Solid-Phase Microextraction-Aided Capillary Zone Electrophoresis-Mass Spectrometry: Toward Bottom-Up Proteomics of Single Human Cells

Abstract

Capillary zone electrophoresis-mass spectrometry (CZE-MS) has been recognized as a valuable technique for the proteomics of mass-limited biological samples (i.e., single cells). However, its broad adoption for single cell proteomics (SCP) of human cells has been impeded by the low sample loading capacity of CZE, only allowing us to use less than 5% of the available peptide material for each measurement. Here we present a reversed-phase-based solid-phase microextraction (RP-SPME)-CZE-MS platform to solve the issue, paving the way for SCP of human cells using CZE-MS. The RP-SPME-CZE system was constructed in one fused silica capillary with zero dead volume for connection via in situ synthesis of a frit, followed by packing C8 beads into the capillary to form a roughly 2 mm long SPME section. Peptides captured by SPME were eluted with a buffer containing 30% (v/v) acetonitrile and 50 mM ammonium acetate (pH 6.5), followed by dynamic pH junction-based CZE-MS. The SPME-CZE-MS enabled the injection of nearly 40% of the available peptide sample for each measurement. The system identified 257 ± 24 proteins and 523 ± 69 peptides (N = 2) using a Q-Exactive HF mass spectrometer when only 0.25 ng of a commercial HeLa cell digest was available in the sample vial and 0.1 ng of the sample was injected. The amount of available peptide is equivalent to the protein mass of one HeLa cell. The data indicate that SPME-CZE-MS is ready for SCP of human cells.

Integrated MICROFASP Method with CZE-Based Fractionation Technique and NanoRPLC-ESI-MS/MS for a Comprehensive Proteomics Analysis of a Submicrogram Sample

“…In contrast, capillary zone electrophoresis (CZE) could separate polar peptides well because its separation mechanism was based on the differences in the electrophoretic mobility of the peptides in the electric field . The advantages of CZE in high-sensitivity proteomics analysis and the good orthogonality between CZE and RPLC have been demonstrated. − Therefore, a combination of CZE and RPLC could greatly improve the protein coverage in proteomics analysis of the mass-limited sample . Yan et al compared the performances of RPLC/CZE-ESI-MS/MS and RPLC/UPLC-ESI-MS/MS two-dimensional (2D) separation platforms using a tryptic digest generated from Xenopus laevis embryos .…”