Adapting an Isobaric Tag-Labeled Yeast Peptide Standard to Develop Targeted Proteomics Assays

Targeted proteomics strategies present a streamlined
hypothesis-driven
approach to analyze specific sets of pathways or disease related proteins.
goDig is a quantitative, targeted tandem mass tag (TMT)-based assay
that can measure the relative abundance differences for hundreds of
proteins directly from unfractionated mixtures. Specific protein groups
or entire pathways of up to 200 proteins can be selected for quantitative
profiling, while leveraging sample multiplexing permits the simultaneous
analysis of up to 18 samples. Despite these benefits, implementing
goDig is not without challenges, as it requires access to an instrument
application programming interface (iAPI), an elution order and spectral
library, a web-based method builder, and dedicated companion software.
In addition, the absence of an example test assay may dissuade researchers
from testing or implementing goDig. Here, we repurpose the TKO11 standardwhich
is commercially available but may also be assembled in-laband
establish it as a de facto test assay for goDig.
We build a proteome-wide goDig yeast library, quantify protein expression
across several gene ontology (GO) categories, and compare these results
to a fully fractionated yeast gold-standard data set. Essentially,
we provide a guide detailing the goDig-based quantification of TKO11,
which can also be used as a template for user-defined assays in other
species.

靶向蛋白质组学（Targeted proteomics）提供了一种简化的假设驱动型研究方法，可用于分析特定通路或疾病相关蛋白质组。goDig是一种基于串联质量标签（TMT, tandem mass tag）的定量靶向测定方法，能够直接从未分级的混合样本中检测数百种蛋白质的相对丰度差异。研究人员可选择特定蛋白质组或多达200种蛋白质的完整通路开展定量分析，同时借助样本多重化技术，可同时完成多达18个样本的同步分析。尽管具备上述优势，但实施goDig并非毫无挑战：其需要访问仪器应用程序编程接口（iAPI, instrument application programming interface）、洗脱顺序与谱文库、基于网页的方法构建工具，以及专用配套软件。此外，缺乏示例测试测定方法可能会阻碍研究人员尝试或实施goDig。在此，我们对市售且可在实验室自行组装的TKO11标准品进行重新利用，将其确立为goDig的实际可用测试测定方法。我们构建了全蛋白质组范围的goDig酵母文库，对多个基因本体（GO, gene ontology）类别中的蛋白质表达进行定量分析，并将所得结果与完全分级的酵母金标准数据集进行对比。简言之，我们提供了一份基于goDig的TKO11定量分析详细指南，该指南也可作为其他物种中用户自定义测定方法的参考模板。