Increasing mass spectrometry throughput using time-encoded sample multiplexing

Area of Science:

• Proteomics
• Analytical Chemistry
• Biotechnology

Background:

• Liquid chromatography-mass spectrometry (LC-MS) offers high sensitivity for analyte quantification but faces throughput limitations.
• Current multiplexing strategies, like plexDIA, increase throughput linearly by multiplexing in the mass domain.
• A need exists for methods that combinatorially scale proteomics throughput beyond linear increases.

Purpose of the Study:

• To develop a novel time-domain multiplexing strategy, termed timePlex, to complement existing mass-domain multiplexing.
• To achieve combinatorial scaling of sample throughput in LC-MS-based proteomics.
• To demonstrate the effectiveness of timePlex for sensitive, high-throughput proteomic analyses.

Main Methods:

• Developed timePlex, a method that staggers and overlaps sample separation periods in the time domain.
• Combined timePlex with plexDIA (mass-domain multiplexing) for orthogonal, combinatorial multiplexing.
• Demonstrated multiplexing of 9 samples using 3-timePlex and 3-plexDIA, and 27 samples using 3-timePlex and 9-plexDIA.

Main Results:

• Achieved a throughput exceeding 500 samples per day with a 27-plex (3-timePlex and 9-plexDIA).
• Demonstrated that timePlex supports sensitive analyses, including single-cell proteomics.
• Showcased the multiplicative increase in throughput by combining time-domain and mass-domain multiplexing.

Conclusions:

• timePlex is an effective label-free multiplexing methodology for LC-MS proteomics.
• The combination of timePlex and plexDIA enables combinatorial scaling of proteomics throughput.
• Projected future throughput increases exceeding 1,000 samples per day with this combined approach.