Quarmby, S. T. | Search bioRxiv

bioRxiv2025

Regarding Emitter Positioning for Nanoflow Electrospray Ionization

Nanoflow electrospray ionization is commonly used for proteomics due to its high sensitivity. Signal intensity, however, is dependent on optimal emitter positioning relative to the mass spectrometer inlet. Here, we characterize the effect of varied emitter positions on peptide signal intensity in all three dimensions using emitters and flows consistent with standard proteomic analyses. We observe improved signal robustness to x/y variations at increasing z distances and demonstrate that positioning within 1 to 2 mm of the optimal location will maintain consistent signal. Signal intensity behavior is consistent across the m/z range, suggesting a certain level of analyte-independence for proteomics analyses. These results provide insight for proteomics researchers using nanoflow LC-MS/MS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/695161v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1a976ccorg.highwire.dtl.DTLVardef@b6627forg.highwire.dtl.DTLVardef@1fca93eorg.highwire.dtl.DTLVardef@cb61f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

SPE-CZE-MS quantifies zeptomole concentrations ofphosphorylated peptides

Capillary zone electrophoresis (CZE) is gaining attention in the field of single-cell proteomics for its ultra-low-flow and high-resolution separation abilities. Even more sample-limited yet rich in biological information are phosphoproteomics experiments, as the phosphoproteome composes only a fraction of the whole cellular proteome. Rapid analysis, high sensitivity, and maximization of sample utilization are paramount for single-cell analysis. Some challenges of coupling CZE analysis with mass spectrometry analysis (MS) of complex mixtures include 1. sensitivity due to volume loading limitations of CZE and 2. incompatibility of MS duty cycles with electrophoretic timescales. Here, we address these two challenges as applied to single-cell equivalent phosphoproteomics experiments by interfacing a microchip-based CZE device integrated with a solid-phase-extraction (SPE) bed with the Orbitrap Astral mass spectrometer. Using 225 phosphorylated peptide standards and phosphorylated peptide-enriched mouse brain tissue, we investigate microchip-based SPE-CZE functionality, quantitative performance, and complementarity to nano-LC-MS (nLC-MS) analysis. We highlight unique SPE-CZE separation mechanisms that can empower fit-for-purpose applications in single-cell-equivalent phosphoproteomics.

systems biology↗