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Li et al. Microbiome Res Rep 2024;3:26 https://dx.doi.org/10.20517/mrr.2023.57 Page 5 of 16

(11) Desalting buffers: Wash buffer [0.1% (v/v) formic acid (FA) in water], elution buffer [0.1% FA in 80%
ACN: 80% (v/v) ACN and 0.1% (v/v) FA in water], acidifying buffer [10% FA: 10% (v/v) FA in water] and
5% (v/v) FA in water was prepared for the desalting procedure.

(12) TMT labeling solutions: 100 mM tetraethylammonium bromide (TEAB) in 20% ACN: 10% (v/v) 1M
TEAB, 20% (v/v) 100% ACN and 70% (v/v) HPLC-grade water was mixed to prepare the TEAB solution.

0.8% hydroxylamine in 100 mM TEAB (quencher): 10% (v/v) 1M TEAB and 90% (v/v) HPLC-grade water
were mixed to prepare 100 mM TEAB water solution. Then, 1.6% (v/v) 50% hydroxylamine was mixed with
98.4% (v/v) 100 mM TEAB water solution to prepare the quencher.

Medium preparation
[19]
Culture media was consisted of the following composition : 2.0 g/L peptone water, 2.0 g/L yeast extract,
0.5 g/L L-cysteine hydrochloride, 2 mL/L Tween 80, 5 mg/L hemin, 10 μL/L vitamin K1, 1.0 g/L NaCl,
0.4 g/L K HPO , 0.4 g/L KH PO , 0.1 g/L MgSO ·7H O, 0.1 g/L CaCl ·2H O, 4.0 g/L NaHCO , 4.0 g/L
2
4
4
4
2
2
2
3
2
porcine gastric mucin, 0.25 g/L sodium cholate, and 0.25 g/L sodium chenodeoxycholate. Follow detailed
procedures in Supplementary Table 2 for medium preparation.
Automated digestion deck set up
A deck layout of the Hamilton Nimbus 96 automation system containing five plate/reservoir plate locations,
two tip racks, a thermo block, and a small-volume reservoir location (for DTT and IAA) was set up for
automated digestion. The plate locations contained four sample plates and one reservoir plate for trypsin-
Tris-HCl solution. Two tip racks held tips to be used in adding DTT and IAA, and trypsin-Tris-HCl
solution, respectively.
Automated desalting deck set up
A deck layout containing seven plate/reservoir locations and two tip racks was set up for automated
desalting. The plate locations contained one sample plate, one elution plate, two sample washing plates, and
three reservoir plates for 100% ACN, 0.1% FA and 80% ACN + 0.1% FA solutions, respectively. The tip racks
held pipette tips and reverse-phase (RP) desalting columns, respectively.

Collecting and processing stool samples
An anaerobic chamber was used for sample processing and culturing. The anaerobic chamber contained 5%
H , 5% CO and balanced with N . We used a palladium catalyst to react O , and Thermo Scientific
TM
2
2
2
2
TM
Oxoid Resazurin Anaerobic Indicator to make sure the chambers remain anaerobic. We used an in-house
prepared buffer to collect fecal samples. Fecal samples were prepared to a 20% (w/v) slurry in sterile, pre-
reduced 1X PBS (pH 7.4) containing 10% (v/v) glycerol and 1 mg/mL L-Cysteine. In addition, the
commercial GutAlive kit was compared for its performance in preserving live microbiota at room
temperature following the manufacturer’s instruction. The stool samples were processed through four
different approaches, i.e., gauze filtration, 100 µm vacuum filtration, 100 µm spin tube filtration, and 100 g
spin. Detailed protocols for collecting and processing stool samples, as well as processing of stool samples
for long-term live microbiota biobanking , are presented in Supplementary Methods 1 and 2. Note that
[35]
institutional ethical approval must be obtained, ensuring all samples are collected with informed written
consent and in accordance with relevant guidelines. In this study, the protocol for human stool sample
collection (# 20160585-01 H) was approved by the Ottawa Health Science Network Research Ethics Board at
the Ottawa Hospital, Ottawa, Canada. Four healthy individual’s microbiomes (V54, V55, V56 and V57, with
ages of 53, 30, 33, and 50 years old) were included. The inclusion criteria were healthy individuals who are
18-65 years of age. Exclusion criteria for participation included the presence of irritable bowel syndrome,