title: Combining protein identification and quantification: C-terminal isotope-coded tagging using sulfanilic acid.
abstract: 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.
authors.affiliation: Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Panchaud
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Guillaume
authors.forename: Michael
authors.initials: M
authors.lastname: Affolter
authors.forename: Fabien
authors.initials: F
authors.lastname: Robert
authors.forename: Philippe
authors.initials: P
authors.lastname: Moreillon
authors.forename: Martin
authors.initials: M
authors.lastname: Kussmann
mesh_terms: D002364:Caseins / Q000737:chemistry; D003198:Computer Simulation; D003627:Data Interpretation, Statistical; D030562:Databases, Protein; D006868:Hydrolysis; D007202:Indicators and Reagents; D007553:Isotope Labeling; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013373:Substance P / Q000737:chemistry; D013425:Sulfanilic Acids / Q000737:chemistry*
chemical_list: D002364:Caseins; D007202:Indicators and Reagents; D010455:Peptides; D011506:Proteins; D013425:Sulfanilic Acids; D013373:Substance P