Robust dimethyl-based multiplex-DIA doubles single-cell proteome depth via a reference channel

Area of Science:

• Proteomics
• Mass Spectrometry
• Single-cell analysis

Background:

• Single-cell proteomics is crucial for understanding biological heterogeneity but faces limitations in depth, throughput, and robustness.
• Existing methods struggle to provide comprehensive protein-level insights from individual cells.

Purpose of the Study:

• To develop a streamlined, multiplexed workflow for enhanced single-cell proteomics.
• To improve proteomic depth, throughput, and robustness in single-cell protein analysis.
• To apply the new workflow to identify proteomic signatures in tumor microenvironments for precision oncology.

Main Methods:

• Development of a streamlined multiplexed data-independent acquisition (mDIA) workflow.
• Automated dimethyl labeling for bulk or single-cell samples.
• Lys-N digestion enabling five-plex quantification at MS1 and MS2 levels.
• Utilization of a reference channel with the RefQuant algorithm for enhanced quantification.
• Integration of mDIA with spatial proteomics for increased throughput.

Main Results:

• The mDIA workflow achieves automated, complete dimethyl labeling without loss of proteomic depth.
• Five-plex quantification is enabled at both MS1 and MS2 levels.
• The RefQuant algorithm confidently quantifies twice as many proteins per single cell compared to previous methods.
• The workflow supports routine analysis of 80 single cells per day.
• Combined mDIA and spatial proteomics increased Deep Visual Proteomics throughput seven-fold for microdissection and four-fold for MS analysis.

Conclusions:

• The developed mDIA workflow significantly enhances proteomic depth and throughput for single-cell analysis.
• This advancement enables confident quantification of more proteins per cell, improving biological insights.
• Application to melanoma revealed proteomic signatures within tumor microenvironments, highlighting potential for precision oncology.