双反相色谱串联平行反应监测靶向定量蛋白质组方法的建立

Establishment of dual reverse phase chromatography combined parallel reaction monitoring for targeted quantitative proteomics

摘要:

近年来质谱技术的持续进步促进了靶向蛋白质组的发展，在多反应监测 (Multiple reaction monitoring，MRM) 靶向蛋白质组定量方法的基础上衍生出平行反应监测 (Parallel reaction monitoring，PRM)技术。该技术靶向定量灵敏度和通量更高，重现性也更好，但其对于高复杂性样本在定量速度和深度上均存在一定的局限性。改善PRM定量的色谱方法包括：色谱柱的优化，增加色谱柱内径、降低柱长；色谱洗脱条件的优化，提高液相洗脱流速、缩短洗脱时间；最终建立了一种简单高效的双反相色谱串联PRM靶向蛋白质组定量平台。该平台使用150 μm内径和8 cm长的短色谱柱，在800 nL/min的高流速下和35 min有效洗脱梯度内，可实现对293T全细胞裂解液蛋白样本中多达400条低丰度肽段的快速定量。该研究优化了PRM靶向定量方法，将有利于PRM技术的推广，尤其为低丰度蛋白的精准定量提供了一种技术选择。

关键词:双反相色谱，平行反应监测，靶向定量

Abstract:

Steady improvement in mass spectrometers technology has transformed the targeted proteome analysis into a new stage. Parallel reaction monitoring (PRM) technology has evolved from the basic multiple reaction monitoring (MRM) targeted proteomics methods in recent years. PRM performs with a higher sensitivity, throughput and reproducibility in targeted quantification, however its limitations in effectiveness and accurate quantification of samples with higher complexity still remain unsolved. In this study through improving the chromatographic conditions of PRM we established a simple and robust platform for targeted proteomic quantification. The newly established PRM system is equipped with columns with increased inner diameter (150 μm) and decreased total length (8 cm); faster liquid phase elution rate (800 nL/min) and shortened elution gradient (35 min). These modifications enable PRM platform to combine with dual reverse phase chromatography, to quantify up to 400 low abundance peptides in human 293T cells whole cell extract. Our findings would benefit the promotion of PRM technology, especially providing a technical option for accurate quantification of low abundance proteins.

Key words:dual reverse phase chromatography, parallel reaction monitoring (PRM), targeted quantification