Comparison of Fused-Core with 3 µm, 5 µm Porous Silica Particles Used in Capillary Columns by LC–MS/MS for Proteomic Study

• Main Authors: Tzu-Wei Tseng, 曾子瑋
• Other Authors: Sung-Fang Chen
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/07806783833603865406

碩士 === 國立臺灣師範大學 === 化學系 === 100 === Capillary liquid chromatography coupled with electrospray tandem mass spectrometry（LC-ESI-MS/MS）plays an important role in proteome analysis by means of shotgun proteomics. More recently, a fused-core technology is developed as an alternative of 3 µm or smaller particle packing for HPLC separation. It enables faster separation with sufficiently high resolution with lower system pressure. Fused-core particles are more narrow size distributions than fully porous particles, and consisting of thick porous shell. The conception is that a packing particle with a narrow particle size distribution allows a more homogeneous packing, which reduces the eddy diffusion contribution to band broadening and thus improves the separation efficiency of the column. Thick porous shell also provides better mass transfer for phase partitioning, especially for large molecules. In this study, lab-made reversed-phase capillary columns were used for separation of protein digest with tandem MS（Q-TOF）analysis. The effects of column length and particle size on the efficiency of separation were investigated. Three different lengths of 3 µm、5 µm solid porous C18 and 2.7 µm fused-core columns（75 µm x 50 mm, 75 µm x 100 mm, and 75 µm x 200 mm）were tested using tryptic peptides, which generated from BSA and RAW 264.7 secretome protein. Three different gradient conditions were also applied for protein identification on each column. A column packing with 2.7 µm fused-core particles was investigated and it is found that column with fused-core particles packing provided higher efficiency than that with 3 µm and 5 µm porous particles. Also, more unique peptides were identified in the same column length with identical gradient condition. It was further applied for peptide/protein identification on RAW 264.7 secretome study.