A Multipoint Validation of Quantification in Capillary Electrophoresis Mass Spectrometry Proteomics: Isobaric Multiplexing with Tandem Mass Tags

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 (MS²), which is effectively remedied using simultaneous precursor selection (SPS) MS³. 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 MS² and SPS-MS³ strategies were benchmarked against the nanoLC SPS-MS³ gold standard. We found electrophoresis-correlative (Eco) ion sorting to order peptides into high-flux transients of nominally isobaric m/z values (Δm/z < 1–2 Th). While the MS² approach struggled with ratio distortion, the SPS-MS³ robustly eliminated them for both separations. The reproducibility and accuracy proved indistinguishable between CE and nanoLC using MS² or SPS-MS³ 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.