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运用液质联用、两种串联质谱对融合蛋白FP3的氨基酸全序列测定,确证其一级结构。将样品还原烷基化后,通过胰蛋白酶酶解蛋白,PNGase F去除多肽混合物中糖肽的糖基化,将去糖后的总肽用于液质联用系统,通过液相分离后,运用Q-TOF和线性离子阱两种串联质谱测定各个肽段的b,y碎片离子,分析测定融合蛋白FP3的氨基酸全序列。通过LC-ESI-Q-TOF完成了融合蛋白FP3的76%氨基酸序列测定,通过LC-ESI-Trap完成余下24%氨基酸序列测定。液质联用、串联质谱法测定蛋白质氨基酸序列快速、灵敏、准确,是对重组蛋白结构分析和确证的重要手段。
The sequence of the amino acid sequence of the fusion protein FP3 was determined by LC-MS and MS / MS, and its primary structure was confirmed. After reducing and alkylating the sample, the glycosylation of the glycopeptide in the polypeptide mixture is removed by trypsin digestion of the protein, PNGase F, and the total peptide after sugar removal is used for the LC / MS system. After the liquid phase separation, Q-TOF and linear ion trap were used to determine the b, y fragment ions of each peptide. The complete amino acid sequence of the fusion protein FP3 was analyzed and determined. The 76% amino acid sequence determination of the fusion protein FP3 was done by LC-ESI-Q-TOF and the remaining 24% amino acid sequence determination was done by LC-ESI-Trap. Liquid-liquid chromatography-tandem mass spectrometry is a fast, sensitive and accurate method to determine the amino acid sequence of protein, which is an important method for the structural analysis and confirmation of recombinant proteins.