Offset Mass Carrier Proteome Improves Quantification of Multiplexed Single Cell Proteomics.

Area of Science:

• Proteomics
• Mass Spectrometry
• Cellular Biology

Background:

• Multiplexed single-cell proteomics by mass spectrometry (scpMS) offers high throughput.
• Current scpMS methods use isobaric labels and carrier proteomes, which can limit peptide sampling and quantitative accuracy.
• High carrier proteome levels interfere with single-cell peptide ion detection.

Purpose of the Study:

• To address the limitations of carrier proteomes in scpMS.
• To introduce a novel method, triggered by offset mass acquisition for scpMS (toma-scpMS), for improved quantitative accuracy.
• To enable robust single-cell proteome analysis even with high carrier proteome concentrations.

Main Methods:

• Developed toma-scpMS utilizing nonisobaric tags for the carrier proteome.
• Implemented a custom data acquisition scheme in inSeqAPI for real-time carrier peptide identification.
• Triggered offset quantification scans for single-cell samples based on carrier peptide detection.

Main Results:

• toma-scpMS separates carrier and single-cell proteomes at the precursor level.
• The method allows for separate isolation, fragmentation, and quantitation.
• Demonstrated increased robustness to high carrier proteome levels and superior quantitative accuracy compared to traditional scpMS.

Conclusions:

• toma-scpMS enhances the accuracy and robustness of single-cell proteomics.
• This new method overcomes critical challenges associated with carrier proteome interference.
• toma-scpMS represents a significant advancement for high-throughput single-cell proteomic analysis.