Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO 2 microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO 2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO 2 . We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented. Molecular & Cellular Proteomics 4: 873-886, 2005.Phosphorylation is among the most widespread post-translational modifications in nature, and it has been estimated that more than 30% of the proteins in a given mammalian cell at some point during their expression are phosphorylated (1). Phosphorylation and dephosphorylation of proteins regulates a large number of biological processes such as signal transduction (2), molecular recognition and interaction, and other cellular events. A fundamental understanding of these biological processes at the molecular level thus requires a characterization of the phosphorylated sites in the proteins. It is therefore essential to develop sensitive and selective methods for this task.A wide variety of methods are known for characterization of phosphorylated proteins. The most widely used have been peptide sequencing using Edman degradation combined with 32 P labeling. This method is well established and very robust but has several limitations. For example, in Edman degradation the peptides have to be separated before the analysis using liquid chromatography. This decreases the overall sensitivity and increases analysis time, and it is therefore not well suited for analysis of complex samples.Recently a number of MS-based strategies have been developed that are relatively sensitive and in many cases easier to perform than Edman degradation with respect to handling complex mixtures (e.g. Ref.3). The increased sensitivity is especially needed for low stoichiometric phosphorylation. However, presently ...
The action of tissue Transglutaminase (TGase) on specific protein-bound glutamine residues plays a critical role in numerous biological processes. Here we provide evidence for a new role of this enzyme in the common, HLA-DQ2 (and DQ8) associated enteropathy, celiac disease (CD). The intestinal inflammation in CD is precipitated by exposure to wheat gliadin in the diet and is associated with increased mucosal activity of TGase. This enzyme has also been identified as the main target for CD-associated anti-endomysium autoantibodies, and is known to accept gliadin as one of its few substrates. We have examined the possibility that TGase could be involved in modulating the reactivity of gliadin specific T cells. This could establish a link between previous reports of the role of TGase in CD and the prevailing view of CD as a T-cell mediated disorder. We found a specific effect of TGase on T-cell recognition of gliadin. This effect was limited to gliadin-specific T cells isolated from intestinal CD lesions. We demonstrate that TGase mediates its effect through an ordered and specific deamidation of gliadins. This deamidation creates an epitope that binds efficiently to DQ2 and is recognized by gut-derived T cells. Generation of epitopes by enzymatic modification is a new mechanism that may be relevant for breaking of tolerance and initiation of autoimmune disease.
Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization were extracted from the literature for a nonredundant set of 2750 human proteins. Almost all the information was obtained manually by biologists who read and interpreted >300,000 published articles during the annotation process. This database, which has an intuitive query interface allowing easy access to all the features of proteins, was built by using open source technologies and will be freely available at http://www.hprd.org to the academic community. This unified bioinformatics platform will be useful in cataloging and mining the large number of proteomic interactions and alterations that will be discovered in the postgenomic era.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF) is becoming a standard tool in mass spectrometry in general and protein analysis in particular.
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