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Page 6 of 16 Li et al. Microbiome Res Rep 2024;3:26 https://dx.doi.org/10.20517/mrr.2023.57

inflammatory bowel disease, or diabetes diagnosis; antibiotic use or gastroenteritis episode in three months
preceding collection; use of pro-/pre-biotic, laxative, or anti-diarrheal drugs in the last month preceding
[35]
collection; or pregnancy . Written consents were obtained from all participants.

Microbiome culturing and treatment
In the anaerobic chamber, 1 mL culture media and 100 µL stool samples were added into each well of a
square-well 96-deepwell plate using a 96-channel liquid handler (e.g., epMotion® 96) (or can use a multi-
channel pipette). 2 mg/mL kestose was added into the culture media for the kestose treatment groups.
Samples were mixed sufficiently before covering the wells tightly with a perforated silicone gel mat and
sealed around using lab tape to prevent popping up due to gas production in the culture. The deepwell
plates were shaken at 500 rpm on an orbital shaker for 18 h at 37 °C.

Microbial cell washing
After culturing, culture plates were centrifuged at 3,000 g for 45 min at 4 °C. The microbial cell-free
supernatant was then removed (the supernatant can be collected for pH, metabolome and/or exosome
analyses). The pellets were resuspended in 1 mL cold PBS buffer using a 96-channel liquid handler (or a
multi-channel pipette). If samples cannot be sufficiently resuspended using pipettes, firmly cover the plate
with a silicon gel mat and vortex the plate at 2,000 rpm to mix well. The plates were then centrifuged again
at 3,000 g, 45 min, 4 °C. After adding 1 mL cold PBS buffer to resuspend the pellet again, the plates were
centrifuged at 300 g, 5 min, 4 °C to pellet debris. The supernatant was next carefully transferred into a new
deepwell plate. The above cell washing steps were repeated for another two rounds, before removing the
supernatant and storing the plates at -80 °C before cell lysis.

Microbial cell lysis and protein double-precipitation
150 μL lysis buffer was added to each of the wells containing microbial cell pellets using a 96-channel liquid
handler. The pellets were resuspended before being transferred to a 96-well PCR plate. The PCR plate was
covered with strip lids and subjected to sonication with a cup-horn ultra-sonicator (QSonica, cat. no.
Q700MPXC) at 10 kHz, 10s-on and 10s-off cycle for 20 min (i.e., total sonication of 10 min), with a
recirculating chiller set to 8 °C to prevent overheat of samples. After sonication, transfer the cell lysate into a
1.2 mL 96-well cluster tube plate, and add 800 μL ice-cold protein precipitation solution. Cover with cluster
lids and mix well.

Samples were precipitated overnight at -20 °C. After the first precipitation, the sample plates were
centrifuged at 3,000 g, 45 min, 4 °C. After carefully removing the supernatant, 150 μL protein resuspension
buffer was added to the pellets before being mixed at 2,000 rpm using a vortex mixer, until protein pellets in
all wells were fully suspended. Next, 800 μL ice-cold protein precipitation solution was added for a second
precipitation at -20 °C overnight.

Protein digestion
The sample plates were centrifuged at 3,000 g, 45 min, 4 °C and the supernatant in each well was carefully
removed. 100 μL protein resuspension buffer was added to each well and mixed at 2,000 rpm using a vortex
mixer, until protein pellets in all wells were fully suspended. Protein concentrations were determined using
the DC Protein Assay kit following the manufacturer’s instructions. Protein samples were next diluted to
reach a concentration of 1 μg/μL using the protein resuspension buffer.

Protein digestion was then performed using an automated liquid handler (Hamilton Nimbus 96, cat. no.
OPP041219): first, 10 μL 0.1 M DTT solution was added to each well of 100 μL samples, and incubated at
56 °C, 800 rpm for 30 min. Next, after cooling the plates to room temperature, 10 μL 0.2 M IAA solution