氨基酸-稳定同位素稀释质谱法用于合成肽段准确含量分析

建立了氨基酸同位素稀释液相色谱-串联质谱法准确测定合成肽段绝对含量的方法。实验中对合成肽段的纯度进行了表征,色谱纯度表征结果为99%以上,质谱纯度为90%以上。在肽段溶液中加入13C标记的氨基酸后进行酸溶液水解时间的优化,水解后的氨基酸直接经液相色谱分离和质谱检测,结果表明肽段中的被测氨基酸在150 ℃、6 mol/L HCl溶液水解4～6 h就可以达到水解平衡。每个肽段选择两个或两个以上的被测氨基酸,测得随机选择的5种合成肽段的绝对含量为62.07%～88.18%,测定结果的相对标准偏差小于8%,相对误差小于5%,均满足定量要求。除常用的被测氨基酸苯丙氨酸、缬氨酸、异亮氨酸外,还考察了选择赖氨酸和精氨酸作为被测氨基酸的可行性,实验结果表明增加精氨酸为被测氨基酸是可行的,从而进一步增加了方法的普适性。该方法的建立避免了色谱法定量时氨基酸衍生化处理带来的副反应影响及操作繁琐等问题,提高了肽段含量测定的准确度和精密度,为肽段含量的准确测定提供了一种新的方法。

Accurate quantification of synthetic peptides by amino acid-stable isotope dilution mass spectrometry

An approach for quantification of peptides by hydrolysis followed by reversed-phase liquid chromatography (RPLC)-stable isotope dilution mass spectrometry (MS) is presented. The purities of all the determined peptides were greater than 99% by using RPLC and over 90% detected by MS, indicating that the method was workable. The hydrolysis conditions optimized were 4-6 h with 6 mol/L HCl at 150 degrees C. Two or more amino acids were chosen to ensure the accuracy of the determined results. The content range of peptides was determined to be 62.07% - 88.18% with the relative standard deviations less than 8% and relative errors less than 5%. Besides Phe, Val and Ile which were commonly used for the analysis of peptide contents, Arg as another amino acid for peptide quantification can be chosen to enhance the popularity of the method. In a word, application of this method for direct determination of peptide content can avoid the side effects in the derivatization of amino acids and the tedious operation in liquid chromatography. This will improve the precision and accuracy of the method and provide an alternative for peptide quantification.