Qualitative and quantitative analysis of wheat gluten proteins by liquid chromatography and electrospray mass spectrometry Gianfranco Mamone 1,2 , Pasquale Ferranti 1,3 *, Lina Chianese 3 , Laura Scafuri 2 and Francesco Addeo 1,3 1 Instituto di Scienze dell'Alimentazione, via Roma 52/c I-83100 Avellino, Italy 2 Associazione Italiana Celiachia, Milano, Italy 3 Dipartimento di Scienza degli Alimenti, Universita Á degli Studi di Napoli Federico II, 80055 Portici, Italy SPONSOR REFEREE: Dr S Daolio, CNR, Padova, Italy Based on analysis by liquid chromatography/electrospray ionisation mass spectrometry, we have developed a new method for fast and sensitive ®ngerprinting of gliadins and glutenins in wheat ¯our. Using this procedure the two protein fractions from seven durum wheat varieties have been analysed by high resolution high performance liquid chromatographic separation coupled to accurate determination of molecular mass. In this way, the molecular mass of the single components from both gliadin and glutenin fractions were measured and more than forty components were detected for each fraction indicating a high heterogeneity. Although the chromatographic pro®les were similar, the molecular masses of protein components with similar retention times among the varieties were often different. The difference ranged from a few mass units corresponding to single amino acid substitution(s) up to thousands implying peptide deletion or insertion along the protein chain. Two components representing about a half of the gliadin fraction, e.g. g 2 - and g 3 -gliadin, were identi®ed through the N-terminal sequence and molecular mass determination. We suggest the use of the high level and the molecular mass of these gliadin components as markers to detect traces of wheat in gluten-free food preparations for celiac patients. Copyright # 2000 John Wiley & Sons, Ltd. Received 20 March 2000; Accepted 22 March 2000 In recent years, research has witnessed two main directions in the study of gluten proteins aimed at (i) finding relationships between individual alleles coding for gliadins, high (HMW) or low molecular weight (LMW) glutenin subunits, and the viscoelastic dough properties of wheat derived products such as pasta and bread; 1±4 and (ii) identifying prolamins and derived peptides involved in celiac disease, a physical condition in which the small intestine of genetically predisposed individuals is reversibly damaged. The gluten proteins are conventionally subdi- vided into gliadins and glutenins representing up to 80% of wheat proteins. These protein fractions are each currently identified according to gel electrophoretic mobility or through 2D co-ordinates. 5 Gliadins, sub-divided into a, b, g, and ! fractions, are alcohol soluble components representing about half a gluten protein. 6,7 Several amino acid sequences of gluten proteins are available from the cDNA 8 sequence, whereas little has been done at the protein level. 9,10 It has been found previously that the quality of pasta good can be determined by the presence in the flour of g-45 or g-42 gliadins, respectively, 11 although the effective property was shown to be linked to the concomitant presence of some glutenin components. 12 The structural analysis of gliadins or glutenins is limited by the extraordinary heterogeneity of the relative fractions, each being constituted by at least 20±30 electrophoretic components. HPLC efficiency in fractionating gluten proteins has been evaluated by means of gel isoelectric focusing in a second dimension as a discriminating technique. 9 Some components were constituted by a single band whereas a number of fractions were still cross- contaminated. 13 Most gliadins have a mass within a 30±40 kDa range, though !-gliadin can reach 80 kDa. 14 Glutenins are as heterogeneous as gliadins and present in the native form mass figures ranging from 300 kDa to a million. 2,14±17 Glutenin subunits are constituted by both LMW (30±40 kDa) and HMW (65±90 kDa). 14 The subunits are linked by a series of inter- and intramolecular disulphide bonds and possibly by other cross-links whose chemical nature has yet to be ascertained. 18,19 Recently, mass spectrometry has gained greater impor- tance as an alternative method for the characterisation of high molecular mass proteins, even in complex mixtures. In particular, matrix-assisted laser desorption/ionisation time- of-flight mass spectrometry (MALDI-TOFMS) has been applied to characterise wheat a-gliadins and HMW glutenin subunits. 20±22 This methodology has been extended to the direct analysis of bread and durum wheat gliadins and glutenin subunits. 23 MALDI-TOFMS in combination with artificial neural network analysis has also been used to classify the wheat gliadin fraction. 24 MALDI analysis of proteins, although considered fast and specific, presents however a high mass resolution still poor compared with *Correspondence to: P. Ferranti, Centro Internazionale di Servizi di Spettrometria di Massa, via P. Castellino, 111 80131 Napoli, Italy. E-mail: ferranti@unina.it Copyright # 2000 John Wiley & Sons, Ltd. RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 14, 897±904 (2000)