| Summary: | 碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 95 === Abstract
Molecular recognition plays an important regulator in cellular
activities. Mapping the interaction sites of protein is of great interest since
it contributes much to our understanding of the mechanisms of molecular
recognition and provides the basis for rational vaccine design. We
employed Nanoprobe-Based Affinity Mass Spectrometry (NBAMS) to
rapidly and accurately map small molecule/protein interaction sites. To
demonstrate the general applicability of our approach in tackling of small
molecule/protein interaction, three biomolecular interaction systems,
mannose/Con-A, haparin/A27Laa, and inhibitors/α 1,2-L-fucosidase
were investigated in this study.
In the first part of this thesis, the mannose/Con-A interaction will be
used the assay optimization, kinetic study, and investigation of detection
limit. Our result shows our nanoscale probe facilitating the fast
interaction of mannose and Con A; the reaction time can be completed
within 15 min with detection limit of 15 ng. For epitope mapping, the
carhohydrate-binding peptides were identified with various proteases
(trypsin, chymotrypsin, and Glu-C), consistent with reported binding sites
by X-ray crystallization analysis. Moreover, the subtle structure change of
Con-A/mannose in metal environment can be observed. For
harparin/A27Laa system, the peptide at m/z 2694.6 (residue 1-32) of
A27Laa was identified, which contains basic strip of 12 residues
responsible for binding to cell-surface heparan sulfates. Although A27Laa
lost its C-terminal, we found the epitope can be bound to the
heparin-MNPs in our approach. For α1,2-L-fucosidases/inhibitor system,
IV
we probed the structural differences of the epitopes for three
fuconojirimycin inhibitors:
Inhibitor1:(2R,3S,4S,5S)-2-(aminomethyl)-6-methylpiperidine-3,4,5-triol
Inhibitor2: 5-(3-aminopropoxy)-2-methylpiperidine-3,4-diol
Inhibitor3:1-(3-aminopropyl)-2-methylpiperidine-3,4,5-triol
and the effects of different length of linker conjugated on the surface of
MNPs. The change of binding epitopes is consistent to the structure
modeling of α1,2-L-fucosidase complexed with different inhibitor.
Moreover, we found that the effect of digestion time is critical for the
epitope mapping.
Given the flexibility of nanoprobe derivation, adaptation to various
proteases, and the high rapid and high sensitivity analysis, we believe our
approach hold great promise in probing binding epitopes of small
molecule and proteins.
|
|---|
