Quantitative Top-Down Proteomics by Isobaric Labeling with Thiol-Directed Tandem Mass Tags

While identification-centric (qualitative) top-down proteomics (TDP) has seen rapid progress in the recent past, the quantification of intact proteoforms within complex proteomes is still challenging. The by far mostly applied approach is label-free quantification, which, however, provides limited multiplexing capacity, and its use in combination with multidimensional separation is encountered with a number of problems. Isobaric labeling, which is a standard quantification approach in bottom-up proteomics, circumvents these limitations. Here, we introduce the application of thiol-directed isobaric labeling for quantitative TDP. For this purpose, we analyzed the labeling efficiency and optimized tandem mass spectrometry parameters for optimal backbone fragmentation for identification and reporter ion formation for quantification. Two different separation schemes, gel-eluted liquid fraction entrapment electrophoresis × liquid chromatography–mass spectrometry (LC–MS) and high/low-pH LC–MS, were employed for the analyses of either Escherichia coli (E. coli) proteomes or combined E. coli/yeast samples (two-proteome interference model) to study potential ratio compression. While the thiol-directed labeling introduces a bias in the quantifiable proteoforms, being restricted to Cys-containing proteoforms, our approach showed excellent accuracy in quantification, which is similar to that achievable in bottom-up proteomics. For example, 876 proteoforms could be quantified with high accuracy in an E. coli lysate. The LC–MS data were deposited to the ProteomeXchange with the dataset identifier PXD026310.尽管以鉴定为导向的（定性）自上而下蛋白质组学（top-down proteomics, TDP）近年来发展迅猛，但复杂蛋白质组内完整蛋白质变体（proteoforms）的定量分析仍颇具挑战。目前应用最为广泛的定量策略为无标记定量（label-free quantification），但该方法的多重检测能力有限，且与多维分离联用时会面临诸多问题。同量异位素标记（isobaric labeling）作为自下而上蛋白质组学（bottom-up proteomics）中的标准定量方法，可有效规避上述局限。本研究介绍了巯基靶向同量异位素标记（thiol-directed isobaric labeling）在定量自上而下蛋白质组学中的应用。为此，我们对标记效率展开分析，并优化了串联质谱（tandem mass spectrometry）参数，以实现最优的主链裂解以完成蛋白质鉴定，同时保障报告离子生成以实现定量分析。我们采用两种不同的分离策略：凝胶洗脱液相组分捕获电泳 × 液相色谱-质谱联用（liquid chromatography–mass spectrometry, LC–MS）以及高/低pH液相色谱-质谱联用，分别分析大肠杆菌（Escherichia coli, E. coli）蛋白质组与混合的大肠杆菌/酵母样品（双蛋白质组干扰模型），以探究潜在的比例压缩现象。尽管巯基靶向标记会对可定量的蛋白质变体引入偏倚——仅能实现含半胱氨酸的蛋白质变体的定量，但本方法的定量准确性优异，可达到与自下而上蛋白质组学相当的水平。例如，在大肠杆菌裂解液中可高精度定量876个蛋白质变体。本研究的液相色谱-质谱联用数据已提交至蛋白质组交换计划（ProteomeXchange），数据集标识符为PXD026310。