Protein biochemistry

2.2.5.1. SDS-PAGE

Sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) serves to separate proteins by their molecular weight in an electric field. SDS is an anionic detergent that binds to the protein surface masking the charge of the protein and covers the protein with a negative charge. Thus, the protein moves in an electric field to the anode. The speed of movement depends on the size of the protein: the smaller the

2.2 Methods protein, the faster it moves through the gel. Before the protein sample was loaded onto the SDS gel the proteins were denaturated with 2x Laemmlibuffer containing β-mercaptoethanol and heated for 5 min at 95°C. SDS gel consisted of a separating gel and a collecting gel having a lower percentage of polyacrylamide and served to concentrate the sample. As a protein marker, prestained PageRuler™ was used. The electrophoresis was run with a constant voltage of 120 V for 90 min.

2.2.5.2. Western Blot Analysis

Proteins that were separated in an SDS-PAGE were transferred on a polyvinylidene difluoride (PVDF) membrane by electrophoresis and target proteins were immunologically detected. For the protein transfer, the wet blot method was used. Due to the global negative charge of the denaturated proteins in the SDS-PAGE, the proteins move to the positive pole in an electric field, thereby proteins were 1:1 horizontally transferred from the SDS gel onto the PVDF membrane. On the anode side of the cassette, one sponge and two Whatman paper wetted in transfer buffer were placed, followed by the PVDF membrane which was activated with methanol for 1 min, washed with water and incubated with transfer buffer before. On top of the membrane, the SDS gel was placed, which was shortly incubated in transfer buffer before, followed by two Whatman paper and a sponge, both wetted with transfer buffer. The cassette was closed and placed in the electrophoresis chamber, filled with transfer buffer. The transfer was run overnight at 4°C with a constant voltage of 20 V per membrane/gel-sandwich. Free binding sites were blocked on the membrane with WB blocking solution for 1 h at RT to avoid unspecific antibody reaction. Membrane was incubated in primary antibody for 90 min at RT or overnight at 4°C, depending on the antibody and washed three times with TBS-Tween 20 (TBS-T). Next, the membrane was incubated with a horseradish peroxidase-coupled secondary antibody for 60 min at RT and washed three times with TBS-T and finally once with TBS. Target protein on the membrane was detected and developed on X-ray film after incubation of the membrane with a two-component ECL solution for 3 min in the dark. The ECL solution was prepared directly prior to use by mixing both components Enhanced Luminal Reagent Plus and Oxidizing Reagent Plus in a 1:1 ratio.

2.2 Methods

2.2.5.3. Mass spectrometry

Mass spectrometry (MS) is an analytical method to determine the mass-to-charge (m/z) ratio of ionized molecules in a sample. The output is a mass spectrum that blots the m/z ratios against their intensities. Here, this method was used to analyze the protein composition of mucus produced by mucosoid cultures. The sample preparation and LC-MS/MS analysis were performed by Monika Schmid from the MPIIB core facility Mass spectrometry.

2.2.5.3.1. Sample preparation

The mucus samples were prepared according to the FASP method (Wisniewski et al.

2009) and following modifications (Rodriguez-Pineiro et al. 2013). Samples were diluted with 200 μl 6 M guanidinium hydrochloride in 0.1 M Tris/HCl pH 8.5 (GuHCl) and 20 µL of 5 protein standard mixture (10 pmol/protein) were added. Cysteines were reduced by adding 30 μl 0.1 M DTT (Sigma-Aldrich D0632) at 60°C for 20 min and samples were transferred into Microcon YM-30 kDa centrifugal filter units. After each step filter units were centrifuged at 10 000 x rpm for 10 to 15 min at RT. 200 µL 6 M GuHCl were added and samples were alkylated with 100 μL 0,05 M iodoacetamide (IAA, Sigma-Aldrich I6125) in 6 M GuHCl at RT for 20 min in the dark. After washing twice with 100 μL 6 M GuHCl and twice with 100 μL 50 mM ammonium bicarbonate (ABC) the proteins were digested with 0.2 μg sequencing-grade modified trypsin (Promega V5111) in 40 μL 50 mM ABC overnight at 37°C with agitation (Thermomixer 500 rpm). In a new collection tube, samples were washed twice with 40 µL 50 mM ABC followed by acidification with TFA to 0.5% (vol/vol). ABC was evaporated by adding 50 µL 60 % acetonitrile/0.1 % TFA and dried at 45°C (Microconcetrator 5301, Eppendorf).

