The incongruity of validating quantitative proteomics using western blots
The incongruity of validating quantitative proteomics using western blots
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使用蛋白质印迹验证定量蛋白质组学的不一致性
DOI：
10.1038/s41477-022-01314-8
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发表时间：
2022
期刊：
影响因子：
18
通讯作者：
Uhrig, R. Glen
中科院分区：
文献类型：
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作者：
Mehta, Devang;Ahkami, Amir H.;Walley, Justin;Xu, Shou-Ling;Uhrig, R. Glen
类似于古老的审查要求定量PCR验证的RNA测序数据，几乎每个研究人员谁使用蛋白质组学技术已在一个时间或另一个被要求提供“蛋白质印迹验证”的质谱为基础的蛋白质丰度数据。我们认为，这一要求表明，植物生物学界对近年来基于质谱的蛋白质组学领域在成本、灵敏度和可靠性方面的非凡改进缺乏认识。在这里，作为定量植物蛋白质组学不同领域的一组专家，我们解释了为什么定量蛋白质组学数据的蛋白质印迹验证既不必要又无效。此外，我们邀请植物科学界的同事更新他们对定量质谱作为一种灵敏可靠的蛋白质鉴定和定量方法的看法。定量蛋白质组学，在其许多风味，为植物科学研究人员提供了在一个实验中定量数千种蛋白质的能力，其精度和可靠性远远高于蛋白质印迹法。实验室内和实验室间的研究都建立了蛋白质定量的重现性，有针对性的1，2和非靶向（特别是数据独立的采集）蛋白质组学3，4。相比之下，蛋白质印迹法由于凝胶内分离、膜转移和非特异性抗体结合的固有随机性而具有高失败率。此外，这些过程与使用鲁米诺基荧光试剂的印迹显影的可变性相结合，使蛋白质印迹结果充其量是半定量的。基于质谱法的蛋白质定量具有高度可重复性1-4并且可靠，因为定量通常基于每个蛋白质的多个光谱和肽的测量，并且伴随着每个肽和蛋白质的统计置信度测量。
Similar to the age-old reviewer request for quantitative PCR validation of RNA-sequencing data, nearly every researcher who uses proteomics technologies has at one time or another been asked by a reviewer to provide ‘western blot validation’of their mass-spectrometry-based protein abundance data. We believe that this request demonstrates a lack of awareness amongst the plant biology community about the extraordinary improvements in cost, sensitivity and reliability that the field of mass-spectrometry-based proteomics has made in recent years. Here, as a group of experts in different domains of quantitative plant proteomics, we explain why western blot validations of quantitative proteomics data are both unnecessary and invalid. Furthermore, we invite our colleagues in the plant science community to update their perception of quantitative mass spectrometry as a sensitive and reliable method of protein identification and quantification. Quantitative proteomics, in its many flavours, offers plant science researchers the ability to quantify thousands of proteins in a single experiment with much greater precision and reliability than western blotting. Both intra-and inter-laboratory studies have established the reproducibility of protein quantification by both targeted 1, 2 and untargeted (particularly data-independent acquisition) proteomics 3, 4. By contrast, western blotting has a high rate of failure owing to the inherent stochasticity of in-gel separation, membrane transfer and unspecific antibody binding. Furthermore, these processes—in conjunction with variability in blot development using luminol-based chemiluminescent reagents—render western blot results semiquantitative at best. Mass-spectrometry-based protein quantification is highly reproducible 1–4 and reliable because quantification is typically based on the measurement of multiple spectra and peptides per protein and is accompanied by statistical confidence measures for each peptide and protein.