LC-MS/MS法对融合蛋白FP3的氨基酸全序列测定
Measurement of the amino acid sequence for the fusion protein FP3 with LC-MS/MS
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摘要:
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。
Abstract:The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3. Seventy-six percent of full amino acid sequence of the fusion protein FP3 was measured by LC-ESI-Q-TOF with the remaining 24% completed by LC-ESI-Trap. As LC-MS and tandem mass spectrometry are rapid, sensitive, accurate to measure the protein amino acid sequence, they are important approach to structure analysis and identification of recombinant protein.
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