title: Characterization of deamidation of barstar using electrospray ionization quadrupole time-of-flight mass spectrometry, which stabilizes an equilibrium unfolding intermediate.
abstract: Deamidation of asparaginyl residues is a common posttranslational modification in proteins and has been studied extensively because of its important biological effects, such as those on enzymatic activity, protein folding, and proteolytic degradation. However, characterization of the sites of deamidation of a protein has been a difficult analytical problem. In this study, mass spectrometry has been used as an analytical tool to characterize the deamidation of barstar, an RNAse inhibitor. Upon incubation of the protein at alkaline pH for 5 h, intact mass analysis of barstar, using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI QToF MS), indicated an increase in the mass of +2 Da, suggesting possible deamidation of the protein. The sites of deamidation have been identified using the conventional bottom-up approach using a capillary liquid chromatography connected on line to an ESI QToF mass spectrometer and top down approach by direct infusion of the intact protein and fragmenting inside MS. These chemical modifications are shown to lead to stabilization of an unfolding intermediate, which can be observed in equilibrium unfolding studies.
authors.affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.
authors.forename: Santosh Kumar
authors.initials: SK
authors.lastname: Jha
authors.forename: Putchen Dakshinamoorthy
authors.initials: PD
authors.lastname: Deepalakshmi
authors.forename: Jayant B
authors.initials: JB
authors.lastname: Udgaonkar
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001669:Biochemical Phenomena; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D058767:Protein Unfolding; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; C057438:barstar protein, Bacillus amyloliquefaciens; D014357:Trypsin