Quantitative Label-Free Single-Cell Proteomics on the Orbitrap Astral MS.

Area of Science:

• Proteomics
• Mass Spectrometry
• Cellular Biology

Background:

• Single-cell proteomics by mass spectrometry (scp-MS) offers deep biological insights but faces sensitivity limitations.
• Current instrumentation struggles with the low signal from single-cell samples, approaching detection boundaries.

Purpose of the Study:

• To evaluate the Orbitrap Astral mass spectrometer's enhanced sensitivity for deep proteome profiling in low-input and single-cell samples.
• To optimize data acquisition methods for maximum sensitivity in scp-MS.
• To assess the quantitative accuracy of measurements from low-input samples.

Main Methods:

• Conducted a comprehensive survey of data acquisition parameters to identify optimal settings for sensitivity.
• Performed quantitative accuracy assessments against known values.
• Generated and analyzed scp-MS data from cultured cell lines and primary bone marrow samples.
• Explored protein covariation to assess the capture of protein complex information.

Main Results:

• The Orbitrap Astral demonstrated enhanced capacity for deep proteome profiling from low-input to single-cell levels.
• Optimized acquisition methods significantly improved sensitivity.
• Quantitative measurements showed good accuracy.
• Differences in proteome coverage were observed across different sample types.
• Protein covariation analysis successfully captured information on known protein complexes.

Conclusions:

• The Orbitrap Astral mass spectrometer significantly advances scp-MS capabilities by enhancing sensitivity and quantitative accuracy.
• This technology facilitates deeper proteome coverage and provides insights into cellular phenotypes and protein complexes from low-input samples.