title: Rapid determination of oxidized methionine residues in recombinant human basic fibroblast growth factor by ultra-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry with in-source collision-induced dissociation.
abstract: The primary structure of the deteriorated recombinant human basic fibroblast growth factor (rhbFGF) was determined by ultra-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) with in-source collision-induced dissociation (CID). The rhbFGFs before and after treatment with hydrogen peroxide (H(2)O(2)) were separated using an ACQUITY UPLC BEH300 C18 column (1.7 microm, 150 mm x 2.1 mm i.d.) with a gradient elution of a mixture of water/acetonitrile containing 0.1% formic acid. The separated proteins were then detected by a SYNAPT High Definition Mass Spectrometry system (SYNAPT-MS). Two methionine (Met) residues in the rhbFGF structure were oxidized to Met-sulfoxide (Met-O) in 0.03% H(2)O(2) at pH 2.0. As the result, three peaks, except for the peak of rhbFGF, appeared on the chromatogram. The three proteins corresponding to each peak were estimated as the denatured rhbFGFs including the Met-O residue(s) with TOF-MS. Furthermore, the position of the Met-O residue(s) was efficiently identified by UPLC/ESI-QTOF-MS using the in-source CID technique. The proposed method seems to be very useful for the structural elucidation of proteins, because the oxidized Met residues in rhbFGF were easily and rapidly identified.
authors.affiliation: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, and Global COE Program, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Ohkubo
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Inagaki
authors.forename: Jun Zhe
authors.initials: JZ
authors.lastname: Min
authors.forename: Daiki
authors.initials: D
authors.lastname: Kamiya
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Toyo'oka
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
chemical_list: D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D008715:Methionine