Critical Steps in an Environmental Metaproteomics Workflow

AbstractEnvironmental metaproteomics is a rapidly advancing field that provides insights into the structure, dynamics, and metabolic activity of microbial communities. As the field is still maturing, it lacks consistent workflows, making it challenging for non‐expert researchers to navigate. This review aims to introduce the workflow of environmental metaproteomics. It outlines the standard practices for sample collection, processing, and analysis, and offers strategies to overcome the unique challenges presented by common environmental matrices such as soil, freshwater, marine environments, biofilms, sludge, and symbionts. The review also highlights the bottlenecks in data analysis that are specific to metaproteomics samples and provides suggestions for researchers to obtain high‐quality datasets. It includes recent benchmarking studies and descriptions of software packages specifically built for metaproteomics analysis. The article is written without assuming the reader's familiarity with single‐organism proteomic workflows, making it accessible to those new to proteomics or mass spectrometry in general. This primer for environmental metaproteomics aims to improve accessibility to this exciting technology and empower researchers to tackle challenging and ambitious research questions. While it is primarily a resource for those new to the field, it should also be useful for established researchers looking to streamline or troubleshoot their metaproteomics experiments.