Synthetic Knockout Protein Standard for Evaluating Interference in Tandem Mass Tag-Based Proteomics

Isobaric labeling is widely used for unbiased, proteome-wide
studies,
and it provides several advantages, such as fewer missing values among
samples and higher quantitative precision. However, ion interference
may lead to compressed or distorted observed ratios due to the coelution
and coanalysis of peptides. Here, we introduced a synthetic KnockOut
standard (sKO) for evaluating interference in tandem mass tags-based
proteomics. sKO is made by mixing TMTpro-labeled tryptic peptides
derived from four nonhuman proteins and a whole human proteome as
background at different proportions. We showcased the utility of the
sKO standard by exploring ion interference at different peptide concentrations
(up to a 30-fold change in abundance) and using a variety of mass
spectrometer data acquisition strategies. We also demonstrated that
the sKO standard could provide valuable information for the rational
design of acquisition strategies to achieve optimal data quality and
discussed its potential applications for high-throughput proteomics
workflows development.

同量异位素标记（Isobaric labeling）技术被广泛应用于无偏倚的全蛋白质组研究，且具备多项优势，例如样本间缺失值更少、定量精度更高。然而，肽段的共洗脱与共分析会引发离子干扰（ion interference），导致观测到的比值被压缩或扭曲。本研究中，我们提出了一种合成敲除标准（synthetic KnockOut，简称sKO），用于评估基于串联质量标签（Tandem Mass Tags，TMT）的蛋白质组学中的离子干扰情况。该sKO标准通过按不同比例混合经TMTpro标记的、源自四种非人类蛋白质的胰蛋白酶肽段，以及作为背景的全人类蛋白质组制备而成。我们通过探究不同肽段浓度（丰度变化最高可达30倍）下的离子干扰情况，并结合多种质谱数据采集策略，验证了sKO标准的实用性。此外，我们还证明了sKO标准可为合理设计采集策略以获取最优数据质量提供重要参考，并讨论了其在高通量蛋白质组学工作流开发中的潜在应用场景。