Development of an Integrated Pipeline for Profiling Microbial Proteins from Mouse Fecal Samples by LC–MS/MS

Metaproteomics
is one approach to analyze the functional capacity
of the gut microbiome but is limited by the ability to evenly extract
proteins from diverse organisms within the gut. Herein, we have developed
a pipeline to optimize sample preparation of stool obtained from germ-free
(GF) mice that were gavaged a defined community of 11 bacterial strains
isolated from the human gut. With 64% more proteins identified, bead-beating
was confirmed to be an indispensable step for the extraction of bacterial
proteins, especially for Gram-positive bacteria. Bacterial enrichment
from mouse fecal samples was further optimized by evaluating three
different methods: (1) a high-speed differential centrifugation (HCE)
or (2) a low-speed differential centrifugation (LCE) and (3) a filter-aided
method (FA). The HCE method was associated with dramatic loss of bacteria
and 71% less recovery of bacterial proteins than the LCE method. Compared
with LCE, the FA method also showed dramatic loss of the amount of
bacteria recovered and decreased protein identifications from Gram-positive
bacteria in the stool samples. Ultimately, LCE may provide an alternative
and complementary method for enriching bacteria from small amounts
of mouse fecal samples, which could aid in investigating bacterial
function in health and disease.