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Page 4 of 16 Li et al. Microbiome Res Rep 2024;3:26 https://dx.doi.org/10.20517/mrr.2023.57

filtration (Vaccum), 100 µm spin tube filtration (Spin), and 100 g spinning (100 g) workflows, were tested to
evaluate whether any of the methods may have an impact on the initial microbiome, cultured microbiome
and microbiome responses. A commercial kit GutAlive® was evaluated following the manufacturer’s manual
for its performance on live microbiome sample collection (0 day) and preservation at room temperature for
24 and 72 h before being processed using the 100 µm vacuum filtration protocol. We also examined the
storing efficacy of our in-house PBS-glycerol buffer by comparing samples processed immediately after
collection (0 day) with those stored for 24 and 72 h at 4 °C.

Reagents and stock solutions
(1) Reagents and consumables: reagents and consumables for gut microbiome culturing, metaproteomic
sample processing, and TMT labeling are listed in detail in Supplementary Information 1.

Culture medium stock solutions
(2) Culture medium stock solutions: stock solutions of the culture medium were prepared following details
described in Supplementary Table 1.

(3) 1M Tris-HCl stock solution, pH = 8.0: 12.11 g of Tris base was weighed and added to 80 mL of ddH O.
2
While being mixed on a magnetic stirrer, pH was adjusted to 8.0 using HCl. Top up the solution to 100 mL
using ddH O and double check the pH.
2

TM
(4) TMT aliquot stock plates : the TMT11plex reagents (5 mg per channel) were equilibrated to room
[34]
temperature, then 300 μL of anhydrous acetonitrile (ACN) was added to each tube of the reagents and the
mixture was allowed to dissolve for 5 min with occasional vortexing. Each of the reagents was transferred to
15 mL Falcon tubes, and 4,700 μL anhydrous ACN was added to each of the 15 mL tubes. Mix thoroughly.
Aliquot the TMT reagents to 96 well plates (50 μL per well); each 11plex should be arranged in order in a
row. 4 C Freeze-dried stock plates were stored at -80 C.
o
o
(5) Microbial cell lysis buffer: cell lysis buffer contained 8 M urea and 4% sodium dodecyl sulfate (SDS) in
100 mM Tris-HCl (pH = 8.0). For every 50 mL lysis buffer, one Roche cOmplete tablet was added and
TM
TM
sonicated to dissolve. Or use a cOmplete mini tablet for every 10 mL of lysis buffer. Lysis buffer must be
freshly prepared.

(6) Protein precipitation solution: precipitation solution of 50%:50%:0.1% (v/v/v) of acetone: ethanol:acetic
acid solution was prepared and stored at -20 °C for at least overnight before use.

(7) Protein resuspension buffer: 6 M urea in 100 mM Tris-HCl (pH 8).

(8) 0.1 M dithiothreitol (DTT) solution: 77 mg of DTT powder weighed into 5 mL ddH O was prepared
2
freshly before use (or prepare in advance and store solution at -80 C).
o
(9) 0.2 M iodoacetamide (IAA) solution: 185 mg of IAA powder weighed into 5 mL ddH O was prepared
2
freshly before use (or prepare in advance and store solution at -80 C).
o
(10) Trypsin solution: 1 mL of 100 mM Tris-HCl buffer containing 2 μg/mL trypsin was prepared for each
sample plate.