“…However, at present, few methods or reagents used in the above four steps have been systematically optimized in TFFB metaproteomics; instead, direct methods are commonly used, such as lysis with certain concentrations of detergents (mostly sodium dodecyl sulfate (SDS) or 3‐((3‐Cholamidopropyl) dimethylammonium)‐1‐propanesulfonate (CHAPS)) combined with grinding, ultrasonication (Kunath et al., 2019), and other methods. The release of proteins from the microorganisms (Gonzalez‐Balderas, Velasquez‐Orta, Valdez‐Vazquez, & Orta Ledesma, 2020) and the choice of extractant/buffer (Bastida, Jehmlich, Torres, & Garcia, 2018), precipitant, and lysate (Miskiewicz & MacPhee, 2019) have crucial effects on protein extraction. For the extraction of proteins in many substrates, the conditions mentioned above must be optimized systematically (Awad & Brueck, 2019; Parkhey, Chandrakar, Naithani, & Keshavkant, 2015; Wohlbrand et al., 2017), and optimal extraction results had been achieved.…”