Solid phase microextraction-aided capillary zone electrophoresis-mass spectrometry: ready for bottom-up proteomics of single human cells

ABSTRACT: Capillary zone electrophoresis-mass spectrometry (CZE-MS) has been recognized as a valuable technique for proteomics of mass-limited biological samples (i.e., single cells) due to its high efficiency and high sensitivity for peptide separation and detection. 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 to use less than 5% of the available peptide material for measurements. Here we present a reversed-phase based solid phase microextraction (RP-SPME)-CZE-MS platform to solve the low sample loading capacity issue of CZE, 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 were captured by SPME, and then 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 measurements. The system identified 257 ± 24 proteins and 523 ± 69 peptides (N=2) 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 available peptide amount is equivalent to the protein mass of two HeLa cells. The data indicates that SPME-CZE-MS offers sufficient sensitivity for SCP of human cells.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Liangliang Sun  

LAB HEAD: Liangliang Sun

PROVIDER: PXD047627 | Pride | 2024-05-01

REPOSITORIES: Pride