Metagenomic approaches focus on
taxonomy or gene annotation but lack power in defining functionality of gut
microbiota. Therefore, metaproteomics approaches have been introduced to
overcome this limitation. However, the common metaproteomics approach uses
data-dependent acquisition mass spectrometry, which is known to have limited
reproducibility when analyzing samples with complex microbial composition. In
this work, we provide a proof-of-concept for data-independent acquisition (DIA)
metaproteomics. To this end, we analyze metaproteomes using DIA mass
spectrometry and introduce an open-source data analysis software package diatools, which enables accurate and
consistent quantification of DIA metaproteomics data. We demonstrate the
feasibility of our approach in gut microbiota metaproteomics using laboratory
assembled microbial mixtures as well as human fecal samples.