Biomedical subjects

Alexandre Panchaud

Publications and source records attributed to Alexandre Panchaud.

3 recordsLinked to original sources

PubMed2006

Differentially isotope-coded N-terminal protein sulphonation: combining protein identification and quantification.

Most proteomic labelling technologies intend to improve protein quantification and/or facilitate (de novo) peptide sequencing. We present here a novel stable-isotope labelling method to simultaneously identify and quantify protein components in complex mixtures by specifically derivatizing the N-terminus of proteins with 4-sulphophenyl isothiocyanate (SPITC). Our approach combines protein identification with quantification through differential isotope-coded labelling at the protein N-terminus prior to digestion. The isotope spacing of 6 Da (unlabelled vs. six-fold 13C-labelled tag) between derivatized peptide pairs enables the detection on different MS platforms (MALDI and ESI). Optimisation of the reaction conditions using SPITC was performed on three model proteins. Improved detection of the N-terminally derivatized peptide compared to the native analogue was observed in negative-ion MALDI-MS. Simpler fragmentation patterns compared to native peptides facilitated protein identification. The 13C-labelled SPITC resulted in convenient peptide pair spacing without isotopic overlap and hence facilitated relative quantification by MALDI-TOF/TOF and LC-ESI-MS/MS. The combination of facilitated identification and quantification achieved by differentially isotope-coded N-terminal protein tagging with light/heavy SPITC represents, to our knowledge, a new approach to quantitative proteomics.

Animals↗

PubMed2006

Combining protein identification and quantification: C-terminal isotope-coded tagging using sulfanilic acid.

Two methods of differential isotopic coding of carboxylic groups have been developed to date. The first approach uses d0- or d3-methanol to convert carboxyl groups into the corresponding methyl esters. The second relies on the incorporation of two 18O atoms into the C-terminal carboxylic group during tryptic digestion of proteins in H(2)18O. However, both methods have limitations such as chromatographic separation of 1H and 2H derivatives or overlap of isotopic distributions of light and heavy forms due to small mass shifts. Here we present a new tagging approach based on the specific incorporation of sulfanilic acid into carboxylic groups. The reagent was synthesized in a heavy form (13C phenyl ring), showing no chromatographic shift and an optimal isotopic separation with a 6 Da mass shift. Moreover, sulfanilic acid allows for simplified fragmentation in matrix-assisted laser desorption/ionization (MALDI) due the charge fixation of the sulfonate group at the C-terminus of the peptide. The derivatization is simple, specific and minimizes the number of sample treatment steps that can strongly alter the sample composition. The quantification is reproducible within an order of magnitude and can be analyzed either by electrospray ionization (ESI) or MALDI. Finally, the method is able to specifically identify the C-terminal peptide of a protein by using GluC as the proteolytic enzyme.

Caseins↗

PubMed2005

Rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by multidimensional protein identification technology.

Direct injection of complex protein mixtures, e.g. those derived from crude biological fluids, is often incompatible with conventional LC supports, because of column clogging and rapid deterioration of chromatographic performance. In this paper, we report the use of restricted access media to rapidly enrich and fractionate human breast milk. This resin, combining size exclusion and anion exchange functionalities, yielded a fraction enriched in soluble CD14 and showing specific sCD14-dependant activity. This fraction was split into five aliquots, which were individually characterized using multidimensional protein identification technology. Reproducibility of the results was addressed by analysing and comparing five datasets using different protein identification tools available within the Sequest software. Furthermore, a comparison of three major releases of the Ensembl human protein database was performed to examine the effect of database updates on our results. We report here the benefit of repeated analysis of aliquots of the same fraction: first to increase the confidence in peptide identification by repeated confirmation in several aliquots; and second to assess experimental reproducibility. We demonstrate furthermore the effect of database modifications on the results and the importance of constantly re-analysing data with new releases to keep them consistent and up to date with the latest protein identities and predictions available.

Amino Acid Sequence↗