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Abstract
Multiplexing quantification using isobaric barcoding has gained traction in single-cell mass spectrometry (MS), both in nano-flow liquid chromatography (nanoLC) and capillary electrophoresis (CE). In nanoLC-MS, ratio compression from isobaric interferences challenges the accuracy of quantification during tandem MS (MS2); this is remedied at the MS3 level, albeit at the expense of a reduction in proteome coverage. In single-cell capillary electrophoresis (CE) electrospray ionization (ESI) MS, electrophoresis-correlative (Eco) ion sorting practically orders ions into narrow mass-to-charge-dependent trends. Despite pioneering targeted and discovery single-cell MS proteomics, practically nothing is known about how close separation of similar m/z values (Eco-sorting) may affect the fidelity of quantification in CE-MS proteomics. This study is dedicated to bridging this gap in our basic knowledge for the sake of accurate proteome quantification, at a time when CE-MS is emerging into the public domain. Leveraging the mouse–yeast two-proteome model, as validated in nanoLC, we systematically characterize the fidelity of quantification in CE-MS. By employing the strategies of both MS2 and MS3 on the same mass spectrometer, we gain valuable insights to interferences within and between the approaches. Briefly, we found CE-MS to yield ~12-fold sensitivity enhancement than nanoLC. Considering +2 charge state, the driver of protein identifications in this study, we find ratio compression to be severe in MS2 in CE-MS (~66.5% interference free index, IFI), but statistically significantly less than in nanoLC (~63.5% IFI). Simultaneous precursor selection MS3 effectively remedied these interferences in CE-MS (~87.0% IFI), statistically indifferently than nanoLC. CE-MS provides comparable, technically actually slightly and significantly better, quantitative performance than the reference standard nanoLC for limited amounts of proteomes, such as single cells and their subcellular organelles.
