Introduction
The new timsMetabo exemplifies multiomics integration by combining unprecedented TIMS separation power for small molecules in metabolomics and lipidomics applications with proven 4D-proteomics performance in a single instrument. It enables analysis from bulk samples to the single-cell level, comprehensive plasma proteome coverage (including ENRICH-iST preparations), and introduces a new TIMS-enabled MS/MS mode (diaMoRE) that expands the dynamic loading range for complex proteomics studies, driving deeper biological insight through true multiomics integration.
Methods
The dda/dia-PASEF and dda/dia-MoRE acquisition routines are enabled on the new timsMetabo platform (Bruker). While achieving highest sensitivity in the previously introduced parallel accumulation mode, with dia-MoRE, ions are progressively introduced into the TIMS analyzer as a function of mobility, thus increasing load and mass range capacity. Instrument tuning and dia- window schemes were optimized per application. Identifications and quantitative reproducibility were evaluated using dilution series of human cell line digests (K562, Promega), an in-house digested triple proteome (HeLa, yeast, E. coli), and human plasma samples (neat and ENRICH) prepared with iST-BCT or ENRICH-iST kits (PreOmics). Separation was performed by nanoHPLC (nanoElute 2, Bruker) at various gradient lengths on 25 cm columns (0.075 mm ID, IonOpticks). Data were processed with Spectronaut 20 (Biognosys).
Results
The novel timsMetabo instrument demonstrated competitive proteomics performance, achieving high sensitivity at the single-cell level—detecting over 3,300 protein groups from just 250 pg of injected K562 peptides—and extensive proteome coverage at higher sample loads, with more than 7,200 protein groups identified from 100 ng. It also delivered accurate and precise quantitation across complex proteome mixtures (HeLa, yeast, E. coli), quantifying approximately 10,000 protein groups, of which 90% showed coefficients of variation (CVs) below 20%. Moreover, the instrument showed strong performance on clinical samples, identifying about 540 protein groups from 200 ng of neat human plasma and over 1,600 from ENRICH samples.