PSMtags improve peptide sequencing and throughput in sensitive proteomics

Mass spectrometry-based proteomics enables comprehensive characterization of proteins, but label-free approaches are challenging to scale to thousands of samples per day (SPD). Multiplexed data-independent approaches (plexDIA) can increase throughput but lack the mass tags to enable it. Here, we systematically screened a library of 576 compounds and identified several small molecules that, when conjugated to peptides, improve their detection and sequence identification. The lead molecule, PSMtag, substantially increases the detection of fragment b-ions, which increases the confidence of sequence identification and enhances de novo sequencing. PSMtags allow 9-plexDIA, using only stable isotopes of carbon, oxygen and nitrogen. As a result, it allows increasing sample throughput for plexDIA workflows. We demonstrate 1,130 SPD with 9-plexDIA from single-cell-level human proteomes, acquiring 27,520 protein datapoints in the same time label-free methods acquire 3,216. Our approach constitutes an expandable framework for developing mass tags to overcome existing limitations in multiplexed proteomics.