A hydrophobic peptide with molecular weight of Da Ward casei

A Edrophonium chloride sale peptide with molecular weight of 5315 Da (Ward, 1998), β-casein f48-91, was observed with low intensity (data not shown) in the sample hydrolysed with GE at 37 °C. The cleavage on C-terminal of Asp with GE was already reported as 1000-fold slower than the cleavage on C-terminal of Glu (Breddam & Meldal, 1992). This is a critical factor of the low intensity identification on f48-91 in the sample hydrolysed with GE at 37 °C. Interestingly, even with low intensity, the sequence was identified with MASCOT score of 30. Therefore, GE cleavage on both Glu and Asp was further demonstrated. There are five phosphorylated serines in β-casein. However, only phosphorylated Ser35 was identified in the samples hydrolysed with GE at 37 and 50 °C, i.e., f32-42 and f32-44 of β-casein. The identification indicates that phosphorylated peptides were less detectable from the non-phosphorylated peptides with iTRAQ-labels. Interestingly, although the phosphorylated peptides with iTRAQ-labels were less detectable in the identification, there is no difference in digesting rates from the ratios of 115/114, 116/114 and 117/114 (representing the ratios of iTRAQ ions intensity labeled on samples enzymatically hydrolysed at 15, 60 and 120 min (in relation to 0 min) respectively) between phosphorylated peptides and non-phosphorylated peptides (Table 1, Table 2), which indicated that phosphorylation would not be affected by the iTRAQ-labeling procedure. Oxidation of Met was a common reaction during heat treatment of proteins and peptides (Li, Schoneich, & Borchardt, 1995). Zhu et al. (Zhu & FitzGerald, 2010) reported that pH-dependent heat effect on methionine oxidation was observed. From Table 1, Table 2, temperature effect was also observed on the oxidation of Met. Three Met containing sequences, i.e., triply oxidised f92-121, singly oxidised f92-100 and singly/doubly oxidised f101-121, were identified in the samples hydrolysed with GE at 50 °C. Singly oxidised f101-121 and doubly oxidised f92-121 were identified in the samples hydrolysed with GE at 37 °C. In the same procedure of the sample treatment, more Met were oxidised in the samples GE hydrolysed at higher temperature, i.e., 50 °C. From the previous report, the Met of f92-100 was identified as non-oxidised. However, three different iTRAQ-labeled sequences of singly oxidised f92-100 were identified in the samples hydrolysed with GE at 50 °C, which suggests that the procedure of the iTRAQ-labeling introduced oxidation to Met. The C-terminal peptides from f132 to f209 contain only single glutamyl residue (E195) and single aspartic acid residue (D184). None peptides in the range was identified with iTRAQ-labels in all samples hydrolysed with GE at 37 and 50 °C in WARP-LC® (Bruker Daltonics). However, iTRAQ-labeled f185-209 was identified in BioTools (Bruker Daltonics). The MASCOT scores were 32 and 18 for the sequence in the sample hydrolysed at 37 and 50 °C, respectively. Interestingly, the intensity results of iTRAQ reporter ions reached maximum at 115 and 116 in the MSMS data of sequences in the sample hydrolysed at 37 and 50 °C, respectively (Fig. 2). The results indicate that the f185-209 was hydrolysed at 37 °C quicker than hydrolysed at 50 °C. It might be a reason that the sequence f185-209 was hydrolysed faster with GE in lower temperature i.e., 37 °C.