Evaluation of the Effect of Trypsin Digestion Buffers on Artificial Deamidation

Nonenzymatic deamidation occurs readily under the condition of trypsin digestion, resulting in the identification of many artificial deamidation sites. To evaluate the effect of trypsin digestion buffers on artificial deamidation, we compared the three commonly used buffers Tris-HCl (pH 8), ammonium bicarbonate (ABC), and triethylammonium bicarbonate (TEAB), and ammonium acetate (pH 6), which was reported to reduce Asn deamidation. iTRAQ quantification on rat kidney tissue digested in these four buffers indicates that artificial Asn deamidation is produced in the order of ammonium acetate < Tris-HCl < ABC < TEAB, and Gln deamidation has no significant differences in all tested buffers. Label-free experiments show the same trend, while protein and unique peptide identification are comparable using these four buffers. To explain the differences of these four buffers in producing artificial Asn deamidation, we determined the half-life of Asn deamidation in these buffers using synthetic peptides containing -Asn-Gly- sequences. It is 51.4 ± 6.0 days in 50 mM of ammonium acetate (pH 6) at 37 °C, which is about 23, 104, and 137 times that in Tris-HCl, ABC, and TEAB buffers, respectively. In conclusion, ammonium acetate (pH 6) is more suitable than other tested buffers for characterizing endogenous deamidation and N-glycosylation.

非酶促脱酰胺作用在胰蛋白酶消化条件下极易发生，易导致大量人工脱酰胺位点被鉴定得到。为评估胰蛋白酶消化缓冲液对人工脱酰胺的影响，我们对比了四种常用缓冲液：Tris-HCl（pH 8）、碳酸氢铵（ABC）、三乙胺碳酸氢盐（TEAB），以及据报道可降低天冬酰胺（Asn）脱酰胺的乙酸铵（pH 6）。对经上述四种缓冲液消化的大鼠肾脏组织开展同位素标记相对和绝对定量（iTRAQ）分析，结果显示人工天冬酰胺脱酰胺的发生程度按乙酸铵 < Tris-HCl < ABC < TEAB的顺序递增，而谷氨酰胺（Gln）脱酰胺在所有受试缓冲液中均无显著差异。无标记定量实验得到了一致的趋势，且四种缓冲液在蛋白质及唯一肽段的鉴定数量上无明显差异。为阐释四种缓冲液在诱导人工天冬酰胺脱酰胺方面的差异，我们采用含有-天冬酰胺-甘氨酸-（-Asn-Gly-）基序的合成肽段，测定了其在各缓冲液中的天冬酰胺脱酰胺半衰期：在37 ℃、50 mM乙酸铵（pH 6）缓冲液中，半衰期为51.4 ± 6.0天，约为Tris-HCl、ABC及TEAB缓冲液的23倍、104倍及137倍。综上，相较于其余受试缓冲液，乙酸铵（pH 6）更适合用于表征内源性脱酰胺作用与N-糖基化。