title: Assessment of solvent residues accessibility using three Sulfo-NHS-biotin reagents in parallel: application to footprint changes of a methyltransferase upon binding its substrate.
abstract: NHS-biotin modification as a specific lysine probe coupled to mass spectrometry detection is increasingly used over the past years for assessing amino acid accessibility of proteins or complexes as an alternative when well-established methods are challenged. We present a strategy based on usage in parallel of three commercially available reagents (Sulfo-NHS-biotin, Sulfo-NHS-LC-biotin, and Sulfo-NHS-LC-LC-biotin) to efficiently assess the solvent accessibility of amino acids using MALDI-TOF mass spectrometry. The same qualitative pattern of reactivity was observed for these three reagents on the THUMPalpha protein at four reagent/polypeptide molar ratios (2 : 1, 6 : 1, 13 : 1, and 26 : 1). Peptide assignment of the detected ions gains in accuracy because of the triple redundancy due to specific increments of monoisotopic mass. These reagents are a good alternative to isotope labeling when using only a single MALDI-TOF mass spectrometer. We observed that hydroxyl groups of serine and tyrosine residues were also modified by these Sulfo-NHS-biotin reagents. The low amount of protein required and the method's simplicity make this procedure accessible and affordable in order to obtain topological information on proteins difficult to purify. This method was used to identify two lysine residues of the TrmG10 methyltransferase from Pyrococcus abyssi that were differentially reactive, modified in the protein but not in the tRNA-protein complex.
authors.affiliation: CEA VALRHO, DSV, iBEB, SBTN, LBSP, Institut de Biologie environnementale et Biotechnologie, Service de Biochimie et Toxicologie Nucléaire, Laboratoire de Biochimie des Systèmes Perturbés, F-30207 Bagnols-sur-Cèze, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Gabant
authors.forename: Julie
authors.initials: J
authors.lastname: Augier
authors.forename: Jean
authors.initials: J
authors.lastname: Armengaud
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D023302:Protein Footprinting / Q000379:methods*; D043903:Pyrococcus abyssi / Q000201:enzymology; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013388:Succinimides / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D019843:Archaeal Proteins; D010455:Peptides; D012997:Solvents; D013388:Succinimides; C065130:sulfo-N-hydroxysuccinimide-biotin; C061443:sulfosuccinimidyl 6-(biotinamido)hexanoate; D014443:Tyrosine; D012694:Serine; D001710:Biotin; D012343:RNA, Transfer; D012359:tRNA Methyltransferases; D014357:Trypsin; D008239:Lysine