JMod: Joint modeling of mass spectra for empowering multiplexed DIA proteomics

The throughput of mass spectrometry (MS) proteomics can be increased substantially by multiplexing that enables parallelization of data acquisition. Such parallelization in the mass domain (plexDIA) and the time domain (timePlex) increases the density of mass spectra and the overlap between ions originating from different precursors, potentially complicating their analysis. To enhance sequence identification and quantification from such spectra, we developed an open source software for Joint Modeling of mass spectra: JMod. It uses the intrinsic structure in the spectra and explicitly models overlapping peaks as linear superpositions of their components. This modeling enabled performing 9-plexDIA using 2 Da offset PSMtags by deconvolving the resulting overlapping isotopic envelopes in both MS1 and MS2 space. The results demonstrate 9-fold higher throughput with preserved quantitative accuracy and coverage depth. This support for smaller mass offsets increases multiplexing capacity and thus proteomic throughput for a given plexDIA tag, and we demonstrate this generalizability with diethyl labeling. By supporting enhanced decoding of DIA spectra multiplexed in the mass and time domains, JMod provides an open and flexible software that enables increasing the throughput of sensitive proteomics.