Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes

The study of low-abundance
proteins is a challenge to discovery-based
proteomics. Mass spectrometry (MS) applications, such as thermal proteome
profiling (TPP), face specific challenges in the detection of the
whole proteome as a consequence of the use of nondenaturing extraction
buffers. TPP is a powerful method for the study of protein thermal
stability, but quantitative accuracy is highly dependent on consistent
detection. Therefore, TPP can be limited in its amenability to study
low-abundance proteins that tend to have stochastic or poor detection
by MS. To address this challenge, we incorporated an affinity-purified
protein complex sample at submolar concentrations as an isobaric trigger
channel into a mutant TPP (mTPP) workflow to provide reproducible
detection and quantitation of the low-abundance subunits of the cleavage
and polyadenylation factor (CPF) complex. The inclusion of an isobaric
protein complex trigger channel increased detection an average of
40× for previously detected subunits and facilitated detection
of CPF subunits that were previously below the limit of detection.
Importantly, these gains in CPF detection did not cause large changes
in melt temperature (Tm) calculations
for other unrelated proteins in the samples, with a high positive
correlation between Tm estimates in samples
with and without isobaric trigger channel addition. Overall, the incorporation
of an affinity-purified protein complex as an isobaric trigger channel
within a tandem mass tag (TMT) multiplex for mTPP experiments is an
effective and reproducible way to gather thermal profiling data on
proteins that are not readily detected using the original TPP or mTPP
protocols.

低丰度蛋白质的研究是基于发现的蛋白质组学所面临的一大挑战。由于使用非变性提取缓冲液，质谱（Mass Spectrometry，MS）相关应用（如热蛋白质组分析（thermal proteome profiling，TPP））在检测完整蛋白质组时面临特定挑战。热蛋白质组分析（TPP）是研究蛋白质热稳定性的强大方法，但其定量准确性高度依赖于稳定一致的检测效果。因此，热蛋白质组分析（TPP）在研究低丰度蛋白质时存在局限性——这类蛋白质往往会因质谱（MS）检测出现随机性或效果不佳的问题。为应对这一挑战，我们将亚摩尔浓度的亲和纯化蛋白质复合物样品作为同量异位触发通道，整合至突变型热蛋白质组分析（mutant TPP，mTPP）流程中，以实现对剪切与多聚腺苷酸化因子（CPF）复合物低丰度亚基的可重复检测与定量。引入同量异位蛋白质复合物触发通道后，此前可被检测到的CPF亚基的检测信号平均提升了40倍，同时也使此前低于检测限的CPF亚基得以被检测到。值得注意的是，CPF检测性能的提升并未对样品中其他无关蛋白质的解链温度（melt temperature，Tm）计算造成显著变化，添加与未添加同量异位触发通道的样品的Tm估算值之间呈现出高度正相关。总体而言，在用于突变型热蛋白质组分析（mTPP）实验的串联质量标签（tandem mass tag，TMT）多重标记体系中引入亲和纯化蛋白质复合物作为同量异位触发通道，是一种高效且可重复的方法，可获取使用原始热蛋白质组分析（TPP）或突变型热蛋白质组分析（mTPP）方案难以检测到的蛋白质的热谱数据。