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During the last decade, the evidences on the relationship between neurodevelopmental disorders and the microbial communities of the intestinal tract have considerably grown. Particularly, the role of gut microbiota (GM) ecology and predicted functions in Autism Spectrum Disorders (ASD) has been especially investigated by 16S rRNA targeted and shotgun metagenomics, trying to assess disease signature and their correlation with cognitive impairment or gastrointestinal (GI) manifestations of the disease. Herein we present a metaproteomic approach to point out the microbial gene expression profiles, their functional annotations, and the taxonomic distribution of gut microbial communities in ASD children. We pursued a LC-MS/MS based investigation, to compare the GM profiles of patients with those of their respective relatives and aged-matched controls, providing a quantitative evaluation of bacterial metaproteins by SWATH analysis. All data were managed by a multiple step bioinformatic pipeline, including network analysis. In particular, comparing ASD subjects with CTRLs, up-regulation was found for some metaproteins associated with Clostridia and with carbohydrate metabolism (glyceraldehyde-3-phosphate and glutamate dehydrogenases), while down-regulation was observed for others associated with Bacteroidia (SusC and SusD family together with the TonB dependent receptor). Moreover, network analysis highlighted specific microbial correlations among ASD subgroups characterized by different functioning levels and GI symptoms. To the best of our knowledge, this study represents the first metaproteomic investigation on the gut microbiota of ASD children compared with relatives and age-matched CTRLs. Remarkably, the applied SWATH methodology allowed the attribution of differentially regulated functions to specific microbial taxa, offering a novel and complementary point of view with respect to previous studies. [Display omitted] • SWATH analysis supported the metaproteomic outcome when comparing ASD and controls. • The gut microbiota of controls differs from both that of ASD and their relatives. • The enhanced functions in the gut microbiota of ASD are ascribed to Clostridia. [ABSTRACT FROM AUTHOR]