HIGH-ACCURACY MOLECULAR-WEIGHT DETERMINATION AND VARIATION CHARACTERIZATION OF PROTEINS UP TO 80-KU BY IONSPRAY MASS-SPECTROMETRY
HIGH-ACCURACY MOLECULAR-WEIGHT DETERMINATION AND VARIATION CHARACTERIZATION OF PROTEINS UP TO 80-KU BY IONSPRAY MASS-SPECTROMETRY
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DOI：
10.1016/1044-0305(91)85005-q
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发表时间：
1991-09-01
影响因子：
3.2
通讯作者：
BELL, AW
中科院分区：
文献类型：
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作者：
FENG, R;KONISHI, Y;BELL, AW
使用具有离子喷雾接口的四极杆质谱仪来测量高达 80,000 u 的蛋白质的分子量 (MW)。 随着统计平均法的仪器校准和高斯曲线拟合法的数据分析的改进，马肌红蛋白的分子量测定精度高达 12 ppm (MW 16,950.4 +/- 0.2 u)。 对 cerato-ulmin 中三种成分的精确 MW 测定表明，两种次要成分分别从主要成分中丢失了氨基酸残基 Ser 和 Ser-Asp（MW 7618.4 +/- 0.2 u）。 单克隆抗体 Fab 片段中八个成分的 MW 分类显示，一组四个成分的 MW 约为 47,540 u，另一组约为 47,640 u。 片段 1 和片段 2 之间的 MW 差异为 100.2 +/- 0.6 u，归因于 Fab C 末端的不均匀切割，可能是由于 1 中多了一个 Thr 所致。发现牛血清白蛋白 (BSA) 的 MW 为 66,431.5 +/- 1.3 u，比计算的序列 MW 高约 164 u，很可能是因为之前报道的 BSA 不正确氨基酸序列。 人血清转铁蛋白的 MW (79,556.8 +/- 1.7 u) 显示比序列 MW 高 4414 u，表明该蛋白质中存在 22.7 个糖单位的糖基化。 牛血清转铁蛋白的复杂性更高（两个主要成分的分子量为 78,030.5 +/- 1.8 和 78,326 +/- 3.3 u）与糖基化的异质性相关。
A quadrupole mass spectrometer with an ionspray interface was used to measure the molecular weight (MW) of proteins up to 80,000 u. With the improvements in instrument calibration by a statistical averaging method and in data analysis by a gaussian curve-fitting method, precision of MW determination as high as 12 ppm was achieved with equine myoglobin (MW 16,950.4 +/- 0.2 u). Exact MW determination of three components in cerato-ulmin revealed that the two minor ones had lost amino acid residues Ser and Ser-Asp, respectively, from the major component (MW 7618.4 +/- 0.2 u). MW classification of eight components in the Fab fragment of a monoclonal antibody revealed that one set of four had MW approximately 47,540 u and the other approximately 47,640 u. The MW difference of 100.2 +/- 0.6 u between fragment 1 and 2, attributed to inhomogeneous cleavage at the Fab C-terminus, was probably due to one additional Thr in 1. The MW of bovine serum albumin (BSA) was found to be 66,431.5 +/- 1.3 u, approximately 164 u higher than the calculated sequence MW, most probably because of the incorrectness in the previously reported BSA amino acid sequence. The MW of human serum transferrin (79,556.8 +/- 1.7 u) was shown to be 4414 u higher than the sequence MW, pointing to a glycosylation of 22.7 sugar units in this protein. The greater complexity in bovine serum transferrin (MW 78,030.5 +/- 1.8 and 78,326 +/- 3.3 u for the two major components) was correlated with the heterogeneity in the glycosylation.