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  • br Introduction As a serine proteinase and a sub

    2020-03-27


    Introduction As a serine proteinase and a sub-family of the chymotrypsin-like proteinase, Glutamyl endopeptidase (GE), which is mainly found in Bacillus species, specifically cleaves negatively charged amino crth2 receptor residues (Glu/Asp) (Yokoi et al., 2001). Madsen et al. (Madsen & Qvist, 1997) reported that GE had better ability of hydrolysis on caseins than on whey proteins. GE was purified from a commercial food-grade proteinase preparation from Bacillus licheniformis, Alcalase™ (Spellman, Kenny, O\'Cuinn, & FitzGerald, 2005). The substrate specificity of GE with bovine β-casein, one of the major proteins in bovine milk accounting for approximately 33% of caseins, has been qualitatively studied (Kalyankar, Zhu, O\'Cuinn, & FitzGerald, 2013). However, the hydrolysis process needs to be further characterised in quantitation analysis. A quantitative analysis on the GE β-casein hydrolysis process can provide more detailed information about how the β-casein is enzymatically hydrolysed by GE. Two isotopic labeling techniques are applied widely in quantitative analysis. The first technology is ICAT, which is using isotopic-labeled cysteine amino acid residues by a 8 Da different tag containing biotinylated reagents (Gygi et al., 1999). However, 15% of total proteins, which didn\'t contain any cysteine amino acid crth2 receptor residues, can\'t be labeled with ICAT technique. Another technique called iTRAQ, is labeled on primary amines on the arginine and lysine side chains and on the N-terminal of the peptides with 2–8 isotope encoded reporter ions, which permits relative quantitation of 2–8 samples simultaneously. Quantitation with iTRAQ methodology is based on the abundance of low mass reporter ions, e.g., m/z 114–117, as iTRAQ4plex reagents, observed in tandem mass spectrometry (MS/MS) fragmentation of iTRAQ-labeled peptides (Treumann & Thiede, 2010). In a single iTARQ analysis, both identification of the peptides and their quantification can be achieved. Proteomics applications of the iTRAQ technique have been reported by several authors (Kristjansdottir and Kron, 2010, Sickmann et al., 2011, Wright et al., 2007). However, quantitative analysis was not widely applied in the field of food chemistry. Quantitative analysis of enzymatic hydrolysis of milk proteins using iTRAQ methodologies is reported on bacterial enzymes released in ripening cheese (Liu, Han, Sun, & Geng, 2014).
    Materials and methods
    Results and discussion Based on the fragments released from CID, i.e., 4-plex iTRAQ reporter ions at 114.1, 115.1, 116.1 and 117.1, iTRAQ-labeled peptides were counted in WARP-LC® 1.2. The sequence coverage is 53.1% and 27.8% of the identified β-casein at 37 and 50 °C, respectively (Table 1, Table 2). The FDR were less than 4%. To improve sequence coverage, sequences of iTRAQ-labeled samples from two parallel experiments were combined (Wright, Chong, Gan, & Pham, 2006). The ratios of 115/114, 116/114 and 117/114 in all 19 peptide fragments, which represented the ratios of concentrations at different sampling intervals, indicated the maximum concentrations were reached at 60 min in the samples hydrolysed with GE at 50 °C (Table 2, Fig. 1). In the samples hydrolysed with GE at 37 °C (Table 1), two peptides (f1-14, f48-91) reached their maximal concentrations at the beginning of sampling, four peptides (f32-42, f92-100, f92-121, f122-131) reached maximal concentrations at 60 min and the rest 10 peptides were still increasing at 120 min, according to the ratios of 115/114, 116/114 and 117/114 in 16 peptide fragments. None of the peptides identified in the GE hydrolysed β-caseins’ samples matched the reported bioactive sequences in BIOPEP database (Dziuba & Dziuba, 2009). A hydrophobic 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.