Sample Preparation, Mass Spectrometry, and Data Analysis for Proteomics.

Bt was cultured in BMM–glucose and BMM–dextran (M_w 9,000 to 11,000) overnight from an initial OD of 0.01. Cells were lysed, and lysates containing 20 µg of protein content were collected for further analysis. Cell lysates were digested with trypsin using the filter-aided sample preparation (57) protocol with minor modifications. Proteins were resuspended in 200 µL of 8 M urea, 100 mM Tris–HCl and deposited on top of Microcon-30K devices. Samples were centrifuged at 9391 g at 20 °C for 30 min. All subsequent centrifugation steps were performed using the same conditions. An additional 200 µL of 8 M urea 100 mM Tris–HCl was added, and devices were centrifuged again. Sample reduction was performed by adding 100 µL of 10 mM TCEP in 8 M urea, 100 mM Tris–HCl on top of filters followed by 60 min incubation at 37 °C with gentle shaking and protected from light. Reduction solution was removed by centrifugation, and filters were washed with 200 µL of 8 M urea, 100 mM Tris–HCl. After removal of the washing solution by centrifugation, alkylation was performed by adding 100 µL of 40 mM chloroacetamide in 8 M urea, 100 mM Tris–HCl, and incubating the filters at 37 °C for 45 min with gentle shaking and protected from light. The alkylation solution was removed by centrifugation, and another washing/centrifugation step with 200 µL of 8 M urea 100 mM Tris–HCl was performed. This last urea buffer washing step was repeated twice followed by three additional washing steps with 100 µL of 5 mM Tris–HCl. Proteolytic digestion was performed overnight at 37 °C by adding 100 µL of Endoproteinase Lys-C and Trypsin Gold in an enzyme/protein ratio of 1:50 (w/w) on top of filters. The resulting peptides were recovered by centrifugation. The devices were then rinsed with 50 µL of 4% trifluoroacetic acid and centrifuged. This step was repeated three times, and peptides were finally desalted on SDB-RPS StageTips (58).

For LC-MS/MS analysis, resuspended peptides were separated by reversed-phase chromatography on a Dionex Ultimate 3000 RSLC nano UPLC system connected in line with an Orbitrap Lumos (Thermo Fisher Scientific, Waltham, USA). A capillary precolumn (Acclaim Pepmap C18, 3 μm-100 Å, 2 cm × 75 μm ID) was used for sample trapping and cleaning. A capillary column (75 μm ID; in-house packed using ReproSil-Pur C18-AQ 1.9 μm silica beads; Dr. Maisch; length 50 cm) was then used for analytical separations at 250 nL/min over 150 min biphasic gradient. Acquisitions were performed through top-speed data-dependent acquisition mode using a 1-s cycle time. First, MS scans were acquired at a resolution of 240,000 (at 200 m/z), and the most intense parent ions were selected and fragmented by high-energy collision dissociation (HCD) with a normalized collision energy (NCE) of 30% using an isolation window of 0.7 m/z. Fragmented ion scans were acquired in the ion trap using a fix maximum injection time of 20 ms, and selected ions were then excluded for the following 20 s.

Protein identification and label-free quantification were performed using MaxQuant 1.6.10.43 (59). The B. thetaiotaomicron Uniprot reference proteome database was used for this search. Carbamidomethylation was set as fixed modification, whereas oxidation (M), phosphorylation (S, T, Y), and acetylation (protein N-term) were considered as variable modifications. A maximum of two missed cleavages were allowed for this search. “Match between runs” was selected. A minimum of two peptides were allowed for protein identification, and the false discovery rate (FDR) cutoff was set to 0.01 for both peptides and proteins. Label-free quantification and normalization was performed by Maxquant using the MaxLFQ algorithm, with the standard settings (60).

In Perseus (61), reverse proteins, contaminants, and proteins only identified by sites were filtered out. Biological replicates were grouped together, and protein groups containing a minimum of two LFQ values in at least one group were conserved. Missing values were imputed with random numbers using a Gaussian distribution (width = 0.3, downshift = 1.8). The two-samples t test was performed to identify the differentially expressed proteins, followed by a permutation-based correction FDR. Significance curves in the volcano plot corresponded to a S₀ value of 1.0 and an FDR cutoff of 0.01.