Comparison of Protein Quantification in a Complex Background by DIA and TMT Workflows with Fixed Instrument Time

Label-free
quantification (LFQ) and isobaric labeling quantification
(ILQ) are among the most popular protein quantification workflows
in discovery proteomics. Here, we compared the TMT SPS/MS3 10-plex
workflow to a label free single shot data-independent acquisition
(DIA) workflow on a controlled sample set. The sample set consisted
of ten samples derived from 10 biological replicates of mouse cerebelli
spiked with the UPS2 protein standard in five different concentrations.
For a fair comparison, we matched the instrument time for the two
workflows. The LC–MS data were acquired at two facilities to
assess interlaboratory reproducibility. Both methods resulted in a
high proteome coverage (>5000 proteins) with low missing values
on
protein level (<2%). The TMT workflow led to 15–20% more
identified proteins and a slightly better quantitative precision,
whereas the quantitative accuracy was better for the DIA method. The
quantitative performance was benchmarked by the number of true positives
(UPS2 proteins) within the top 100 candidates. TMT and DIA showed
a similar performance. The quantitative performance of the DIA data
stayed in a similar range when searching the spectra against a fasta
database directly, instead of using a project-specific library. Our
experiments also demonstrated that both workflows are readily transferrable
between facilities.

无标记定量（Label-free quantification, LFQ）与同重同位素标记定量（isobaric labeling quantification, ILQ）是发现蛋白质组学中最主流的蛋白质定量工作流之一。本研究在受控样本集上，对TMT SPS/MS3 10重标记工作流与无标记单针数据非依赖性采集（data-independent acquisition, DIA）工作流进行了比较。该样本集包含10份样本，均源自小鼠小脑的10份生物学重复，并以5种不同浓度掺入了UPS2蛋白标准品。为保障比较的公平性，我们为两种工作流匹配了相同的仪器上机时长。LC-MS数据在两家实验室完成采集，以评估实验室间重现性。两种方法均实现了较高的蛋白质组覆盖度（>5000种蛋白质），且蛋白质水平的缺失值占比低于2%。TMT工作流可多鉴定出15%~20%的蛋白质，且定量精密度略优；而DIA方法的定量准确度更佳。我们通过前100个候选蛋白中的真阳性（UPS2蛋白）数量对定量性能进行了基准测试，结果显示TMT与DIA的表现相近。当直接将谱图检索至FASTA数据库而非使用项目专属谱图库时，DIA数据的定量性能仍处于相近水平。本实验还证实，两种工作流均可便捷地在不同实验室间迁移。