毛细管反相液相色谱-串联质谱用于肽的鉴定和相对定量分析

建立了一种基于毛细管反相液相色谱-串联质谱联用技术和质谱峰强度数据处理的肽段鉴定和相对定量分析方法。该方法无需对样品中的肽进行化学标记,在对样品进行反相色谱分离和串联质谱分析后,将二级质谱扫描数据进行蛋白质数据库搜索，获得所鉴定肽段的序列、保留时间、质荷比、带电荷数等定性信息；再以此为定位依据，在全扫描质谱数据中提取该肽段对应的离子峰并以该离子峰的峰强度作为定量信息,从而实现对不同样品中的共有肽段进行差异比较分析。以标准蛋白酶解混合肽段为实验对象,以肽段相对强度的相对标准偏差为指标,考察了该方法用于肽段相对定量分析的重现性、检测动态范围以及浓度标准曲线等,为将该方法用于生物样品中内源性肽的差异分析奠定了基础。

Identification and relative quantification of peptides by capillary reversed-phase liquid chromatography-tandem mass spectrometry

A method for the identification and relative quantification of peptides by capillary liquid chromatography ion-trap tandem mass spectrometry (LC-IT-MS/MS) was established. At first, the peptides were automatically identified by correlating the tandem mass spectra with the peptide sequences from a database. After the quantitative information of peptide ions were extracted from the full-scan MS according to the results of database searching, the peak intensities of the identified peptide ions with different charge states were summed together to define the total intensity of the peptide. Then, the peak intensities of the same peptide in the replicate analysis of the same sample were averaged and assigned as the abundance of the peptide. Finally, the abundances of the common peptide in the analysis of different samples were compared. This approach relied on the analytical reproducibility and linearity of signal versus molecular concentration. As a measure of the analytical reproducibility for tryptic peptides sampling, the median of the relative standard deviation of 35% was determined for 50 common peptides from 3 replicate analysis of 200 fmol bovine serum albumin (BSA) digest. The method was further illustrated using digested mixtures of BSA and myoglobin as follows. The BSA digest was gradually diluted while the myoglobin digest was present in the mixtures at constant level. This study revealed that the abundance of the variable BSA peptides increased linearly (trend line r2 > 0.97) with increasing amount from 10 to 1 000 fmol, while the abundances of the constant peptides from myoglobin remained approximately the same. In the present method, chemical derivatization steps are not needed to create an internal standard, as in isotope-coded affinity tag or similar methods. This method provides an alternative approach for differential analysis of peptides in biological samples.