Top-Down Proteomics Analysis of Picogram-Level Complex Samples Using Spray-Capillary-Based Capillary...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Top-down proteomics is crucial for understanding biological systems in mass-limited samples like patient tissues and single cells.
• Existing top-down methods lack the sensitivity required for these challenging sample types.
• Capillary electrophoresis (CE) offers high resolution and low detection limits, making it suitable for mass-limited samples.

Purpose of the Study:

• To develop an ultrasensitive top-down proteomics platform for mass-limited biological samples.
• To enhance the sensitivity of electrospray ionization (ESI)-assisted spray-capillary devices for proteomics.
• To enable the analysis of intact proteins in complex samples down to the single-cell level.

Main Methods:

• Development of a spray-capillary-CE-MS platform incorporating a polyethylenimine (PEI)-coated capillary.
• Optimization of the spray-capillary inner diameter to improve sensitivity.
• Coupling nanodroplet-based sample preparation with the optimized CE-MS platform.

Main Results:

• Detection of over 200 proteoforms from 50 pg of E. coli lysate.
• Achieved high sensitivity, representing a significant advancement in top-down proteomics detection.
• Successfully identified 261 ± 65 and 174 ± 45 intact proteoforms from fewer than 50 HeLa and OVCAR-8 cells, respectively.

Conclusions:

• The modified spray-capillary CE-MS platform enables top-down proteomics analysis of picogram-level samples.
• This technology facilitates meaningful proteomic analysis of mass-limited samples, including single mammalian cells.
• Represents a breakthrough for ultrasensitive top-down proteomics in complex biological systems.