Personal genome machine (pgm)

Fibrinogen was dissolved in deimination buffer (80 mM Tris-HCl, pH 7.6, 8 mM CaCl2, 4 mM DTT) to a concentration of 1 mg/mL. Citrullinated fibrinogen was digested by trypsin at 37 °C overnight, and we mixed 10 μL of the digested solution with 30 μL of trifluoroacetic acid (Sigma-Aldrich). Next, we added 10 μL of 50 mM PGM (Sigma-Aldrich) and left the mixture to act at 37 °C for 3 h. The reaction mixture was dried in a SpeedVac concentrator and resuspended in 10 μL of distilled water. Subsequently, we purified the modified peptides for mass spectrometric analysis using Ziptip (Millipore, Watford, UK).

Calix[4]resorcinol VC10 was synthesized by the reaction of tetra-(bromomethyl)calix[4]resorcinol with monomethylviologen using the procedure described in the literature [26 (link)]. Haloperidol, Hal (Alfa Aesar), D₂O (99.9 atom% D, Carl Roth GmbH), mucin from porcine stomach, and PGM (Type III, bound sialic acid 0.5–1.5%, partially purified powder, Sigma-Aldrich (St. Louis, MO, USA)) were purchased and used without further purification. Deionized, ultrapure water with a resistivity of 18.2 MΩ was generated using a Direct Q-5 UV water purification system from Millipore SAS (Molsheim, France) and was used throughout this work.

P proteins were pretreated with decreasing concentrations of mAbs for 1 h at 37 °C and added to wells coated with pig gastric mucin type III (PGM, Sigma-Aldrich, USA) and incubated for 1 h at 37 °C. Followed by washing steps, mouse anti-GII.17 P domain hyperimmune serum diluted 1:2000 in PBS was added and incubated for 1 h at 37 °C. After washing with PBST, HRP-conjugated goat anti-mouse IgG was added for incubating for 30 min at 37 °C. Blocking rates were calculated by comparing binding levels of in the presence and absence of mAbs. Sera from free P proteins-immunized animals were used as negative controls.

50 mg of PGM (Sigma Aldrich) was mixed with 5 mL gold salt (2.5 mM of AuHCl₄, STREM). The mixture was stirred until the salt was fully dissolved. Next, 2.5 mL of glycine buffer (200 mM pH = 3, 6, and 9) were added to the solution, and the mixture was further stirred for an additional hour for pH stabilization. Fine-tuning of the buffer pH was done by titration of hydrochloric acid or sodium hydroxyl. After the solution was stabilized, it was purged with nitrogen gas and stirred at room temperature in the dark for 72 h. Alternatively, the solution was heated to 70 °C to obtain similar results in a shorter time (< 30 h) for the kinetics and growth study. Next, the sample's residual gold ions were removed by a dialysis (14 kDa, Sigma Aldrich), resulting in AuMu exhibiting a molar weight of larger than 12,400 kDa.

Ten milligram of porcine gastric mucin (PGM; Sigma Aldrich) was suspended in 1 mL milliQ and UV‐killed four times at 100.000 μJoule in a Stratalinker (Stratagene). For fucosidase treatment of mucins with recombinant enzyme, 50 mU of α‐(1–2,3,4,6)‐L‐Fucosidase (FucHS; Megazyme;) was added to the 10 mg/ml mucin solution and incubated for 18 hr at 37°C. To harvest secreted fucosidases of B. fragillis, a 6 ml anaerobic overnight culture of B. fragillis grown in Mega medium at 37°C was concentrated 60 times using 10 K MWCO spinfilters (ThermoFisher Scientific). As a negative control, FNJ (CAS 99212–30‐3, Carbosynth) was added to the secreted fraction with a final concentration of 100 μM to inhibit fucosidase activity. The concentrated secreted fraction was added to the 10 mg/ml mucin aliquot in the ratio 1:8 and incubated for 18 h at 37°C. The 10 mg/ml mucin aliquots were diluted in DMEM medium to obtain a final concentration of 1 mg/ml treated or untreated mucins. Campylobacter jejuni 108 was prepared for growth assays as described above and added to the DMEM medium, containing treated or untreated mucins, at an OD₆₀₀ of 0.01. Growth of C. jejuni was quantified by counting colony‐forming units (CFU) on saponin plates incubated at 37°C under microaerophilic conditions for 24 hr. Statistical analysis was performed using a Student t test.