甲胎蛋白纯品的同位素稀释质谱法测定

Measurement of Alpha-Fetoprotein by Isotope Dilution Mass Spectrometry

建立了基于特征肽段的甲胎蛋白的液相色谱-同位素稀释串联质谱检测方法。选取3条同位素标记的甲胎蛋白特征肽段作为内标,准确称其质量后与酶切后的甲胎蛋白样品定量混合,采取Phenomenex Kinetex 2.6μm C18色谱柱分离,电喷雾三重四极杆串联质谱多反应监测模式(MRM)测定,并对最优酶切条件、酶切效率以及定值结果的不确定度进行了考察和评定,得到的甲胎蛋白标准物质的最终测量结果为(0.329±0.016)mg/g。

Compared with small molecules, it＇s difficult to achieve accurate quantification of protein biomarkers because of their high molecular weight, complex structure and unstable nature. Due to that, alpha-fetoprotein （AFP） reference measurement procedure and high grade standard material has not been established yet. For protein macromolecular compounds, it is usually needed to split the protein into amino acids or peptide segments then use isotope dilution mass spectrometry to achieve accurate quantification. The method using isotope labeled amino acid as internal standard is relatively mature, but it is only suitable for high purity samples, otherwise it will introduce large errors due to the contamination of exogenous substances containing quantitative amino acids. The method using isotope labeled signature peptide as internal standard is more accurate and reliable. Therefore, a method for the determination of AFP by liquid chro- matography-isotope dilution mass spectrometry based on signature peptides was developed. As a potential value assay method for AFP pure certified reference material （CRM）, three isotope labeled peptides of AFP were selected as internal standard, which were added into the digested AFP samples. The samples were separated by PhenomenexKinetex 2.6 μm C18 column, and measured by tandem mass spectrometer equipped with an electrospray ionization source operated in multiple reaction monitoring （MRM） mode. The optimal digestion condition and efficiency of enzymatic hydrolysis were investigated and the determination of uncertainty was calculated and analyzed. The content of the AFP reference material was measured to be （0. 329 ±0. 016） mg/g. The selection of the signature peptides and the optimization of the digestion conditions were key to the experiment. The signature peptides should have high and reproducible response, no missed cleavages and suitable sequence length. The optimization of the digestion conditions need repeated experiments based on a large number of literatures.