The N terminal amino acid sequence was
The N-terminal amino Octyl-α-ketoglutarate sequence was made for STH2. This sequence showed high similarity (100%) with to trypsin-like found in the human airway classified as serine proteinase that also belongs to peptidase family SI and related also with chymotrypsin-like enzymes (Yasuoka et al., 1997). The sequence of SH1 was not determined because the N-terminal portion of the enzyme is blocked.
The molecular mass estimated for serine endopeptidase SH1 (49kDa) and serine thiol endopeptidase (60kDa), added to their localization along of the nephron (distal tubule) (Quinto, 2000), suggest that these enzymes are originated from the kidney and may cleave the filtered kinins by the glomerulus or the kinins that are produced in the kidney, specifically in the distal tubule, where all kallikrein-kinin system are located (Vio, Loyola, & Velarde, 1992).
Introduction Hydrolysed milk proteins are widely applied in food industry for many purposes, e.g., as ingredients in infant formula and in parenteral nutrition (Meisel, 2004, Meisel, 2007, Nongonierma and FitzGerald, 2012, Power et al., 2013). α-Caseins (αs1- and αs2-casein) are major proteins in bovine milk accounting for approximately 38% of overall milk protein. Analysis of the digestion of α-caseins is therefore important for nutritional studies of dairy protein. Glutamyl endopeptidase (GE) is a chymotrypsin-like serine protease, mainly found in Bacillus species, that specifically cleaves peptide bonds between negatively charged amino acid residues (Glu/Asp) (Yokoi et al., 2001). Madsen et al. has reported that GE is more efficient in the hydrolysis of caseins compared to whey proteins (Madsen & Qvist, 1997). The substrate specificity of GE with bovine α-caseins’ preparation has been qualitatively studied (Kalyankar, Zhu, O’Keeffe, O’Cuinn, & FitzGerald, 2013). However, to our knowledge, the hydrolysis process was not quantitatively characterised. A quantitative analysis is necessary to provide more detailed information on the GE α-caseins’ hydrolysis process. Isobaric tag for relative and absolute quantification (iTRAQ) is one of the two pioneering silent isotope incorporation methodologies (Wright, Gan, Chong, & Pham, 2007). Both the identification of proteins/peptides and their quantification can be achieved in a single injection (Sickmann, Burkhart, Vaudel, Zahedi, & Martens, 2011). In food science, the iTRAQ labelling was applied to quantitative proteomic analysis of bacterial enzymes in ripening cheese (Jardin, Molle, Piot, Lortal, & Gagnaire, 2012). However, quantitative analysis of enzymatic digestion of milk proteins using iTRAQ methodologies does not yet appear to have been reported in the literature. This work reports the quantitative investigation of the substrate specificity of GE with bovine α-caseins, the main proteins in bovine milk. GE was purified from Alcalase™ and its specificity was investigated using α-caseins. The hydrolysates of α-caseins digested with GE at different times at 37 and 50°C were quantitatively analysed using iTRAQ technology.
Materials and methods
Results In the 4-plex iTRAQ labelled peptides, the fragments released from CID were the reporter ions at 114.1, 115.1, 116.1 and 117.1m/z. The fragmentation spectrum of f15–30 of αs1-casein is shown by way of example in Fig. 1. The sequence coverage was 56% and 60% for the identified αs1-casein peptides at 37 and 50°C, respectively (Table 1, Table 2). The sequence coverage was 26% and 30% for the identified αs2-casein peptides at 37 and 50°C, respectively (Table 1, Table 2). The false discovery rates (FDR) were less than 5%. The identified sequences of the iTRAQ-labelled samples were combined to provide greater coverage and precise quantification (Wright, Chong, Gan, & Pham, 2006). The sequence coverage data shows that such a high temperature during digestion may increase the extent of hydrolysis, so that the digested peptides are more concentrated and more easily detected and identified during the subsequent LCMS analysis.