2.2.5.3.2. LC-MS/MS Analysis

The peptides were analyzed using a QExactive Plus mass spectrometer (Thermo Fisher Scientific) coupled online to a Dionex UltiMate 3000 RSLC nano system (Thermo Fisher Scientific). After solubilization in 90 μl 2 % (v/v) acetonitrile/0.1 % TFA, 5 μL of each sample was loaded on a C18 PepMap 100 trap column (300 μm x 5mm; 5 μm

2.2 Methods particle size 100 Å pore size; Thermo Fisher Scientific) at a flow rate of 20 μL/min 2 % (v/v) acetonitrile/water containing 0.1% TFA for pre-concentration. Separation was performed using an Acclaim C18 PepMap RSLC column (75μm x 250 mm; 2 μm particle size 100 Å pore size; Thermo Fisher Scientific) at a flow rate of 300 nL/min.

HPLC solvent A was 0.1 % (v/v) FA and peptides were eluted from the column using HPLC solvent B 80 % (v/v) acetonitrile/0.1 % FA starting from 3 %, increasing to 40 % in 85 min, and to 98 % in 5 min. The peptides were analyzed in data-dependent acquisition mode that alternated between one MS scan and 10 MS/MS scans for the most abundant precursor ions. MS scans were acquired over a mass range of m/z 350–

1600 and resolution was set to 70 000. Peptides were fragmented using HCD at 27 % normalized collision energy and measured in the orbitrap at a resolution of 17 500.

2.2.5.3.3. Protein identification

Proteins were identified and quantified using the MaxQuant software (Version 1.626) (Cox and Mann 2008; Tyanova, Temu, and Cox 2016) searching against the SwissProt human sequence database (released 2018_11, 20 412 entries). Searches were performed using the following parameters: max. missed cleavages 2; variable modifications Oxidation (M); Acetyl (Protein N-term); pyro-Glu (Gln) and carbamidomethylation of cysteines as fixed modification. The FDR was set to 0.01 for proteins, peptides, and modified sites.

MaxQuant data analysis results are provided in the electronic appendix EA.2.

2.2.5.4. Cytokine Array (Quantibody®)

Quantibody® (Raybiotech) cytokine arrays are commercially available multiplex ELISA systems based on a glass slide antibody array that allow simultaneous measurement of multiple cytokines. For this assay, the conditioned media of (1) GSC co-cultured mucosoids infected with H. pylori for three days, (2) non-infected counterpart and (3) mucosoids cultured without stromal cells infected and (4) non-infected were used. 160 µL conditioned medium of each condition was collected, centrifuged (1 000 x g, 2 min) and 150 µL were diluted 1:1 with sample diluent of the Quantibody® array kit. The protocol provided by the supplier was followed. Briefly,

2.2 Methods

glass slide was equilibrated in the plastic bag for 30 min at RT followed by air desiccation for two hours at RT. For blocking 100 µl Sample Diluent were added into each well and incubated for 30 min at RT (gentle rocking). The cytokine standard mix dilution series (STD1-7: 200µl Sample Diluent + 100µL Std, negative control) was prepared. After removing the Sample Diluent with a vacuum pump from the wells, 100 µL of standard cytokines or samples were added and incubated for 2 h. Array was washed 5x 5 min with 150 µL 1x Wash Buffer I and 2x 5 min with 150 µL 1x Wash Buffer II both at RT (gentle rocking). The primary antibody was added (80 µL/well) and incubated overnight at 4°C. After washing (5x 5 min 150 µL Wash buffer I, 2x 5 min 150 µL Wash buffer II) the secondary Cy3-antibody was applied (80 µL/well) and incubated overnight at 4°C. Array was washed 5x 5 min with 150 µl 1x Wash Buffer I at RT. The device was disassembled; slide was placed in the slide washer filled with 1x Wash Buffer I and washed with gentle shaking at RT for 15 min followed by washing with 1x Wash Buffer II for 5min. The liquid was decanted and vessel was centrifuged 3 min, 200 x g to dry the slide. The array was imaged with Agilent high-resolution laser microarray scanner (5 µm high-resolution). Afterward, data was extracted by Dr. Hans Mollenkopf and analyzed with the supplier’s analyzing software.