雙通道毛細管電泳質譜界面於蛋白質分析之應用

蛋白質水解胜肽的分離與胺基酸定序在蛋白質體學中已成為蛋白質鑑定上重要的一環。在經過二維膠體分離後的蛋白質鑑定中，目前以利用毛細管液相層析/質譜分析為主，每次分析時間約需60分鐘，但面對複雜蛋白質水解胜肽系統仍會遭遇因過多離子同時進入質譜而損失大量片段資訊的問題，曾有研究者利用多次進樣毛細管電泳並配合質譜設定的方式提升蛋白質序列的涵蓋率，本實驗利用實驗室的雙通道界面，將兩種分離方法或條件同時架設並配合多次進樣的策略，分析蛋白質水解胜肽並進行蛋白質鑑定，如此可免除平衡、沖洗毛細管的手續，以達到快速、方便的分析，最後將不同條件下各次進樣的結果擷取至同一層析圖檔中，經過四次進樣共35分鐘的時間，可將序列涵蓋率提升至85%。

關鍵字

蛋白質分析；毛細管電泳質譜；毛細管電層析質譜；多次進樣；雙通道界面；序列涵蓋率

並列摘要

Separation of protein digested peptides and amino acid sequencing has become a common practice for protein identification in proteomics. Currently, capillary LC/MS is the major technique used to identify the proteins separated by 2-D gel electrophoresis. One HPLC run takes about 60 minutes in analysis of the digested peptides. The HPLC/MS approach suffers from low sequence coverage, because there are too many peptides in one LC peak. Recently, multiple injection CE/MS under the same run utilizing the dynamic exclusion capability of the mass spectrometer has been reported to improve the sequence coverage. In this study, to improve sequence coverage, dual-channel interface developed by our lab was used to combine two separation methods (such as CEC and CE), or two separation conditions (such as different pH) and the multiple injection for analyzing the tryptic digested peptides. By utilizing the two channel interface, there is no need to condition and wash the capillary when different separation conditions were used. The results showed that after four injection and 35 min, sequence coverage can be improved to 85% in the analysis time of bovine serum albumin.

並列關鍵字

protein analysis ； CE-MS ； CEC-MS ； sequence coverage ； dual-channelnterface ； multiple injection

參考文獻

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國際替代計量

雙通道毛細管電泳質譜界面於蛋白質分析之應用

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