Analysis of the Effect of Temperature Changes Combined with Different Alkaline pH on the beta-Lactoglobulin Trypsin Hydrolysis Pattern Using MALDI-TOF-MS/MS

作者：Cheison Seronei Chelulei^*; Brand Janina; Leeb Elena; Kulozik Ulrich

来源：Journal of Agricultural and Food Chemistry, 2011, 59(5): 1572-1581.

DOI：10.1021/jf1039876

• Trypsin hydrolysis
• beta-lactoglobulin
• alkaline pH
• MALDI-TOF-MS(/MS)

Temperature and pH influence the conformation of the whey protein beta-lactoglobulin (beta-Lg) monomer, dimer, and octamer formation, its denaturation, and solubility. Most hydrolyses have been reported at trypsin (EC 3.4.21.4) optimum conditions (pH 7.8 and 37 degrees C), while the hydrolysate mass spectrometry was largely limited to peptides with < 4 kDa. There are few reports on trypsin peptide release patterns away from optimum. This work investigated the influence of alkaline (8.65 and 9.5) and optimum (7.8) pH at different temperatures (25, 37.5, and 50 degrees C) on beta-Lg (7.5%, w/v) hydrolysis. Sample aliquots were drawn out before the addition of trypsin (blank sample) and at various time intervals (15 s to 10 min) thereafter. Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was used to monitor peptide evolution over time with the use of two matrixes: alpha-cyano-4-hydroxycinnamic acid (HCCA) and 2.5-dihydroxyacetophenone (DHAP). Mass analysis showed that the N- and C-terminals (Lys(8)-Gly(9), Lys(100)-Lys(101), Arg(124)-Thr(125), Lys(141)-Ala(142), and Arg(148)-Leu(149)) of beta-Lg were cleaved early (15 s) implying the ease of trypsinolysis at the exposed terminals. Hydrolyses at 25 degrees C and pH 7.8 as well as at 50 degrees C and pH 9.5 were slowed down and ordered. Nonspecific chymotrypsin-like behavior occurred more at higher temperatures (50 degrees C) than at lower ones (25 and 37.5 degrees C). In addition to our earlier work in the acid pH region, it can be concluded that there is potential for controlled hydrolysis outside the trypsin optimum, where different target peptides with predictable biofunctionalities could be produced.

• 出版日期2011-3-9