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
✍ Scribed by Haruya Sato; Naoyuki Yamada; Nobuhisa Shimba; Yoshiyuki Takahara
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 117 KB
- Volume
- 281
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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 1 H 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.