Chaps

C. neoformans H99 (serotype A) and 1841 (serotype D) cells were recovered from 10% skim milk stocks stored at -80°C and grown independently for 48 h in Sabouraud dextrose broth while shaking (80 rpm) at 30°C. Cells were harvested by centrifugation and washed twice with 250 mM sucrose. The pellet was resuspended in lysis buffer [5 mM Tris/HCl pH 7.5, 2.5 mM EDTA, 0.5X protease inhibitor cocktail (Roche, Basel, Switzerland)] additionally containing 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, Cat. No. 1479, Carl Roth, Karlsruhe, Germany) and 50 mM dithiothreitol (DTT, Cat. No. 6908, Carl Roth, Germany). The cell suspension was transferred into a mortar, frozen with liquid nitrogen and homogenized with a pestle twice. Afterwards, homogenates were centrifuged and supernatant was recovered. Protein content was estimated using Bradford reagent (Carl Roth, Karlsruhe, Germany). Proteins were precipitated with 10% trichloroacetic acid over night at -20°C and centrifuged. After removal of the supernatant, the pellet was washed three times with ice-cold acetone and air-dried. The protein pellet was dissolved in a solution containing 7 M urea, 2 M thiourea, and 4% CHAPS and protein content was estimated using Bradford reagent (Carl Roth, Germany).

10⁷ cells per each cell line were collected. Allantoic and amniotic membranes were extracted from 10-day old chicken embryos. All samples were treated as described previously^{37 (link),59 (link)}. Briefly, each cell line was subjected to sonication in the presence of detergent 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS, #10810118001, Roche), reduction in 4 M guanidine-HCl (#24115, Thermo), carboxymethylation, and trypsin (#T0303, Sigma) digestion. The digested glycoproteins were then purified by plus short HLB-Sep-Pak (#186000132, Waters Corp.). N-glycans were released by peptide N-glycosidase F (E.C. 3.5.1.52; #11365177001, Roche) digestion. Released N-glycans were permethylated using the sodium hydroxide procedure and purified by classic short C₁₈-Sep-Pak (#WAT051910, Waters). Permethylated N-glycans were eluted at the 50% acetonitrile fraction.

Immobilised pH gradient (IPG) strips and IPG buffers were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Acrylamide/piperazine-di-acrylamide (PDA) solution (37.5:1, w/v) was purchased from Biosolve Ltd. (Valkenswaard, The Netherlands), and the other reagents for the polyacrylamide gel preparation were acquired from Bio-Rad Laboratories. CHAPS was obtained from Roche Diagnostics (Mannheim, Germany), urea from AppliChem (Darmstadt, Germany), thiourea from Fluka (Buchs, Switzerland), 1,4-dithioerythritol (DTE) and EDTA from Merck (Darmstadt, Germany) and tributylphosphine (TBP) from Pierce Biotechnology (Rockford, IL, USA). All reagents were kept at 4°C.

Salmonella Typhi IMSS-1 and S. Typhi Δcas-CRISPR harboring plasmid pFMTrcleuO-50 were grown in MA medium supplemented with Ap and IPTG (50 μM) to an optical density of 0.6 at 595 nm (OD₅₉₅). Salmonella cultures (100 ml) were pelleted and washed with 1X phosphate-buffered saline (PBS). Cellular proteins were obtained by sonication at 24 kHz for 1 min in the on position and 1 min in the off position, for five cycles at 4°C using a Vibra Cell (Sonics, United States), in the presence of a protease inhibitor (Complete tablets; Roche Diagnostics GmbH, Mannheim, Germany). To further limit proteolysis, protein isolation was performed using phenol extraction (Hurkman and Tanaka, 1986 (link)). To solubilize proteins and to obtain completely denatured and reduced proteins, pellets were dried and resuspended as previously reported (Encarnación et al., 2005 (link)). Prior to electrophoresis, samples were mixed with 7 M urea, 2 M thiourea, 4% 3-[(3-choloamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS; Roche Diagnostics GmbH, Germany), 2 mM tributylphosphine, 2% ampholytes, and 60 mM dithiothreitol.