Single-cell proteomics in complex tissues using microprobe capillary electrophoresis mass spectrometry

Area of Science:

• Proteomics
• Cell Biology
• Developmental Biology

Background:

• Understanding cell-to-cell molecular differences (heterogeneity) is crucial for development and disease.
• High-resolution mass spectrometry (HRMS) typically analyzes large cell populations.
• Advancements enable HRMS application to single cells.

Purpose of the Study:

• To present a protocol for direct proteomic profiling of single cells within complex tissues.
• To enable label-free protein identification and quantification at the single-cell level.
• To explore cellular heterogeneity in embryonic and neuronal cells.

Main Methods:

• Protocol based on microprobe capillary electrophoresis (CE) coupled with HRMS.
• Direct single-cell collection from intact tissues without dissociation.
• Bottom-up proteomics using a custom CE electrospray ionization (ESI)-mass spectrometer.

Main Results:

• Demonstrated proteomic profiling of individual cells in Xenopus embryos, zebrafish, and mouse neurons.
• Identified previously unknown proteomic differences between embryonic cells and neurons.
• Showcased the ability to analyze single cells based on physiological or morphological cues.

Conclusions:

• Microprobe CE-ESI-HRMS enables direct proteomic analysis of single cells in complex biological systems.
• This method reveals novel insights into cellular heterogeneity.
• Complements single-cell transcriptomics for deeper understanding of cellular and developmental processes.