金属标记结合高效液相色谱-选择离子监测质谱的蛋白质绝对定量新方法

建立了金属标记结合高效液相色谱-选择性离子监测质谱(SIM)的蛋白质绝对定量新方法。实验考察了金属标记效率、金属标记的稳定性、标记后肽段的色谱保留和质谱行为、新定量方法的线性范围和准确度。实验结果表明金属标记具有标记效率高,稳定性好,色谱保留行为一致等优点。另外,金属标记-选择离子监测质谱绝对定量方法灵敏度高,其定量限低至1 fmol,线性范围为1~500 fmol,线性范围内R2值大于0.99,具有良好的线性关系;经过测量,标准肽段的回收率为117.01%,说明该方法具有较高的准确度。将该方法应用于腾冲嗜热菌中烯醇酶蛋白的定量分析,相对标准偏差为5.47%,表明该方法的精密度高。以上结果表明该方法可以用于生物样本中的蛋白质的绝对定量分析,为比较简单的生物样本中蛋白质的绝对定量方法提供了一种新的选择。

Metal-tag labeling coupled with high performance liquid chromatography-selected ion monitoring mass spectrometry for absolute quantitation of proteins

A novel method has been established based on metal element chelated tags coupled with high performance liquid chromatography-selected ion monitoring mass spectrometry (HPLC-SIM/MS). The labeling efficiency and stability of metal element chelated tags, the chromatographic retention behavior and MS behavior of the labeled peptides, the linear range and accuracy of this method were examined. The results showed that the metal element chelated tag method has high labeling efficiency and high labeling stability, and the labeled peptides with different kinds of metal tags have consistent chromatographic retention behavior. The method of metal tags coupled with HPLC-SIM/MS has high sensitivity with the limit of quantification (LOQ) up to 1 fmol. The linear range for the method was between 1 fmol to 500 fmol with R²>0.99, which means the method has a good linearity. Moreover, this method had an average recovery of 117.01%. The method was used in the absolute quantitation of a protein enolase in Thermoanaerobacter tengcongensis (TTE) with a relative standard deviation of 5.74%, which means high precision. All the results showed that this method is accurate and reliable for the absolute quantitation of proteins. This gives us an alternative for the quantitative determination of proteins in relatively simple biological samples.