The astounding exhaustiveness and speed of the Astral mass analyzer for highly complex samples is a quantum leap in the functional analysis of microbiomes.

Background: By analyzing the proteins which are the workhorses of biological systems, metaproteomics allows us to list the taxa present in any microbiota, monitor their relative biomass, and characterize the functioning of complex biological systems.

Results: Here, we present a new strategy for rapidly determining the microbial community structure of a given sample and designing a customized protein sequence database to optimally exploit extensive tandem mass spectrometry data. This approach leverages the capabilities of the first generation of Quadrupole Orbitrap mass spectrometer incorporating an asymmetric track lossless (Astral) analyzer, offering rapid MS/MS scan speed and sensitivity. We took advantage of data-dependent acquisition and data-independent acquisition strategies using a peptide extract from a human fecal sample spiked with precise amounts of peptides from two reference bacteria.

Conclusions: Our approach, which combines both acquisition methods, proves to be time-efficient while processing extensive generic databases and massive datasets, achieving a coverage of more than 122,000 unique peptides and 38,000 protein groups within a 30-min DIA run. This marks a significant departure from current state-of-the-art metaproteomics methodologies, resulting in broader coverage of the metabolic pathways governing the biological system. In combination, our strategy and the Astral mass analyzer represent a quantum leap in the functional analysis of microbiomes. Video Abstract.