| Summary: | 碩士 === 國立中興大學 === 化學系所 === 102 === Over the last decade, NanoLC coupling ESI-MS is an important
technology for the study of proteomics. In this work, a straightforward and reliable frit fabrication method is developed for the preparation of nanoLC column. The Stage-Frit is simply fabricated by a C18 Extraction disk with a sol-gel polymerization. The b ack pressu re of a 7 5 μm x 1 0 cm stag e-frit column packed with 1.8 μm C18 beads is regularly low than 185 bar at a flow rate of 300 nl/min with 0.2% formic acid in H2O. That means this tiny C18 beads filled in our Stage-Frit nanoLC column could be operated on a common nanoLC system instead of nanoUPLC system that will totally reduce the cost for the purchase of instrument. Furthermore, the back pressure of 3 μm C18 Stage-Frit column is decreased around 35 bar at the flow rate of 300 nl/min as compared with a commercial available nanoLC column. The Stage-Frit allows long-term continuous flow of the solvent and no significant beads loss or pressure instability was observed during the period. In the analysis of tryptic peptides of bovine serum albumin (BSA) by the common nanoLC system integrated with LTQ-Orbitrap XL mass spectrometer showed that our Stage-Frit nanoLC column had better separation performance than the commercial available nanoLC column. The repeatability of retention time was found to be less than 3.3% (RSD) in nanoLC-ESI-MS experiments. The average peak width at half high for the twelve BSA tryptic peaks was 6.36s. As compared with commercial column, an additional 362 unique peptides corresponding to 91 proteins were identified from human spleen tissue proteins by homemade 1.8 μm C18 nanoLC column. In summary, a novel method was developed for the fabrication of an inexpensive, simple, highly reproducible and durable capillary column frit for nanoLC-ESI-MS analysis. This method will become a routine frit preparation procedure for proteomic laboratories.
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