Correlation between lactosylation and denaturation of major whey proteins: an investigation by liquid chromatography-electrospray ionization mass spectrometry.
The Maillard-reaction-induced lactosylation of the major whey proteins, alpha-lactalbumin (alpha-La) and beta-lactoglobulins (beta-Lg) A and B, occurring upon heating at 70, 80 and 90 degrees C for 1 to 5 h in the presence of lactose excess, was studied by HPLC coupled to electrospray ionization single and tandem mass spectrometry (HPLC-ESI-MS, MS/MS). The presence of significant amounts of mono and bi-lactosylated forms of the three proteins and their increase with heating temperature and time were assessed from MS data. Evidences for a concomitant, significant denaturation, involving partial tertiary structure unfolding, were also obtained in the case of beta-lactoglobulins. A subsequent ESI-MS and MS/MS investigation on the tryptic digests of heated protein solutions exhibiting high percentages of mono and bi-lactosylated forms provided information on lactosylation sites. In particular, the latter were identified both on tryptic and on aspecific peptides, whose unusual relevance (compared to similar studies) was found to be due mainly to heat-induced protein degradation, occurring before protein digestion with trypsin. Among lactosylation sites identified only on tryptic peptides, i.e., those reasonably related to intact protein lactosylation, two lysines residues were found for alpha-La, both located in accessible regions of its tertiary structure. In the case of beta-Lg, besides three sites common to variants A and B (leucine 1, lysines 70, and 75), lysine 69 was found to be lactosylated only in variant B. Its proximity to a critical region of beta-Lg tertiary structure suggests that the difference between the two variants could be ascribed to a different evolution of their conformation upon heating.
Losito
Ilario
I
Dipartimento di Chimica, Università degli Studi di Bari, Via E. Orabona 4, 70126 Bari, Italy. losito@chimica.uniba.it
Stringano
Elisabetta
E
Carulli
Saverio
S
Palmisano
Francesco
F
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007768:Lactalbumin / Q000737:chemistry*; D007785:Lactose / Q000737:chemistry; D008894:Milk Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D000067816:Whey Proteins
D008894:Milk Proteins; D000067816:Whey Proteins; D007768:Lactalbumin; D007785:Lactose