质谱多反应监测技术建立质谱相对定量分析β半乳糖苷酶方法

Quantitative analysis of Beta-Galactosidase using a mass spectrometry-linked MRM technique

目的利用质谱多反应监测（multiple reaction monitoring,MRM）技术,建立不依赖抗体的质谱定量检测蛋白的方法。方法通过skyline软件选择β半乳糖苷酶（Beta-Galactosidase,BG）定量的肽段及母-子离子对并优化;通过5次重复实验计算方法的相对标准偏差RSD（%）（relative standard deviation,RSD）;将BG加入到酵母提取物酶切产物中建立复杂背景下标准曲线。结果在多次实验中反复被鉴定的可用于蛋白定量分析的BG 3个唯一肽段分别为：VDEDQPFPAVPK,IDPNAWVER,GDFQFNISR;5次重复实验MRM检测BG积分峰面积的RSD均小于6%;3个肽段在0-50×10^-6fmol/L的浓度范围内线性相关良好,标准曲线R2值为0.998 52-0.999 55,3个肽段在浓度为0.625×10^-6fmol/L时依然可以检测到稳定的质谱峰,显示了方法的高灵敏度。结论建立了MRM质谱相对定量检测BG的方法,为MRM质谱定量检测血清标志物奠定了基础。

Objective To quantitatively analyze Beta-Galactosidase（BG）using a mass spectrometry-linked multiple reaction monitoring（MRM）technique with no antibody involved. Methods Peptides of BG used for quantitative analysis were selected and the parent-daughter ion pairs were optimized by a skyline software.Relative standard deviation（RSD）was calculated based on five repeated trials and a standard curve under complex background was established by mixing BG with the yeast extract enzyme-digested product. Results Three unique peptides of BG for the protein quantitative analysis identified in the study were VDEDQPFPAVPK,IDPNA WVER,GDFQFNISR.The RSD of integral peak area was found to be less than 6% after 5repeated trials of the method.A nice linear correlation was noticed under the concentration range of 0-50 ×10-6fmol/L for all three peptides with the R2 value of standard curve fluctuating between 0.998 52and 0.999 55.Still,a stable mass peak could be detected even under low peptide concentration of 0.625×10-6 fmol/L,indicating a high sensitivity of the method. Conclusions A new method of mass spectrometrylinked MSM for BG quantitative analysis is developed and confirmed to be consistant,which lays a solid foundation for the quantification of other serum biomarkers.