Papers by Giovanni Chiappetta
Technical advances in proteomics mass spectrometry: identification of post-translational modifications
Clinical Chemistry and Laboratory Medicine, Jun 1, 2009
The importance of post-translational modifications (PTMs) of proteins has become evident in the p... more The importance of post-translational modifications (PTMs) of proteins has become evident in the proteomic era as it plays a critical role in modulating cellular function, and can vary in response to different stimuli thereby tuning cellular mechanisms. Assessment of PTMs on a proteomic scale is a challenging task since they are substoichiometric, transient and reversible. Moreover, the amount of post-translationally modified proteins is generally very small when compared to their unmodified counterparts. Existing methodologies for identification of PTMs essentially relies on enrichment procedure to selectively increase the amount of modified peptides. These procedures need to be integrated with sophisticated mass spectrometric methods to enable the identifications of PTMs. Although the strategies developed so far are not optimal, a number of examples will be given where the combination of innovative separation methods along with advanced mass spectrometric analyses provide positive results. These experiences are leading the way for the next generation of proteomic approaches for identification of a wide range of PTMs.
Mass spectrometry (MS) analysis for detection of immunoglobulins (IG) of the human IgG3 subclass ... more Mass spectrometry (MS) analysis for detection of immunoglobulins (IG) of the human IgG3 subclass is described that relies on polymorphic amino acids of the heavy gamma3 chains. IgG3 is the most polymorphic human IgG subclass with thirteen G3m allotypes located on the constant CH2 and CH3 domains of the gamma3 chain, the combination of which leads to six major G3m alleles. Amino acid changes resulting of extensive sequencing previously led to the definition of 19 IGHG3 alleles that have been correlated to the G3m alleles. As a proof of concept, MS proteotypic peptides were defined which encompass discriminatory amino acids for the identification of the G3m and IGHG3 alleles. Plasma samples originating from ten individuals either homozygous or heterozygous for different G3m alleles, and including one mother and her baby (drawn sequentially from birth to 9 months of age), were analyzed. Total IgG3 were purified using affinity chromatography and then digested by a combination of AspN and trypsin proteases, and peptides of interest were detected by mass spectrometry. The sensitivity of the method was assessed by mixing variable amounts of two plasma samples bearing distinct G3m allotypes. A label-free approach using the high-performance liquid chromatography (HPLC) retention time of peptides and their MS mass analyzer peak intensity gave semi-quantitative information. Quantification was realized by selected reaction monitoring (SRM) using synthetic peptides as internal standards. The possibility offered by this new methodology to detect and quantify neo-synthesized IgG in newborns will improve knowledge on the first acquisition of antibodies in infants and constitutes a promising diagnostic tool for vertically-transmitted diseases.
Several labelling strategies have been developed targeting specific amino acid residues and/or PT... more Several labelling strategies have been developed targeting specific amino acid residues and/or PTMs. Methods specifically tailored for the qualitative and sometimes quantitative determination of PTMs have emerged. Many research groups have focused their attention towards onitrotyrosine residues, developing various methodologies for their identification, while direct quantification has remained elusive. So far the iTRAQ chemistry has been limited to primary amines. Here, we report a new strategy based on the use of iTRAQ reagents coupled to MS analysis for the selective labelling of o-nitrotyrosine residues. This method was proved to lead to the simultaneous localisation and quantification of nitration sites both in model proteins and in biological systems.
Synthetic peptides are widely used in immunological research as epitopes to stimulate their cogna... more Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted b-cell epitopes in nonobese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) 206-214 epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP 206-214 using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP 206-214 . If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.
Methods in Enzymology, 2010
Proteomics, 2009
Several labelling strategies have been developed targeting specific amino acid residues and/or PT... more Several labelling strategies have been developed targeting specific amino acid residues and/or PTMs. Methods specifically tailored for the qualitative and sometimes quantitative determination of PTMs have emerged. Many research groups have focused their attention towards o-nitrotyrosine residues, developing various methodologies for their identification, while direct quantification has remained elusive. So far the iTRAQ chemistry has been limited to primary amines. Here, we report a new strategy based on the use of iTRAQ reagents coupled to MS analysis for the selective labelling of o-nitrotyrosine residues. This method was proved to lead to the simultaneous localisation and quantification of nitration sites both in model proteins and in biological systems.
Bidimensional Tandem Mass Spectrometry for Selective Identification of Nitration Sites in Proteins
Analytical Chemistry, 2007
Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitr... more Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitrite formation during development, oxidative stress, and aging. However, protein nitration might also have biological functions comparable to protein phosphorylation, mainly in redox signaling and in signal transduction. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Existing methodologies essentially rely on immunochemical techniques either using 2D-PAGE fractionation in combination with western blot analyses or exploiting immunoaffinity procedures to selectively capture nitrated proteins. Here we report a totally new approach involving dansyl chloride labeling of the nitration sites that rely on the enormous potential of MSn analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and an MS3 analysis. This new procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures.
Technical advances in proteomics mass spectrometry: identification of post-translational modifications
Clinical Chemistry and Laboratory Medicine, 2009
The importance of post-translational modifications (PTMs) of proteins has become evident in the p... more The importance of post-translational modifications (PTMs) of proteins has become evident in the proteomic era as it plays a critical role in modulating cellular function, and can vary in response to different stimuli thereby tuning cellular mechanisms. Assessment of PTMs on a proteomic scale is a challenging task since they are substoichiometric, transient and reversible. Moreover, the amount of post-translationally modified proteins is generally very small when compared to their unmodified counterparts. Existing methodologies for identification of PTMs essentially relies on enrichment procedure to selectively increase the amount of modified peptides. These procedures need to be integrated with sophisticated mass spectrometric methods to enable the identifications of PTMs. Although the strategies developed so far are not optimal, a number of examples will be given where the combination of innovative separation methods along with advanced mass spectrometric analyses provide positive results. These experiences are leading the way for the next generation of proteomic approaches for identification of a wide range of PTMs.
Analytical and Bioanalytical Chemistry, 2008
Human body fluids have been rediscovered in the post-genomic era as a great source of biological ... more Human body fluids have been rediscovered in the post-genomic era as a great source of biological markers and perhaps as source of potential biomarkers of disease. Recently, it has been found that not only proteins but also peptides and their modifications can be indicators of early pathogenic processes. This paper reports the identification of free phosphopeptides in human fluids using an improved IMAC strategy coupled to iterative mass spectrometry-based scanning techniques (neutral loss, precursor ion, multiple reaction monitoring). Many peptides were detected in the enriched extract samples when submitted to the MS-integrated strategy, whereas they were not detected in the initial extract samples. The combination of the IMAC-modified protocol with selective “precursor ion” and constant “neutral loss” triple quadrupole scan modes confers a high sensitivity on the analysis, allowing rapid phosphopeptide identification and characterization, even at low concentrations. To the best of our knowledge this work represents the first report exclusively focused on the detection of free phosphorylated peptides in biological fluids.
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Papers by Giovanni Chiappetta