Rodriguez Laura G, Lombard-Banek Camille, Quach Vi M, Choi Sam B, Manzini M Chiara, Nemes Peter
Department of Chemistry & Biochemistry, University of Maryland, College Park, Maryland 20742, United States.
Department of Chemistry, The George Washington University, Washington, District of Columbia 20052, United States.
Anal Chem. 2025 May 27;97(20):10901-10909. doi: 10.1021/acs.analchem.5c01832. Epub 2025 May 13.
Multiplexing quantification using isobaric barcoding has gained traction in trace-sensitive and single-cell mass spectrometry (MS), both in nanoflow liquid chromatography (nanoLC) and capillary electrophoresis (CE). In nanoLC-MS, ratio compression from isobaric interferences is known to challenge quantification accuracy during tandem MS (MS), which is effectively remedied using simultaneous precursor selection (SPS) MS. Despite mounting interest in CE-MS for trace-sensitive bottom-up proteomics, the fidelity of multiplexed quantification is unknown using this technology. Here, we address this fundamental knowledge gap by holistically investigating quantification depth, reproducibility, and accuracy using a validated mouse-yeast two-proteome model. CE-based quantification via the MS and SPS-MS strategies were benchmarked against the nanoLC SPS-MS gold standard. We found electrophoresis-correlative (Eco) ion sorting to order peptides into high-flux transients of nominally isobaric / values (Δ/ < 1-2 Th). While the MS approach struggled with ratio distortion, the SPS-MS robustly eliminated them for both separations. The reproducibility and accuracy proved indistinguishable between CE and nanoLC using MS or SPS-MS quantification. CE enhanced the depth of quantification by ∼12-fold. These analytical insights can be used to design trace CE-MS studies with high scientific rigor.
使用等压条形码的多重定量在痕量敏感和单细胞质谱（MS）中受到关注，在纳流液相色谱（nanoLC）和毛细管电泳（CE）中均是如此。在nanoLC-MS中，已知等压干扰导致的比率压缩会在串联质谱（MS/MS）期间对定量准确性构成挑战，而使用同步前体选择（SPS）MS可有效解决这一问题。尽管人们对用于痕量敏感的自下而上蛋白质组学的CE-MS兴趣日增，但使用该技术进行多重定量的保真度尚不清楚。在此，我们通过使用经过验证的小鼠-酵母双蛋白质组模型全面研究定量深度、重现性和准确性，填补了这一基础知识空白。通过MS和SPS-MS策略基于CE的定量与nanoLC SPS-MS金标准进行了对比。我们发现电泳相关（Eco）离子分选可将肽段排列成名义上等压的/值（Δ/<1-2 Th）的高通量瞬态。虽然MS方法存在比率失真问题，但SPS-MS对两种分离方式都能有效消除这些问题。使用MS或SPS-MS定量时，CE和nanoLC之间的重现性和准确性被证明没有差异。CE将定量深度提高了约12倍。这些分析见解可用于设计具有高度科学严谨性的痕量CE-MS研究。
