Regioselective analysis of heat-induced conformational changes of R-lactoglobulin by quantitative liquid chromatography-mass - mass spectrometry analysis of chemical labeling kinetics

R-Lactoglobulin is a main allergen in cow's milk; its allergenicity is strongly impacted by processing. To understand heat-induced epitope-specific effects, the present study analyzed regiospecific conformational changes of heated native R-lactoglobulin variant A (BLG-A). Complementary fluorescence spectroscopy methods indicated two denaturation phases comprising minor sequential conformational changes (25-75 degrees C) and complete transitions (80-90 degrees C). Regioselective conformational changes of BLG-A in the native state (25 degrees C), sequential (70 degrees C) and complete transition (90 degrees C) were determined by quantitative liquid chromatography-mass spectrometry analysis of chemical labeling kinetics covering 14 lysine residues and the N-terminus. Conformational changes in two phases were observed for N-terminus, K8 8 (both N-terminal chain), K 60 (R-sheet C), K 75 (R-sheet D), K 77 (DE loop), K 83 (R-sheet E), K 100 and K 101 (FG loop). The residues K 14 (R-sheet A1), K 47 (R-sheet B), K69 , 69 , K 70 (both R-sheet D), and K 91 (R-sheet F) were not involved in conformational changes.