Title
Quantitation and localisation of (in vitro) transglutaminase-catalysed glutamine hydroxylation using mass spectrometry
Author
Piersma, S.R.
van de Pijpekamp, A.
Wijngaards, G.
Gruppen, H.
Boumans, H.
Centraal Instituut voor Voedingsonderzoek TNO
Publication year
2002
Abstract
A mass spectrometric approach was chosen to quantify and localise in vitro enzymatically modified glutamine (Gln) residues in a glutamine-rich protein. This protein (named dB1), a cloned domain of the high molecular weight wheat glutenin subunit Dx5, was modified by microbial transglutaminase (TGase) using hydroxylamine as the amine donor. Using MALDI-TOF MS (matrix assisted laser desorption ionization-time of flight detection mass spectrometry) it was found that maximally 70% of the 64 Gln residues of dB1 were modified after prolonged incubation with TGase and hydroxylamine. Next, modified dB1 was proteolytically digested and the peptides obtained were subjected to nanospray MS/MS analysis. Using this analysis, the peptides could be identified, and in a second stage of the analysis, a number of modified Gln residues were localised. The results show that indeed some of the Gln residues in dB1 can not be modified by TGase, and that these non-modified Gln are flanked C-terminally by a proline residue. The analysis method described in this article is generic and can be applied to other (in vitro) protein modification products as well. © 2002 Elsevier Science Inc. All rights reserved.
Subject
Biology
HMW gluten
Mass spectrometry
Protein modification
Transglutaminase
Biotechnology
Desorption
Hydroxylation
Laser beams
Mass spectrometry
Molecular weight
Quantitation
Enzymes
Triticum aestivum
To reference this document use:
http://resolver.tudelft.nl/uuid:1c269f00-9080-4948-a58d-2a8e040b8825
DOI
https://doi.org/10.1016/s0141-0229(01)00500-2
TNO identifier
57406
ISSN
0141-0229
Source
Enzyme and Microbial Technology, 30 (3), 266-272
Document type
article