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Page 4 of 17 Wang et al. Microbiome Res Rep 2024;3:39 https://dx.doi.org/10.20517/mrr.2024.21

cup-horn sonicator at 50% amplitude, 10 s pulse on/off cycle, for 20 min active sonication time at 8 °C. The
lysate was then centrifuged at 16,000 g for 10 min at 8 °C to remove any non-lysed debris. Protein-
containing supernatant was transferred to a new tube and protein concentrations were determined using
the Pierce BCA protein assay kit (Thermo Fisher Scientific, Cat#23225) following the manufacturer’s
protocol.

In-solution trypsin digestion
For in-solution trypsin digestion, proteins underwent acetone precipitation by adding 5 volumes of protein
precipitation buffer composed of 50% acetone, 50% acetonitrile (ACN), 0.1% acetic acid, mixed through
inversion and incubated at -20 °C overnight. Samples were spun down at 16,000 g at 4 °C for 25 min and the
supernatant was discarded. The remaining pellet was washed with ice-cold acetone, sonicated with a
QSonica Q700 water-chilled cup-horn sonicator at 50% amplitude for 10 s, and spun down at 16,000 g at
4 °C for 10 min for a total of 3 washes. After briefly air drying, the protein pellets were resuspended in 6 M
urea in 50 mM ammonium bicarbonate (ABC) buffer (pH 8) for trypsin digestion. Protein concentrations
were determined using the Pierce BCA protein assay kit, and 100 µg of protein lysates of each sample were
reduced with 10 mM 1,4-dithiothreitol (DTT) incubated at 850 rpm at 56 °C for 30 min and alkylated with
20 mM iodoacetamide (IAA) for 40 min at room temperature protected from light. Samples were diluted in
50 mM ABC buffer to a final urea concentration of 0.6 M and then digested with 4 µg MS-grade Trypsin
(Promega, Cat#V511B, lot: 0000575718) / 100 µg of protein incubated at 850 rpm at 37 °C for 20 h. The
reaction was stopped by adding formic acid (FA) to acidify the samples to pH 2-3 prior to desalting, as
described below.

Filter-aided sample preparation digestion
For filter-assisted digestion methods, two different molecular weight cut-off filters were used, namely filter-
aided sample preparation (FASP)-10kDa and FASP-3kDa (Merck Millipore Ltd). For each sample, 100 µg of
protein lysate was directly diluted to 200 µL of 8 M urea in 100 mM Tris-HCl buffer and added to the pre-
rinsed FASP columns. SDS was diluted twice using 200 µL of 8 M urea in 100 mM Tris-HCl buffer each
time. Reduction was performed by adding 8 M urea in 100 mM Tris-HCl buffer containing a final
concentration of 20 mM DTT and incubated at 850 rpm at 37 °C for 30 min. After removing the flow-
through, samples were then alkylated by 20 mM IAA at room temperature in the dark for 30 min. To
remove excessive IAA, an additional 200 µL 20 mM DTT in 8 M urea in 100 mM Tris-HCl buffer was
added, incubated at room temperature for 2 min, and eluates were discarded. The column was then washed
with 8 M urea in 100 mM Tris-HCl buffer once and 100 mM Tris-HCl buffer for four times prior to adding
200 µL 100 mM Tris-HCl buffer containing 4 µg of trypsin (1 µg trypsin : 25 µg protein input). The trypsin
digestion was performed by shaking at 850 rpm at 37 °C for 20 h. Peptides were eluted by spinning at
16,000 g at room temperature for 20 min followed by additional elution with 200 µL fresh 100 mM Tris-HCl
buffer. Both eluents were combined and acidified to a pH of 2-3 using 10% (v/v) FA for desalting.

Desalting
Desalting was performed using C18 columns (Thermo Scientific, Cat#89870). Columns were activated by
adding 100% ACN, centrifuging at 100 g for 1 min for a total of 3 times, and then equilibrated with 0.1%
(v/v) FA, centrifuging at 300 g for 2 min for 2 times. Samples were loaded to the column and centrifuged at
300 g to remove flow-through until all samples were loaded. The desalting column was washed with 0.1%
(v/v) FA for 2 times. Desalted peptides were then eluted with 100 µL 80% (v/v) ACN / 0.1% (v/v) FA buffer
for two times by centrifuging at 100 g for 1 min for each elution. The eluates containing desalted tryptic
peptides were then dried on a centrivap (Labconco, Cat#7810010) and stored at -20 °C until LC-MSMS
analysis.