比較Fused-Core與3 µm、5 µm多孔性靜相材料在毛細管管柱中搭配液相層析串聯式質譜儀於蛋白質體之研究

Abstract

毛細管管柱液相層析搭配電噴灑串聯式質譜儀在蛋白質體學的研究中扮演著非常重要的角色，近來在於HPLC的分離上，fused-core技術的發展替代了3 µm或者更小的填充材料，此技術在快速分離的環境下，仍然保有好的解析度（resolution）以及較低的系統壓力；fused-core粒子特殊的合成方式，相較於全多孔球型粒子可以得到較窄的粒徑散佈，可以較為均勻地填充至管柱當中，降低渦流擴散（eddy diffusion）提升管柱分離效率，較短擴散路徑可以加快靜相間質量傳輸的速率以降低波峰寬度。 在本篇研究中，使用實驗室自製的逆相毛細管管柱分離經胰蛋白酶水解之胜肽，搭配串聯式質譜儀（Q-TOF）分析；研究管柱長度以及填充材料粒徑對於分離的影響，三種不同粒徑大小的填充材料3 µm、5 µm porous C18與 2.7 µm fused-core分別填充至毛細管管柱中（75 µm x 50 mm, 75 µm x 100 mm, and 75 µm x 200 mm），分別以BSA與RAW 264.7 secretome 樣品所水解的胜肽來進行測試，在蛋白質鑑定中每個管柱皆使用三種不同液相層析的梯度進行分析。 由實驗結果顯示，fused-core相較於3 µm和5 µm填充管柱提供了較佳的分離效率，在相同的長度與梯度環境下，鑑定到較多的unique peptides，同時進一步應用於鑑定RAW 264.7 secretome的胜肽及蛋白質鑑定上。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.