Kameoka Daisuke, Ueda Tadashi, Imoto Taiji
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-3-1 Maidashi, Higashi-ku, Fukuoka 812-8582.
J Biochem. 2003 Jul;134(1):129-35. doi: 10.1093/jb/mvg120.
A method was established for evaluating Asn deamidation and Asp isomerization/racemization. To detect the subtle changes in mass that accompany these chemical modifications, we used a combination of enzyme digestion by endoproteinase Asp-N, which selectively cleaves the N-terminus of L-alpha-Asp, and MALDI/TOF-mass spectrometry. To achieve better resolution, we employed digests of (15)N-labeled protein as an internal standard. To demonstrate the advantages of this method, we applied it to identify deamidated sites in mutant lysozymes in which the Asn residue is mutated to Asp. We also identified the deamidation or isomerization site of the lysozyme samples after incubating them under acidic or basic conditions.
建立了一种评估天冬酰胺脱酰胺作用和天冬氨酸异构化/消旋作用的方法。为了检测伴随这些化学修饰的质量细微变化，我们结合使用了天冬氨酸内肽酶Asp-N进行酶切，该酶可选择性切割L-α-天冬氨酸的N端，以及基质辅助激光解吸电离飞行时间质谱（MALDI/TOF-MS）。为了获得更好的分辨率，我们采用（15）N标记蛋白的酶切产物作为内标。为了证明该方法的优势，我们将其应用于鉴定天冬酰胺残基突变为天冬氨酸的突变溶菌酶中的脱酰胺位点。我们还在酸性或碱性条件下孵育溶菌酶样品后，鉴定了其脱酰胺或异构化位点。
