超過500個 (大約占2％) 人類基因編碼為蛋白酶 (proteases) 或肽酶(peptidases)，這些功能不明確的蛋白酶或肽酶，其生理受質 (substrates) 大多未知，且不易以傳統分析方法鑑定，然而二肽基肽酶-4 (DPP-4) 已被認為是糖尿病的新興治療標的。在本研究中，以DPP-4、液相層析串聯式質譜 (LC-MS/MS) 結合二甲基穩定同位素標記來大規模篩選肽酶之外源性受質。使用三種胃腸道蛋白酶水解牛奶蛋白以建立胜肽庫 (peptide library)。胜肽混合物分成兩等分，一等分標記為輕原子；另一等分在DPP-4預培養後以重原子標記，之後將兩等分合併後以LC-MS/MS分析。經過DPP-4預培養裂解導致重原子標記之胜肽訊號減少或消失者被認為是DPP-4受質。
結果顯示，從牛奶蛋白水解物鑑定的975個胜肽中篩選出41個受質候選胜肽，並使用Web Logo分析其序列保守性。自N端第二個胺基酸為Proline 與Alanine是受DPP-4裂解之保守序列。使用了6種合成肽LPLSLLK (LK-7)、FALPQYLK (FK-8)、RPKHPIKHQGLPQE (RE-14)、YPELFR (YR-6)、VPQLEIVPN (VN-9)、VPYPQRDMPIQA (VA-12)來確認它們與DPP-4的反應。LK-7、YR-6、VN-9被DPP-4迅速地裂解，自N端釋放二肽且觀察到LSLLK、ELFR、QLEIVPN的片段，還觀察到FK-8、VA-12自N端裂解兩次，分別得到產物LPQYLK、QYLK和YPQRDMPIQA、QRDMPIQA的片段，6種合成肽在DPP-4的切位以及其裂解速率與D/H 比值呈現一致的趨勢。然而VN-9有較佳的DPP-4抑制活性，其IC50值為217.3 ±9.99 µM，抑制型態為真實底物 (real substrate)。有趣的是，還發現當P1和P1’為A和V時，DPP-4難以裂解出二肽。我們相信，DPP-4的外源性受質或抑制劑的發現將有利於降血糖藥物的開發。

More than 500 (around 2%) of human genes are encoded as proteases or peptidases that have still not yet understood clearly about their functions. According to previous publications, the inhibition of dipeptidyl peptidases- 4 (DPP-4) is considered an emerging therapeutic target for diabetes. In this study, DPP-4, liquid chromatography－tandem mass spectrometry (LC-MS/MS) combined with stable-isotope dimethyl labeling were used to screen the exogenous peptidase substrates on a large scale. After hydrolyzing milk protein using three gastrointestinal proteases, the resulting peptides were used to generate the peptide library. The peptide mixture was divided into two parts. One part was labeled with hydrogen (H) atom and used as the control (without DPP-4 treatment); while the remaining one was incubated with DPP-4 following by deuterium (D) atom labeling. After that, both samples were combined and analyzed by LC-MS/MS. After DPP-4 pre-incubation, the reduction or disappearance of the peptide signal of heavy-atoms (D/H < 1) was regarded as a DPP-4 substrate candidate.
The results showed that 41 of the 975 peptides identified from the milk protein hydrolysate were assumed as DPP-4 substrate candidates and their sequence conservation was analyzed using Web Logo. Proline and alanine at the second position from the N-terminal were the most common sites for DPP-4 cleavage. The six peptides LPLSLLK (L-7), FALPQYLK (FK-8), RPKHPIKHQGLPQE (RE-14), YPELFR (YR-6), VPQLEIVPN (VN-9) and VPYPQRDMPIQA (VA-12) were synthesized and analyzed their reactivities towards DPP-4. Among them, LK-7, YR-6 and VN-9 were cleaved rapidly from the N-terminus by DPP-4 to release dipeptides, and their products of LSLLK, ELFR, and QLEIVPN were also observed, respectively. Moreover, FK-8 and VA-12 was cleaved two times to release its products as LPQYLK, QYLK and YPQRDMPIQA, QRDMPIQA, respectively. The reactivity confirmation of six synthetic peptides towards DPP-4 indicated that they were readily cleaved by DPP-4 and their cleavage rate showed a similar trend with the D/H ratio values observed in the above-mentioned experiment. Furthermore, the peptides inhibitory activities against DPP-4 were also evaluated. Among them, the VN-9 showed the best DPP-4 inhibitory activity, with an IC50 value of 217.3 ± 9.99 µM, and its inhibition type was characterized as a real substrate. Interestingly, we found that DPP-4 is difficult to cleave the peptide when its P1 and P1' residue are A and V respectively. We believe that the discovery of DPP-4’s exogenous substrates or inhibitors can benefit the development of blood glucose-lowering agents.

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