基于质谱的蛋白质绝对定量研究策略和建议

科学家已经研究获得了大量的蛋白质/多肽潜在生物标志物，这些生物标志物需要经过验证和确证才能进一步转化到临床应用。针对蛋白质/多肽的绝对定量研究在标志物验证和确证过程中起到关键作用。传统的蛋白质定量方法，如酶联免疫吸附试验（ELISA）技术存在蛋白质抗体难以获得、不同抗体批次之间存在差异、基于抗体的检测存在交叉反应等问题。近年来，随着质谱仪器性能的提升，质谱法已经广泛应用于蛋白质大分子以及代谢物小分子定量研究领域，基于质谱法的蛋白质/多肽定量方法也得到发展，其中靶向蛋白质组学技术，如SRM/MRM、PRM和MRM3技术结合标准品可对目标蛋白质/多肽进行精确多重定量。因此，基于靶向质谱法的蛋白质/肽段绝对定量的策略应运而生。根据标准品不同，质谱绝对定量方法包括基于合成肽段、基于合成完整蛋白以及基于标记试剂。本文详细介绍了基于质谱法的蛋白质/多肽绝对定量策略的研究进展，对比各种策略的优势和限制，总结其在生物医学研究领域的应用。同时，还总结了目前蛋白质绝对定量面临的挑战，并根据已有研究报道对提高定量的精确度，促进蛋白质/多肽定量技术向临床转化提出建议，包括对标准品肽段的选择、处理、储存，标准曲线的建立，以及对整个流程严格的质控方法。

Strategies and Recommendations for the Absolute Quantification of Target Proteins Based on LC-MS/MS

Scientists have identified a large amount of protein/peptide biomarker candidates based on high throughput proteomics research in recent years.In order to transform the novel biomarkers from research to clinical utility,the accurate determination of the amount of protein and peptides is essential.Traditional immunoassays like ELISA holds lots of restrictions with regarding to the difficulty to obtain antibodies of mutant proteins,the existence of cross-activity,the low specificity for some candidate proteins,and the discrepancy between production batches.Owning to the great development of LC-MS/MS,technologies based on LC-MS/MS have been widely applied in quantification research of proteins and metabolites.Targeted proteomics technology such as SRM/MRM,PRM and MRM 3 combined with protein/peptide standards can offer the advantage of accurate quantitation of interesting proteins or peptides.Many MS-based strategies of absolute quantification for proteins/peptides spring up,including strategies based on peptide standards,intact proteins and labeling reagents.Here,the current progress and the challenge of LC-MS/MS based absolute quantification strategies were summarized in detail,and the advantages and limitations of various strategies were compared.The applications in biomedical research of these strategies based on LC-MS/MS were also listed.Of course,some recommendations,as peptide standards choosing,processing,storage,the construct of standard curve and strict control of the pipeline according to published research,were currently available surrounding the improvement of quantification accuracy,and the technical transformation from research to clinic.We hope these summaries and advice would be helpful for choosing proper strategy according to the research purpose and the accurate quantification of proteins or peptides.