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

Reversed-phase HPLC CRP 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-RPLC, 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 H-1 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. (C) 2000 Academic Press.