Research paper
Quantitation and localisation of (in vitro) transglutaminase-catalysed glutamine hydroxylation using mass spectrometry

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Abstract

1A 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.

Introduction

Enzymatic modification of proteins results in changes in protein structure, and may therefore result in changes of the functional properties, such as solubility, gelation, emulsion formation and stabilisation [1]. These functional properties are of importance to the behaviour of proteins in a food matrix [2]. Enzymatic modification of proteins can, therefore, be used to improve properties like gel strength and emulsion stability [3]. Transglutaminase (TGase) is one of these enzymes, a review covering modification of food proteins using TGase can be found in [4].

Transglutaminase (protein-glutamine γ-glutamyltransferase, E.C. 2.3.2.13) catalyses acyl-transfer reactions in which the γ-carboxamide group of protein-bound glutaminyl residues is the acyl donor. Depending on the acceptor molecule three different reactions can be discerned. First, the ϵ-amino group of lysine residues can serve as substrate to generate inter- and intramolecular ϵ-(γ-glutamyl)-lysine isopeptide bonds. This is the most common type of TGase catalysed reaction, resulting in protein crosslinking. Secondly, other primary amines like the protein N-terminal α-amino group and small primary amines can serve as nucleophiles. This second reaction is termed the attachment reaction. The third reaction catalysed by TGase, in the absence of NH2 donors, in aqueous solution is glutamine deamidation, resulting in the conversion of glutamine into glutamic acid [5]. The TGase catalysed protein crosslinking reaction has been studied most extensively [4], while research into the attachment reaction has been hampered by protein crosslinking as side reaction. A protein which does not contain any (accesible) lysine residues cannot be crosslinked by TGase, and is therefore very suited to study the attachment reaction seperately. Here we have chosen a small, soluble, primary amine as substrate to study the attachment reaction. The TGase catalysed attachment reaction of primary amine hydroxylamine (HA) to glutamine is shown in Fig. 1. The reaction produces ammonia and hydroxylated glutamine at the γ-NH2 of the carboxamide moiety (and is accompanied by a mass increase of 16 Da). Upon attachment of HA to a protein by TGase, the extent of modification and the sites of modification have to be determined in order to correlate protein modifications to changes in protein structure and functional properties.

Naturally occurring, (in vivo) post-translational protein modifications such as glycosylation, phosphorylation and prenylation, are widely studied using mass spectrometric techniques to characterise their nature, the extent of modfication, and the site of modification (reviewed in [6]). The same approach can be used for analysis of in vitro (enzymatically) modified proteins. Mass spectrometry can both be used for determination of the extent of modification (overall mass increase) as well as localisation of modified glutamine residues at the amino acid level. After the TGase modification reaction, MALDI-TOF MS (matrix assisted laser desorption ionisation–time of flight detection mass spectrometry [7]) can be used to monitor the mass increase of the whole protein and thus the extent of modification. Upon proteolytic digestion, peptides can be localysed based on their molecular mass and specific fragmentation pattern [8]. Modified glutamines can be identified in peptides by (nano)ESI (electrospray ionization) MS/MS. Using this combined MALDI and ESI-MS approach, one can monitor the modification reaction in time at the amino-acid sequence level.

In order to study the TGase catalysed glutamine hydroxylation reaction, a protein was chosen that is rich in glutamines and which does not contain lysines. The model protein dB1 (Fig. 2) used in this study is a cloned and bacterially expressed soluble 160 amino-acid protein fragment of Dx5, a high molecular weight subunit of wheat glutenin [9]. The central domain of high molecular weight (HMW) glutenin subunits represents 60–80% of their amino-acid sequence, varies from 500–700 residues, and has a highly repetitive sequence. The cloned domain consists of three consensus peptides PGQGQQ, GYYPTSPQQ and GQQ [9], [10], [11]. The repetitive domain is thought to consist of a series of β-turns which fold into a β-spiral structure [9], [10], [11]. The dB1 protein contains 64 glutamine residues but no lysines.

In this paper we describe the above mentioned methodology of mass spectrometric analysis of the dB1 fragment modified with HA by TGase. The method is illustrated by MALDI MS analysis of the attachment reaction as a function of time and an example of MS/MS analysis of tryptic/chymotryptic peptides of dB1 after modification.

Section snippets

Materials

Recombinant dB1 was produced in E. coli cultures and was purified as reported previously [9]. Microbial transglutaminase (TGase) was isolated from fermentation cultures of Streptoverticilium mobaerense as reported previously [12]. Hydroxylamine and sinapinic acid (4-hydroxy-3-methoxy cinnamic acid) were purchased from Sigma and sequencing grade trypsin and chymotrypsin were purchased from Roche Biochemicals.

TGase catalysed modification of dB1

A solution of dB1 (1 mg/ml, 59 μM) was incubated with 6 mM hydroxylamine (HA) and 0.5 μM

Modification of dB1 as function of time

The MALDI mass spectra of dB1 samples after different incubation times in the presence of HA and TGase are shown in Fig. 3A. In panel a (t = 0 min) a single mass peak of 16915 Da is shown, corresponding to the intact, unmodified dB1 protein [9]. Upon incubation the average molecular mass increases with time. A Gaussian distribution of masses with 16 Da increments is observed, this incremental mass difference corresponds to attachment of a single HA moiety (Fig 3A, panels b-f). The extent of

General conclusions

Mass spectrometry can be applied to in vitro protein modification products in order to localise modified amino acids at the sequence level. This approach is of value for food protein research, for correlation of modifications at the amino acid level to (altered) functional properties. MALDI-MS is the preferred technique for determining the overall extent of protein modification. Nanospray MS/MS is the technique of choice for localisation of modifications at the amino acid level. Using this

Acknowledgements

This work was financially supported by the Dutch Ministry of Economic Affairs through the Innovation Oriented Programme (IOP) Industrial Proteins.

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