Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3

Mass spectrometry (MS) coupled toisobaric labeling has developed rapidly into a powerful strategy for high-throughput protein quantification. Sample multiplexing and exceptional sensitivity allow for the quantification of tens of thousands of peptides and, by inference, thousands of proteins from multiple samples in a single MS experiment. Accurate quantification demands a consistent and robust sample-preparation strategy. Here, we present a detailed workflow for SPS-MS3-based quantitative abundance profiling of tandem mass tag (TMT)-labeled proteins and phosphopeptides that we have named the streamlined (SL)-TMT protocol. We describe a universally applicable strategy that requires minimal individual sample processing and permits the seamless addition of a phosphopeptide enrichment step (“mini-phos”) with little deviation from the deep proteome analysis. To showcase our workflow, we profile the proteome of wild-type Saccharomyces cerevisiae yeast grown with either glucose or pyruvate as the carbon source. Here, we have established a streamlined TMT protocol that enables deep proteome and medium-scale phosphoproteome analysis.

质谱（Mass Spectrometry, MS）与同重同位素标记联用技术已快速发展成为高通量蛋白质定量的强大策略。样本多重标记与优异的灵敏度可实现单次质谱实验中对多份样本内数万个肽段的定量分析，并据此推定数千个蛋白质的表达水平。精准定量需依托一套稳定可靠的样本制备方案。本文详细报道了一种基于SPS-MS3的定量丰度分析流程，用于分析经串联质谱标签（Tandem Mass Tag, TMT）标记的蛋白质与磷酸化肽段，我们将其命名为精简型SL-TMT实验方案。该策略具备普适性，仅需极少量单样本处理步骤，且可无缝集成磷酸化肽段富集步骤（"mini-phos"），与深度蛋白质组分析流程几乎无偏差。为验证该流程的实用性，我们对以葡萄糖或丙酮酸为碳源培养的野生型酿酒酵母（Saccharomyces cerevisiae）的蛋白质组进行了分析。综上，我们建立了一套精简型TMT实验方案，可同时实现深度蛋白质组与中等规模磷酸化蛋白质组分析。