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  • Compared with qualitative analysis by Kalyankar et

    2019-08-13

    Compared with qualitative analysis by Kalyankar et al. (2013), less sequences were identified in iTRAQ-labelled analysis. It is not surprising with the finding that each of the identified peptide includes the sequence in every sampling point. Glu(51)-Tyr(52) of αs2-casein was observed to have been cleaved in the α-caseins digested with GE at 37 and 50°C. This result was not observed in the previous qualitative analysis. The iTRAQ-labelled αs2-casein f43–50 was also identified in the samples digested at 50°C. However, the MASCOT score was only 12 for the peptide, therefore, this sequence was not listed in Table 1. Even though the sequence was below the reliable limit for the MASCOT score, the identification of this peptide at 50°C is supportive evidence that the Glu-Glu bond may also be hydrolysed with GE probably at a slower rate. Theoretically expected short peptides (<6 amino smad inhibitor residues) obtained on GE digestion of α-caseins have already been reported by Kalyankar et al. (2013). However, only αs1-casein f(15–18), VLNE, was identified an iTRAQ-label using a manual search approach. This would indicate that short peptide sequences were not amenable to iTRAQ labelling and/or short iTRAQ labelled peptides were less detectable with the LCMS method employed herein.
    Conclusion The results presented herein indicate that large peptides were generated earlier than small sequences during GE hydrolysis of α-caseins. Furthermore, incubation temperature had an important effect in promoting the hydrolysis. The time for the iTRAQ-labelling process needs to be optimised since the results herein demonstrate that incubation for 1h at room temperature as presented in the manufacturer’s protocol was insufficient for complete labelling of some peptides to LCMS detection. The results herein indicate that phosphorylated peptides were less sensitive to LCMS detection than non-phosphorylated peptides. However, there was no distinct difference in digestion rates between phosphorylated and non-phosphorylated peptides compared by the ratio of the iTRAQ ions. In Glu-Glu-Glu-Tyr, hydrolysis of Glu-Tyr was preferred over hydrolysis of Glu-Glu. The conclusion by Kalyankar et al. (2013) that Glu residues at the P1’ position were poorly preferred was further demonstrated.
    Acknowledgements
    Introduction Bioactive peptides are regulated by specific peptidases, which hydrolyze them. The cyclic amino acid proline is present in several biologically active peptides and plays a critical phyisiological role by protecting these peptides from proteolytic degradation [1], [2]. Prolyl endopeptidase (EC 3.4.21.26) (PEP) is a member of the serine peptidase group, classified under the prolyl oligopeptidase family, enzymes which have the ability to cleave peptides at internal proline residues [2]. Initially discovered in the human uterus [3], PEP activity and expression has been demonstrated throughout human and other mammalian tissues [4], [5], [6], [10], [11]. Although it has been described as a predominantly cytosolic enzyme, it has become increasingly clear that PEP is also found in cell membranes [5], [7], [8], [9]. The activity of PEP is higher in developing than in adult tissues [8]. In addition, its nuclear localization in proliferating (peripheral) tissues seems to link this enzyme in proliferation and differentiation of the tissues [10]. Altered patterns of the expression and catalytic function of pepetidases may contribute to several disease processes, neoplastic smad inhibitor transformation and tumor progression included [13], [14]. However, most efforts to understand the physiopathological roles of PEP have hitherto focused on non neoplastic diseases. Thus, PEP has been linked to a variety of neuropsychiatric disorders, such Alzheimer\'s disease, amnesia, depression, schizophrenia, anorexia and bulima, as well as other diseases such as coeliac sprue, hypertension and a variety of infectious diseases [1], [2], [6]. PEP and PEP inhibitors have been proposed as potential therapeutic agents for some of these disorders [1], [6], [15], [16], [17].