麩氨酸上的羧酸化轉譯後修飾對於凝血過程中所參與的蛋白扮演極為重要的角色。於本篇論文中，我們藉由γ-羧基麩氨酸作用區與鈣離子具有高度親和力的生物特性，而開發出一個利用化學沉澱的方法來純化含有γ-羧基麩氨酸修飾的胜肽片段，並稱此方法為親和性鈣離子純化法。此方法對於γ-羧基麩氨酸修飾的胜肽有其專一性的純化作用，實驗結果顯示此純化方法可提高質譜上γ-羧基麩氨酸修飾的胜肽的離子訊號強度十倍以上。於方法中我們額外添加碳酸氫鈉，使其提供碳酸根陰離子基團來提升γ-羧基麩氨酸修飾的胜肽的共沉澱。
我們利用親和性鈣離子共沉澱法，成功在標準的第九號凝血因子蛋白中純化出γ-羧基麩氨酸修飾的胜肽，並於液相層析串聯式質譜儀中鑑定出位於胺基端上的六段胜肽鏈，具有十二個γ-羧基麩氨酸的轉譯後修飾位點。
本方法亦應用於生物樣品的純化。於腹腔中積水樣品，此方法成功地純化出第九號凝血因子蛋白中的γ-羧基麩氨酸修飾胜肽片段，並偵測到三個γ-羧基麩氨酸的轉譯後修飾位點，證實了此法亦可運用生物樣品中蛋白γ-羧基麩氨酸修飾的偵測。
我們進一步應用此方法於具更複雜基質干擾的人類紅血球樣品中凝血因子之偵測，我們在人類紅血球中額外添加18 pmole的第九號凝血因子標準品，以測試此方法是否能運用於高度基質干擾的樣品中，結果顯示成功地在紅血球裡偵測到第九號凝血因子蛋白中的γ-羧基麩氨酸修飾胜肽片段，亦偵測到三個γ-羧基麩氨酸的轉譯後修飾位點，證實了此法亦可使用於高度基質干擾的蛋白樣品中。

Carboxylation at the gamma position of glutamic acid (Gla) is an important post-translational modification required for the activity of vitamin K-dependent (VKD) proteins. Detection and identification of Gla modifications using mass spectrometry, however, is difficult due to many problems including ion suppression effects by negative charges. We developed a method using calcium affinity precipitation method to enrich the gamma-carboxyglutamic acid (Gla) peptides which can be subsequently detected by electrospray-mass spectrometry.
In this approach, we used calcium ion to co-precipitate gamma-carboxyglutamate paptides which can form stable six-ring neutral complexes with calcium ion via its two carboxylic acid functional groups. This approach had low nonspecific bindings with the non-Gla modified peptides, and resulted in a 10-fold of signal enhancement. In addition to calcium ion, NaHCO3 was added to increase the recovery yield for Gla peptides.
Our results indicate that the 12 carboxylation sites at the N-terminal region of coagulation Factor IX can be well characterized by LC-MS/MS using the proposed method for enrichment. Moreover, the method can be successfully applied to identify three carboxylation sites on the endogenous coagulation Factor IX in human ascites fluid. For highly complicated sample matrix like human plasma, we spiked 18 pmole Factor IX standards in the 60μL human plasma and used this method to precipitate Factor IX. The result indicated that the three carboxylation sites were able to be successfully identified.

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