In-Depth Metaproteomics Analysis of Oral Microbiome for Lung Cancer

The human oral microbiome correlates with numerous diseases,including lung cancer.Identifying the functional changes by metaproteomics helps understand the disease-related dysbiosis,yet characterizing low-abundant bacteria is challenging.Here,we developed a free-flow isoelectric focusing electrophoresis-mass spectrometry-(FFIEF-MS-)based metaproteomics strategy to reduce host interferences and enrich low-abundant bacteria for in-depth interpretation of the oral microbiome.With our method,the number of interfering peptides decreased by 52.87%,whereas the bacterial peptides and species increased by 94.97%and 44.90%,respectively,compared to the conventional metaproteomics approach.We identified 3647 bacterial proteins,which is the most comprehensive oral metaproteomics study to date.Lung cancer-associated bacteria were validated among an independent cohort.The imbalanced Fusobacterium nucleatum and Prevotella histicola and their dysregulated functions in inhibiting immune response and maintaining cell redox homeostasis were revealed.The FFIEF-MS may serve as a valuable strategy to study the mechanisms between human diseases and microbiomes with broader applications.