Investigation On Interactions Of Pseudomenas Cepacia Lipase And Insulin With Small Molecules By Mass Spectrometry

Protein is a kind of important biomacromolecule. The relationship of its function and structure, interaction with small molecule and performance enhancement in non-conventional reaction media are continuous hotspots of investigation. Currently, the interaction of protein with small molecule was studied by researchers through chemical modification and molecular biotechnology and the biological function was improved on the basis of these techniques. However, assay of the structure and mechanism is still not so perfect and the function improvement is also not so satisfied. In this work, the relationship of structure and function, reaction mechanism and structure analysis were studied by the combination of residue-specific modification, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization mass spectrometry (ESI-MS) and molecular dynamics (MD) simulation.Firstly, the effect of chemical modification on the p-NPP (p-nitrophenyl palmitate) catalytic activity and enantioselectivity (E value) towards secondary alcohol of Pseudomenas cepacia lipase (PcL) was studied. Five pairs of secondary alcohol ester with different acyl and alcohol moieties were designed and synthesized. And the correlation between the structural differences of substrates and enantioselectivity was revealed. PcL was modified by four specific reagents respectively at the aqua-hexane interface, and the modification sites were identified by MALDI-TOF-MS. The results obtained from MD demostrated that modification of Tyr29 which located in the catalytic cavity of PcL had a significant impact on the E value. The strong steric hindrance of acetyl and iodine ion to the groups on the chiral center of the substrates is responsible for the E value improvement. The enhancement of hydrophobicity on the surface of the lipase due to the sidechain replacement of Asp with uncharged hydrophobic groups also improved the E value. In addition, the catalytic activity of EDA-PcL was increased by about 6 folds. And conformation change which was induced by the modification is responsible for the catalytic activity improvement.The interation of insulin from porcine pancreas and antitumor drug, cisplatin, was investigated afterwards. The effect of insulin/cisplatin molar ratio, pH, protein configuration and reaction time on the reaction was studied by MALDI-TOF-MS. The results showed that the reaction was time-dependent and the other three factors also played significant roles in this reaction. The presence of cisplatin resulted in the rupture of the disulfide bond, especially under the condition of high concentration and long rection time. Further analysis by MALDI-TOF-MS and ESI-ion trap/time-of-flight mass spectrometry (ESI-IT-TOF-MS) allowed the identification of platinated mono-, di- and triadducts in the incubations under different conditions. Firstly, the MALDI laser induced dissociation (LID) results indicated that one platinum bound to insulin B chain. Then, platinum binding sites were identified by ESI collision induced dissociation (CID) as N-terminus (B chain), cysteine7 (B chain) and cysteine19 (B chain) residues.Finally, the reactions of matrices in MALDI and cysteine, RCDAA, RCEAA and insulin from porcine pancreas were studied. MALDI-TOF-MS and ESI-MS were utilized to investigate the mechanism of this reaction. The MALDI-TOF-MS results indicated that α,β-unsaturated acid reacted with biological samples containing sulfhydryl under alkaline condition. ESI-MS results demostrated that the reaction occurred in liquid phase, not in the MALDI-TOF mass spectrometer. The results of ESI-tandem mass spectrometry demonstrated that sulfhydryl-containing biological samples covalently attached to a,p-unsaturated acid, not just associated together.