SPE-CZE-MS Quantifies Zeptomole Amounts of Phosphorylated Peptides

Capillary zone electrophoresis
(CZE) is gaining attention in the
field of single-cell proteomics for its ultralow-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 electropherographic time scales. 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.