Trifluoroacetic acid tfa

Milli-Q water was generated
from a Merck Milli-Q IQ 7003 system (Darmstadt, DE). Pronase and trastuzumab
were obtained from Roche (Woerden, NL). α-(1-2,3,4,6)-l-Fucosidase was obtained from Megazyme (Ayr, U.K.). Chloroacetamide
(CAA), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), Tris, trypsin,
Lys-C, sodium deoxycholate (SDC), sodium acetate, and formic acid
(FA) were obtained from Merck (Darmstadt, DE). Acetonitrile and 0.1%
FA were obtained from Biosolve (Valkenswaard, NL). Egg yolk powder
was obtained by freeze-drying egg yolks from eggs obtained from SPAR
(Waalwijk, NL). Cotton string was obtained from Kruidvat (Renswoude,
NL). Trifluoroacetic acid (TFA) was obtained from Honeywell International
Inc. (Charlotte, NC).

In this study, the medical masks were produced by Wenner company (China), which is sterilization, synthetic blood no penetration under16Kpa, bacterial filtration efficiency under 95%, and particle (The diameter is 0.075 ± 0.02 μm) filtration efficiency under 30%. Nutrient agar (NA) is purchased from hepobio (Shandong province, China). Phosphate-Buffered dilution water is obtained from Land Bridge (Beijing, China). Bacterial Test Standard (BTS) (Mass Pure Grade), and Matrix alpha-cyano-4-hydroxycinnamic acid (HCCA) (Mass Pure Grade) for MALDI-TOF-MS are purchased from Bruker (Germany). Formic acid (FA) (High performance liquid chromatography, HPLC grade), Standard solvent (SS) (Mass Pure Grade) contained pure water 475 μL, trifluoroacetic acid (TFA) 25 μL, and acetonitrile 500 μL are obtained from HoneyWell (Germany).
Biological safety cabinet used in this research is purchased from AirTECH (Jiangsu province, China). Microbiological incubator is obtained from Zhicheng (Shanghai, China). Bruker MALDI Biotyper used in this study is purchased from Bruker (Germany). 16SrRNA sequencing service is provided by the NOVOGene Co. (Beijing, China).

Protein digestion was performed as already reported with few modifications [44 (link)]. Briefly, 400 µg of proteins for each sample were diluted with ammonium bicarbonate (NH₄HCO₃, Sigma-Aldrich, ≥99.0%, Darmstadt, Germany) buffer solution at 50 mM and with RapiGest^TM SF Surfactant (Waters Corporation, Milford, MA, USA) at a final concentration of 0.1%. The proteins were reduced by adding DL-dithiothreitol (DTT) (Sigma-Aldrich, St. Louis, MO, USA, ≥99.5%) at a final concentration of 40 mM and incubated for 45 min at 56 °C; carbamidomethylation reaction was carried out by 55 mM iodoacetamide (IAA) (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min in the dark.
Trypsin (Trypsin from the porcine pancreas, Sigma-Aldrich, St. Louis, MO, USA) was added to the sample for protein digestion at an enzyme/protein ratio of 1:100 and incubated overnight at 37 °C. The digestion was stopped by adding trifluoroacetic acid (TFA) (Honeywell, Seelze, Germany) at a final concentration of 0.5% to reach an acidic pH (<2). RapiGest^TM SF surfactant removal was operated by incubating the samples for 30 min at 37 °C followed by centrifugation at 13,000× g rpm for 10 min. The supernatant which contained the peptides was finally collected and analysed.