title: Unique substrate specificities of two adjacent glutamine residues in EAQQIVM for transglutaminase: identification and characterization of the reaction products by electrospray ionization tandem mass spectrometry.
abstract: Reversed-phase HPLC (RP-HPLC) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) were used to characterize the transglutaminase (TGase)-catalyzed dual modification of a peptide (EAQQIVM, named FibN) with monodansylcadaverine (MDC). The synthesized FibN peptide, which was derived from the N-terminal sequence of fibronectin, was used as the substrate for a guinea pig liver TGase (G-TGase). The time course of incorporation of MDC into FibN, detected by RP-HPLC, indicated two separate fluorescent product peaks. ESI-MS analysis of the isolated fractions indicated that products represented MDC-incorporated FibN molecules in molar ratios of 1:1 ((MDC)-FibN) and 2:1 ((MDC)2-FibN). A sequence analysis of MDC-FibN, using ESI-MS/MS, showed that the first modified residue in FibN was mainly Gln3. The kinetic analysis of MDC incorporation suggested that dual incorporation would occur by mainly one route. A one-dimensional 1H NMR comparison of MDC-FibN and unmodified FibN suggested that the first incorporation of MDC at Gln3 altered the substrate reactivity of the Gln4 residue in FibN for the G-TGase-catalyzed reaction. Thus, a detailed analysis of the peptide products using RP-HPLC and ESI-MS/MS should provide a powerful tool for exploring the mechanism of the substrate requirements of TGases.
authors.affiliation: Central Research Laboratories, Ajinomoto Company, Inc., Kawasaki-shi, Kanagawa, Japan. haruya_satou@ajinomoto.com
authors.forename: H
authors.initials: H
authors.lastname: Sato
authors.forename: N
authors.initials: N
authors.lastname: Yamada
authors.forename: N
authors.initials: N
authors.lastname: Shimba
authors.forename: Y
authors.initials: Y
authors.lastname: Takahara
mesh_terms: D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000737:chemistry; D005973:Glutamine / Q000378:metabolism; D006168:Guinea Pigs; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D010455:Peptides; D005973:Glutamine; D011503:Transglutaminases; C008542:monodansylcadaverine; D002103:Cadaverine