| Summary: | 碩士 === 國立中正大學 === 化學暨生物化學研究所 === 100 === Protein phosphorylation modulates a variety of molecular and cellular functions. Although mass spectrometry has been emerged as a powerful tool in determination of phosphorylation site, the enrichment of phosphopeptides is indispensable prior to mass spectrometric analysis owing to low abundances, poor ionization efficiency, and heterogeneous type of phosphopeptides. The objective of the study is to enhance the detection sensitivity of phosphopeptide in LC-MS/MS by (1) fabrication of the electrospray emitter with small diameter and (2) complementary metal-directed immobilized metal ion affinity chromatography (IMAC) method.
Detection sensitivity in ESI-MS is dependent on the quality of the electrospray, which relies on the physical properties of the nanoelectrospray emitter. In the first part of the thesis, we attempted to increase the phosphopeptide detection sensitivity by reducing the inner diameter (ID) and outer diameter (OD) of nanoelectrospray emitters. By the use of laser based micropipette puller system, we devised a robust fabrication protocol on the nanoelectrospray emitter with smaller orifice (ID: 4.89 ± 1.26 μm and OD: 6.18 ± 1.81 μm), compared to the conventional emitter of 14.90 ± 6.33 μm (ID) and 62.34 ± 25.00 μm (OD). To improve the sample loss problem due to the presence of metal frit in the LC interface, the emitter can be directly interfaced with column. We further fabricated C18-packed column to facilitate the LC-MS/MS analysis. Based on the two peptides, m/z 671.34 and m/z 550.28, from beta-galactosidase, the preliminary results show that packing with 1.9 μm C18 into the 25 cm column has efficient separation of the two peptides locating at similar retention time.
Immobilized metal ion affinity chromatography (IMAC), based on the coordination of metal ion with phosphate group, is one of the most commonly used approaches for the enrichment of phosphopeptides. Previous literatures have shown that different metal ions can provide different affinity for heterogeneous phosphopeptides. Though the Fe3+ is often used in IMAC, the use of other metal ions have not been explored. In the second part of the thesis, we aim to develop a sequential elution strategy based on the metal-directed IMAC. Towards more comprehensive profiling of the phosphoproteome, we have used Ga3+-IMAC, Al3+-IMAC, and Fe3+-IMAC according the order to increase the number of the phosphoproteomic identification. Optimization on the enrichment specificity were achieved with the use of acetic acid, acetonitrile for Ga3+-IMAC, Al3+-IMAC , respectively. On the model study on Raji B cell, a total of 3081 phosphopeptide can be identified by ESI-Q-TOF MS without other fractionation method. Comparing with single Fe3+-IMAC, 1.5-fold increase on the number of phosphopeptide can be obtained by sequential IMAC elution. The result demonstrated that this strategy improve the identification coverage for phosphoproteomics profiling.
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