L-Kynurenine During the course of enzymatic digestion
During the course of enzymatic digestion, protein is digested into large peptides and these large peptides are then further digested into small fragments. αs1-Casein f15–39, which contains 25 amino L-Kynurenine residues was the longest iTRAQ-labelled peptide identified in the samples digested with GE at 50°C. It was seen that the level of this peptide decreased during sampling. However, the intensity of the same sequence in the samples digested at 37°C increased up to 60min incubation and then decreased at 120min presumably due to further hydrolysis of this peptide. The ratio of iTRAQ-labels, 115/114, 116/114 and 117/114, were 0.82, 0.53 and 0.31 for the samples digested at 50°C, respectively, and were 2.57, 3.00 and 1.28 for the samples digested at 37°C, respectively. αs1-Casein f31–55, another long iTRAQ-labelled peptide with 25 amino acid residues, was only identified in the GE digested samples at 37°C. The ratio of iTRAQ-labels, 115/114, 116/114 and 117/114, were 1.54, 1.48 and 0.44, respectively, which indicates that the peptide reached its highest concentration after 15min digestion. For the GE digested samples at 50°C, fragments of αs1-casein f31–55, i.e., f31–39 and f40–55, were identified, which indicated that αs1-casein f31–55 was cleaved so rapidly at peptide bond of Glu(39)-Leu(40) in the GE digested samples at 50°C. The αs1-casein f31–55 could not be detected and identified by LCMS analysis. The findings also indicate that temperature is an important factor in the digestion, e.g., incubation at 50°C enhanced the rate of the GE digestion process. Based on the ratio of iTRAQ reporter ions (Table 1, Table 2), the concentration of 18 and 17 peptides from 27 and 28 identified peptides reached their maximal values after 120min digestion in the samples digested at 50°C and 37°C, respectively. Only two peptides (αs1-casein f15–39 and αs2-casein f13–23, which were specifically cleaved by GE), reached their highest concentration at 0min in the samples digested at 50°C. This indicates that these peptides were generated immediately after GE addition prior to sampling. The fact that 67% of the peptides identified in the samples digested at 50°C had not reached a plateau suggests that in the iTRAQ-based quantitative analysis, protein digestion should be continued for a longer time in order to reach its maximal concentration of peptides even at the high incubation temperature, i.e., 50°C. In the iTRAQ application protocol (Applied Biosystems), labelling of protein digests with the iTRAQ™ Reagents is recommended for 1h at room temperature. However, peptides with partially and fully labelled iTRAQ were identified in the samples of GE digested both at 50°C and 37°C, i.e., αs1-casein f71–89, f119–141 and αs2-casein f64–84 in the samples digested at 50°C, and αs1-casein f19–39 and f90–96 and αs2-casein f134–145 in the samples digested at 37°C. This result indicates that 1h incubation at room temperature may not be a sufficient duration for completion of the iTRAQ-labelling process. Further work needs be performed to find the optimal incubation time to fully label all peptides in GE digests of α-caseins. Table 3 presents the number of phosphorylated serines and phosphorylated peptides from αs1- and αs2-casein identified in the iTRAQ-labelled α-caseins’ digests at 37 and 50°C, respectively. These results of identification suggest that phosphorylated peptides were less sensitive than the non-phosphorylated peptides to LCMS detection with iTRAQ-labels. Interestingly, there was no distinct difference in digestion rates between phosphorylated and non-phosphorylated peptides (Table 1, Table 2) in GE digests of α-caseins. Several peptides with reported bioactive sequences in BIOPEP database (Dziuba & Dziuba, 2009) were identified in the GE digested α-caseins’ samples, i.e., αs1-casein f90–96, f142–148, and f193–199. The αs1-casein f90–96, f142–148 were both identified in the samples digested with GE at 37 and 50°C. The αs1-casein f193–199 was only identified in the samples digested with GE at 50°C. Further research may be carried out on the bioactivity ability, e.g., ACE inhibitory, with these samples.