隨著蛋白質藥物的興起，尤其是利用抗體來攜帶小分子藥物，更使得藥物發展有很大的躍進，而對於藥物的效用與品質上的監管，務必得從蛋白質的高階結構來探討，才能確保其活性與功能。目前有文獻提出利用標記方法結合質譜來分析蛋白質結構，像是氫氘交換質譜、氫氧自由基腳印拓取(Hydroxyl radical footprinting)等等，都能用來分析蛋白質其高階結構特性，或是應用在偵測蛋白質與配體鍵結的區域與活性，在氫氘交換質譜中，可以較為廣泛的分析各個胺基酸結構資訊，但只限於胜肽骨架，而不能了解胺基酸側鏈的變化，因此本篇利用還原胺化反應針對帶有胺基的N端與離胺酸(Lysine)的蛋白質進行標記，根據不同的反應時間與還原劑反應濃度，進行標記後再將抗體還原成重鏈、輕鏈，並以液相層析分離進入質譜，能夠初步判斷還原胺化反應對蛋白質的標記數量，可以發現二甲基標記在短時間內就能達到較高的反應效率；另外將標記過的蛋白質以由下而上的質譜方法來處理成多段胜肽，同樣以液相層析分離來簡化數據的複雜度，可以進一步分析抗體上個別的離胺酸修飾程度，並且在此篇中能觀察到抗體上大部分的離胺酸位點，也藉此判斷其與溶液的接觸程度。除此之外，在本篇中同時針對原始狀態的與加熱過的蛋白質進行標記，以比較蛋白質經過加熱後其結構的變化，發現加熱過後蛋白質可能失去摺疊，而使離胺酸更加裸露，而當加熱時間變長時，也能發現某些位點可能是聚集的位置，因此經由二甲基的標記也能了解離胺酸在加熱後發生的改變。

Antibody-based protein drugs are under intensive development by pharmaceutical industries in recent years. The higher order structure (HOS) and exposed active sites of proteins may affect the drug stability and efficacy. H/D exchange is a popular MS-based method to characterize HOS of proteins. However, the data revealed by H/D exchange are mainly for the dynamics of the peptide backbone and do not give information about the structure of side chain residues. Here, we applied stable isotope dimethyl labeling method towards surface lysine residues of a protein drug and coupled the labeling method with both the intact protein measurement and bottom-up based MS approach to characterize the reactivity and solvent accessibility of lysine residues on proteins. By systematic studies of the reaction time and the concentration of reactants, we showed the labeling is rapid with high yield, allowing the accessment of a wide range of reactivity of almost all lysine residues of an antibody drug. We expect this method will be a very useful complementary approach to H/D exchange for resolving HOS of proteins by LC-MS.

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