Characterization and Optimization of Multiplexed Quantitative Analyses Using High-Field Asymmetric-Waveform Ion Mobility Mass Spectrometry

Multiplexed, isobaric tagging methods are powerful techniques to increase throughput, precision, and accuracy in quantitative proteomics. The dynamic range and accuracy of quantitation, however, can be limited by coisolation of tag-containing peptides that release reporter ions and conflate quantitative measurements across precursors. Methods to alleviate these effects often lead to the loss of protein and peptide identifications through online or offline filtering of interference containing spectra. To alleviate this effect, high-Field Asymmetric-waveform Ion Mobility Spectroscopy (FAIMS) has been proposed as a method to reduce precursor coisolation and improve the accuracy and dynamic range of multiplex quantitation. Here we tested the use of FAIMS to improve quantitative accuracy using previously established TMT-based interference standards (triple-knockout [TKO] and Human-Yeast Proteomics Resource [HYPER]). We observed that FAIMS robustly improved the quantitative accuracy of both high-resolution MS2 (HRMS2) and synchronous precursor selection MS3 (SPS-MS3)-based methods without sacrificing protein identifications. We further optimized and characterized the main factors that enable robust use of FAIMS for multiplexed quantitation. We highlight these factors and provide method recommendations to take advantage of FAIMS technology to improve isobaric-tag-quantification moving forward.多重同量异位素标记技术是提升定量蛋白质组学通量、精密度与准确度的高效手段。然而，定量的动态范围与准确度常会受到携带标记肽段共分离现象的限制：此类肽段会释放报告离子，混淆不同前体间的定量检测结果。为缓解此类问题所开发的方法，通常需通过在线或离线过滤含干扰的质谱图，这往往会造成蛋白质与肽段鉴定数量的损失。为缓解该效应，高场不对称波形离子迁移谱（high-Field Asymmetric-waveform Ion Mobility Spectroscopy, FAIMS）被提出作为可减少前体共分离、提升多重定量准确度与动态范围的技术手段。本研究采用已建立的基于串联质量标签（Tandem Mass Tag, TMT）的干扰标准品，即三重敲除（triple-knockout, TKO）与人-酵母蛋白质组资源库（Human-Yeast Proteomics Resource, HYPER），测试了FAIMS对定量准确度的提升效果。我们发现，FAIMS可稳健提升高分辨率二级质谱（high-resolution MS2, HRMS2）与同步前体选择三级质谱（synchronous precursor selection MS3, SPS-MS3）两类方法的定量准确度，且不会降低蛋白质鉴定数量。本研究进一步优化并表征了可实现FAIMS在多重定量中稳定应用的核心影响因素。我们重点梳理了这些因素，并针对后续利用FAIMS技术优化同量异位素标记定量的实验提供了方法学建议。