timePlex: Increasing mass spectrometry throughput using time-encoded sample multiplexing

Liquid chromatography-mass spectrometry (LC-MS) can enable precise and accurate quantification of analytes at high-sensitivity, but the rate at which samples can be analyzed remains limited. Throughput can be increased by data-independent-acquisition (DIA) using short separation times and by multiplexing samples in the mass domain with plexDIA; yet multiplexing along one dimension scales throughput only linearly with plex. To enable combinatorial scaling of proteomics throughput, we developed a complementary DIA strategy that multiplexes samples in the time domain, termed `timePlex'. timePlex staggers and overlaps the separation periods of individual samples. This strategy is orthogonal to isotopic multiplexing, which enables combinatorial multiplexing in mass and time domains, and thus multiplicatively increased throughput. We demonstrate this with a 27-plex (3-timePlex and 9-plexDIA) exceeding 1,200 samples (4M protein datapoints) per day. These results establish timePlex as a framework for multiplexing both label-free and labeled samples that supports sensitive and accurate protein quantification and combinatorial scaling.