ELISA for detecting gliadins quantitatively in hydrolysed foods. Regarding the extraction procedure, we did not find any significant interference from components of the UPEX solution at the concentration used. In addition, the UPEX solution extraction was compatible with the R5 western blot and mass spectrometry techniques. The competitive R5 ELISA we developed, combined with the UPEX solution described here, is a very useful tool for detecting and quantifying gluten in any kind of food samples, including heat-treated and/or hydrolysed ones.

Surfing transcriptomic landscapes. A step beyond the annotation of chromosome 16 proteome

The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study.

Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue

Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I–associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.

A molecular basis for the presentation of phosphorylated peptides by HLA-B antigens

As aberrant protein phosphorylation is a hallmark of tumor cells, the display of tumor-specific phosphopeptides by Human Leukocyte Antigen (HLA) class I molecules can be exploited in the treatment of cancer by T-cell-based immunotherapy. Yet, the characterization and prediction of HLA-I phospholigands is challenging as the molecular determinants of the presentation of such post-translationally modified peptides are not fully understood. Here, we employed a peptidomic workflow to identify 256 unique phosphorylated ligands associated with HLA-B*40, -B*27, -B*39, or -B*07. Remarkably, these phosphopeptides showed similar molecular features. Besides the specific anchor motifs imposed by the binding groove of each allotype, the predominance of phosphorylation at peptide position 4 (P4) became strikingly evident, as was the enrichment of basic residues at P1. To determine the structural basis of this observation, we carried out a series of peptide binding assays and solved the crystal structures of HLA-B*40 in complex with a phosphorylated ligand or its nonphosphorylated counterpart. Overall, our data provide a clear explanation to the common motif found in the phosphopeptidomes associated to different HLA-B molecules. The high prevalence of phosphorylation at P4 is dictated by the presence of the conserved residue Arg62 in the heavy chain, a structural feature shared by most HLA-B alleles. In contrast, the preference for basic residues at P1 is allotype-dependent and might be linked to the structure of the A pocket. This molecular understanding of the presentation of phosphopeptides by HLA-B molecules provides a base for the improved prediction and identification of phosphorylated neo-antigens, as potentially used for cancer immunotherapy.

Comparative Analysis of the Endogenous Peptidomes Displayed by HLA-B*27 and Mamu-B*08: Two MHC Class I Alleles Associated with Elite Control of HIV/SIV Infection

Indian rhesus macaques are arguably the most reliable animal models in AIDS research. In this species the MHC class I allele Mamu-B*08, among others, is associated with elite control of SIV replication. A similar scenario is observed in humans where the expression of HLA-B*27 or HLA-B*57 has been linked to slow or no progression to AIDS after HIV infection. Despite having large differences in their primary structure, it has been reported that HLA-B*27 and Mamu-B*08 display peptides with sequence similarity. To fine-map the Mamu-B*08 binding motif and assess its similarities with that of HLA-B*27, we affinity purified the peptidomes bound to these MHC class I molecules and analyzed them by LC-MS, identifying several thousands of endogenous ligands. Sequence analysis of both sets of peptides revealed a degree of similarity in their binding motifs, especially at peptide position 2 (P2), where arginine was present in the vast majority of ligands of both allotypes. In addition, several differences emerged from this analysis: (i) ligands displayed by Mamu-B*08 tended to be shorter and to have lower molecular weight, (ii) Mamu-B*08 showed a higher preference for glutamine at P2 as a suboptimal binding motif, and (iii) the second major anchor position, found at PΩ, was much more restrictive in Mamu-B*08. In this regard, HLA-B*27 bound efficiently peptides with aliphatic, aromatic (including tyrosine), and basic C-terminal residues while Mamu-B*08 preferred peptides with leucine and phenylalanine in this position. Finally, in silico estimations of binding efficiency and competitive binding assays to Mamu-B*08 of several selected peptides revealed a good correlation between the characterized anchor motif and binding affinity. These results deepen our understanding of the molecular basis of the presentation of peptides by Mamu-B*08 and can contribute to the detection of novel SIV epitopes restricted by this allotype.

PCA-4248, a PAF receptor antagonist, inhibits PAF-induced phosphoinositide turnover

The effect of a new PAF (platelet activating factor; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoryl-choline) receptor antagonist, PCA-4248 (2-phenylthio)ethyl-5-metoxycarbonyl-2,4,6-trimethyl-1, 4-dihydropyridine-3-carboxylate), on phosphoinositide turnover evoked by PAF was investigated. PAF treatment resulted in an increased 32P incorporation into phosphoinositides and phosphatidic acid in rabbit platelets. Treatment with PCA-4248 abolished both effects in a dose-dependent manner, 10 microM being the most effective dose. In thrombin stimulated platelets, phosphoinositide turnover was not influenced by PCA-4248. In addition, PAF caused a rapid and significant increase in protein phosphorylation. Thus, PAF treatment resulted in a marked phosphorylation of two proteins of 47 kDa and 20 kDa. Treatment with PCA-4248 resulted in an inhibition of these actions. Serotonin secretion evoked by PAF was also inhibited by PCA-4248. It is concluded that PCA-4248 antagonizes the PAF effects by acting as a competitive antagonist at the PAF receptor level as evidenced from binding studies.

INVOLVEMENT OF CYCLIC GMP IN THE MODE OF ACTION OF A NEW ANTITHROMBOTIC AGENT PCA-4230; INHIBITION OF THE PLATELET CYCLIC GMP DEPENDENT PHOSPHODIESTERASE

The effect of PCA-4230, a new dihydropyridine derivative with a potent antithrombotic activity, on cyclic nucleotide phosphodiesterase in platelets was studied. PCA-4230 inhibited (54%) cyclic GMP hydrolytic activity of a platelet cytosolic fraction, whereas it did not affect that of cyclic AMP. Results suggested that PCA-4230 inhibited a cyclic GMP-dependent phosphodiesterase, known as cGB PDE or type V, on a purified enzyme from rabbit platelets by a non-competitive-uncompetitive type inhibition. In addition, PCA-4230 potentiated the increase in both cyclic GMP and cyclic AMP levels evoked by sodium nitroprusside. Furthermore, PCA-4230 and forskolin caused a synergistic effect in cyclic AMP, and also potentiated the phosphorylation of 50 kDa and 22 kDa proteins, reported as substrates of cyclic GMP- and cyclic AMP-dependent protein kinases that are related to the inhibition of platelet functions. Finally, PCA-4230 also potentiated the forskolin- and sodium nitroprusside-inhibited serotonin release evoked by thrombin, probably related to the increased cyclic nucleotide level.