In Vitro Metabolic Labeling of Intestinal Microbiota for Quantitative Metaproteomics

Abstract

Intestinal microbiota is emerging as one of the key environmental factors influencing or causing the development of numerous human diseases. Metaproteomics can provide invaluable information on the functional activities of intestinal microbiota and on host-microbe interactions as well. However, the application of metaproteomics in human microbiota studies is still largely limited, in part due to the lack of accurate quantitative intestinal metaproteomic methods. Most current metaproteomic microbiota studies are based on label-free quantification, which may suffer from variability during the separate sample processing and mass spectrometry runs. In this study, we describe a quantitative metaproteomic strategy, using in vitro stable isotopically ((15)N) labeled microbiota as a spike-in reference, to study the intestinal metaproteomes. We showed that the human microbiota were efficiently labeled (>95% (15)N enrichment) within 3 days under in vitro conditions, and accurate light-to-heavy protein/peptide ratio measurements were obtained using a high-resolution mass spectrometer and the quantitative proteomic software tool Census. We subsequently employed our approach to study the in vitro modulating effects of fructo-oligosaccharide and five different monosaccharides on the microbiota. Our methodology improves the accuracy of quantitative intestinal metaproteomics, which would promote the application of proteomics for functional studies of intestinal microbiota.

Metaproteomic responses ofin vitrogut microbiomes to resistant starches: the role of resistant starch type and inter-individual variations

“…The treatment with the positive control FOS showed 16 COG categories significant changed (total abundance of their protein constituents compared to the blank control). This was consistent with our previous experiments with FOS [24]. The corn starch-treated microbiomes only had three significantly changed COG categories whereas microbiomes treated with the three RS showed a total of 12 significantly changed COG categories of which three COG categories were only changed in the presence of RS.…”

“…In this study, we investigated the functional and taxonomic changes in individual microbiomes exposed to different type and sources of RS using in vitro culturing and metaproteomics. The effects of RS on the microbiomes were milder than the changes observed with FOS, which is known to induce taxonomic and functional changes in the human gut microbiome in vitro [22][23][24].…”

“…Briefly, stool microbiomes from seven healthy individuals were cultured for 24 hr in the presence of RS or controls using our previously described method [21] (Figure 1). The microbiomes were treated with RS2(Hi Maize 260), RS3(Novelose 330), RS4(Fibersym RW), a non-resistant starch control (corn starch (CS)), a positive control (fructo-oligosaccharide (FOS) known to consistently and markedly shift the in vitro gut microbiome [22][23][24]) or a blank control containing no compounds. The samples were then subjected to metaproteomic analysis as previously described [21].…”

An in vitro model maintaining taxon-specific functional activities of the gut microbiome

“…1:1 (w/w) sodium salt forms of cholic acid (CA) and chenodeoxycholic acid (CDCA), and (2) a commercialized 1:1 (w/w) mixture of sodium salt forms of CA and deoxycholic acid (DCA). The commercialized bile salts mixture has been adopted in a majority of gut microbiome culture media 13,14,[27][28][29][30][31][32][33] . The ratio of Firmicutes to Bacteroideteshas been extensively used to characterize microbiomes 34 .…”

Metaproteomic analysis of human gut microbiota: where are we heading?

“…Concerning gut microbiota, label-free quantification is the most common strategy adopted in the aforementioned metaproteomic studies, but has limited accuracy. The most recent take on quantitative metaproteomics of human gut microbiota is the application of metabolic labeling for improved peptide quantification [52]. This newly introduced method hold the potential to facilitate future metaproteomic study.…”