标志物研究中常用蛋白组学质谱方法的定量准确性评估

Quantitative Accuracy Evaluation of Mass Spectrometry Based Proteomics Methods Commonly Used in Biomarker Research

在疾病发生过程中,蛋白质的表达水平、修饰状态或相互作用模式的变化有可能反映病理状态或疾病进展,因此蛋白质常被用作疾病标志物.基于质谱的蛋白质组学发现了大量的潜在疾病标志物,这些标志物及其质谱检测方法能否进一步推广至临床检测,定量准确性和稳定性是关键.本文合成了32条标准肽段,对数据非依赖性采集(DIA)、多反应监测(MRM)及平行反应监测(PRM) 3种有潜力在临床质谱方面大规模应用的定量方法进行了比较和评估.将肽段稀释成4个不同的浓度来模拟不同丰度的实际样本,用以上3种质谱方法采集数据后,比较了肽段发现率、标准曲线线性、日间和日内精密度及保留时间(RT)漂移等情况,总结了3种定量方法各自的优缺点和潜在应用方向.结果表明,MRM定量方法最适合临床应用,在于其优异的日间日内稳定性、灵敏度和定量精度;PRM适合科研方向靶向定量,因其拥有最高灵敏度和较高稳定性定量精度;而DIA由于高通量而依赖于软件和算法解析,定量数目多的同时,对于特定肽段定量准确性比PRM/MRM差.

In the progression of diseases,changes in protein expression levels,modification states,or interaction patterns may reflect pathological conditions or disease advancement,making proteins commonly used as disease biomarkers.Mass spectrometry-based proteomics has identified numerous potential disease biomarkers.However,these biomarkers and their mass spectrometry detection methods need to have high quantitative accuracy and stability for further clinical application.This study synthesized 32 standard peptides and evaluated three quantitative methods:data independent acquisition(DIA),multiple reaction monitoring(MRM),and parallel reaction monitoring(PRM),for their potential in large-scale clinical applications in mass spectrometry.We simulated actual samples at four different concentrations to assess peptide discovery rates,standard curve linearity,inter-day and intra-day precision,and retention time shift using the aforementioned three mass spectrometry methods.The results indicate that MRM quantification is most suitable for clinical applications due to its good stability,sensitivity,and quantitative accuracy.PRM is suitable for targeted quantification in research settings,which can offer high sensitivity and stability.Finally,despite its high throughput nature,DIA demonstrates inferior quantitative accuracy for specific peptides compared to PRM and MRM.