EuPA 2013 Scientific meeting, St Malo, France, 14-17 october 2013 Tracking of the chemical modifications occurring during the dry heating of -lactalbumin Gulzar M.1,2, Bouhallab S.1,2, Briard-Bion V.1,2, Jardin J.1,2, Croguennec T.1,2 INTRODUCTION: Dry heating is commonly used in food and pharmaceutical industries to decontaminate heat-sensitive samples such as egg white or therapeutic protein products. Under these conditions, thermal stability of proteins is increased and the changes in protein structure are limited. However, it was noted that the functional properties of the dry heated proteins are modified: for instance lysozyme, a poor foaming agent, gives stable foams after dry heating even if the secondary and tertiary structure of the protein are not affected. This discovery expends the use of dry heat treatment for functionalizing a wide variety of food proteins. A clear picture of dry-heat induced modifications of proteins is necessary especially for those dedicated to the formulation of infant formula. Mass spectrometry was used to assess such modifications on -lactalbumin, a food model protein. MATERIAL AND METHODS: -lactalbumin adjusted to pH 6.5 and water activity aw = 0.23 was dry heated at 100°C for up to 24 hours and analyzed on a hybrid Q-TOF mass spectrometer QStar XL (MDS Sciex, Toronto, Canada). Entire mass of -lactalbumin was investigated by ESI-MS using a nano ESI sample source. Analysis of hydrolytic peptides was performed using a nano-LC system equipped with a PepMP100 C18 column and coupled to the mass spectrometer. RESULTS: One mass loss of 18 Da (H2O) was observed on the N-term trypsin peptide (m/z of 600.3 Da instead of 618.3 Da). Its sequencing indicates that the modification was on the N-term glutamic acid (Fig 2). The formation of a pyroglutamic acid during dry heating is assumed (Fig 3). -lactalbumin is the predominant protein in human milk. Dry heat treatment traps a fraction of -lactalbumin molecules into non-native monomers. These non-native monomers were characterized by the loss of one or two water molecules per protein molecules. Fig 2: N-terminal -lactalbumin tryptic peptide sequencing Fig 1: mass spectrum -lactalbumin before and after dry heating The second mass loss of 18 Da occurred on the peptide 54-71 (Fig 4). LC-MS/MS analysis indicated that the modification was on D64. This was highlighted by the presence of a fragment Y8 at 880.47 Da (The release of a water molecule at D63 would give a fragment Y8 at 898.4 Da). The formation of an internal cyclic imide is suggested (Fig 5). Fig 3: Cyclisation of the N-terminal glutamic acid into pyroglutamic acid. Fig 5: Cyclisation of internal aspartyl residue D64 Fig 4: sequence of the pepsin peptide 54-71 CONCLUSION: Mass spectrometry is a method of choice to identify very small changes in the structure of proteins. Beyond the change in the functional properties targeting by dry heating, the potential impact of observed modifications on the nutritional properties and digestibility of dry heated proteins must also be considered. Further details in: Gulzar M., Bouhallab S., Jardin J., Briard-Bion V., Croguennec T. (2013) Structural consequences of dry heating on a-La and b-Lg at pH 6.5. Food Research international, (51) 899-906. 1, Agrocampus Ouest, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France 2, INRA, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France - www.rennes.inra.fr