Porcine trypsin

Twenty-five micrograms of total protein diluted in 45 µL 50 mM NH₄HCO₃ were reduced using dithioerythritol (DTE) at a final concentration of 5 mM for 30 min at 37°C. Cysteins were blocked using iodoacetamide (final concentration 15 mM) at room temperature for 30 min in the dark. Proteins were first digested for 4 h at 37°C using 250 ng LysC (FUJIFILM Wako Pure Chemicals, Osaka, Japan) and then overnight at 37°C with 500 ng porcine trypsin (Promega). Five micrograms of peptides diluted in 0.1% formic acid (FA) were injected into an Ultimate 3000 (Thermo Scientific) nano-chromatography system and transferred to a trap column (PepMap 100 C18, 100 µm × 2 cm, 5 µM particles, Thermo Scientific) at a flow rate of 20 µL/min of solvent A (0.1% FA). Peptides were separated at 250 nL/min (column: PepMap RSLC C18, 75 µm × 50 cm, 2 µm particles, Thermo Scientific) with a 160 min gradient from 5% solvent A to 25% solvent B (0.1% FA in acetonitrile) and a subsequent 10 min gradient from 25 to 40% solvent B. For MS acquisition a Q Exactive HFX (Thermo Scientific) instrument and a top 15 data-dependent method was used. Ion spray voltage was set to 2.2 kV and MS spectra were acquired at a resolution 60,000 (mass-range: 350–1600). The resolution for MS/MS was set to 15,000.

The samples were separately resuspended in PBS extraction buffer containing 8 M of urea, 10 mM of dithiothreitol (DTT), benzonase, and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were determined by the Qubit fluorimetric detection method. The proteins were diluted in water to a concentration of 1 of nmol/μL, and reduced by the addition of dithiothreitol (DTT) to a final concentration of 10 mM and incubation for 30 min. The proteins were alkylated prior to digestion by the addition of iodoacetamide (IAA) to a final concentration of 40 mM, and incubation for 30 min in the dark at room temperature. To quench the reaction, DTT was added to a final concentration of 5 mM. Porcine trypsin (1:50, w/w) (Promega, Fitchburg, WI, USA) was added, and the mixture was incubated overnight at room temperature. The resulting peptide mixtures were desalted with hydrophilic–lipophilic-balanced solid phase extraction (Waters) and peptides eluted with 1 mL of 70% (v/v) acetonitrile and 1% (v/v) trifluoroacetic acid (TFA). Two biological replicates and two technical replicates were acquired for each condition.

All reagents used in this study are analytical or HPLC/MS grade. PhosStop, cOmplete EDTA-free, and pepstatin were acquired from Roche Applied Science (Germany). Nuclease Mix, Destreak Reagent, IPG Buffer pH 4–7, and 7 cm Immobiline^® Drystrips pH 4–7 were obtained from GE Healthcare (UK). The phosphoprotein stain Pro-Q^® Diamond (PQD) and the PeppermintStick^™ phosphoprotein molecular weight markers were purchased from Life Technologies (CA, USA). The whole proteome Coomassie Brilliant Blue stain, BlueSafe (CBB) and the protein molecular weight markers NZYColour Protein Marker II were acquired from NZYTech (Portugal). Porcine trypsin was acquired from Promega Corporation (WI, USA). Seeds from Zea mays inbred line B73 used in this study were amplified in our greenhouse over the years. Original seeds were kindly provided by Dr. Christoph Peterhansel (University of Hannover, Germany).

Excised SDS-PAGE gel-bands of rLifA^C1480A were incubated in 50 mM ammonium bicarbonate containing porcine trypsin (Promega) in a trypsin:lymphostatin ratio of ~1:30, overnight at 32 °C. Peptides were identified by MALDI mass spectroscopy on an ultrafleXtreme™ mass spectrometer (Bruker) using an α-cyano-4-hydroxycinnamic acid matrix. A peptide mass map was generated from spectral data using Compass DataAnalysis 4.4 software (Bruker). Peptide masses were searched against the National Center for Biotechnology Information database of non-identical protein sequences (NCBInr) with the MASCOT search engine,⁵⁰ (link) mass tolerance of 10 ppm (Matrix Science). Peptide masses were also compared to the sequence of full-length LifA from EPEC E2348/69 (Protein Prospector software).12 (link), 13 (link), 51 The instrument was calibrated prior to each data acquisition and an internal calibration performed on the digest products of porcine trypsin.

One milligram of porcine tubulin (lyophilized powder >99% pure; Cytoskeleton, Denver, CO, catalog number T240) was dissolved in 3.6 ml of 20 mm Tris/Cl buffer, pH 8.5, containing 0.01% sodium azide to give 0.275 mg of tubulin/ml. This was mixed with 2.5 μl of 0.3 m chlorpyrifos oxon (Chem Service, West Chester, PA, catalog number MET-11459B) in ethanol to make 1.5 mm CPO. The reaction was incubated for 8 days at 24 °C. Tubulin remained in solution. Excess CPO was separated from 50 μl of protein by size exclusion chromatography on a Prob Quant G50 micro-column (GE Healthcare catalog number 28-9034-08) while simultaneously changing the buffer to 20 mm ammonium bicarbonate, pH 8. CPO-tubulin in 20 mm ammonium bicarbonate was digested with 0.5 μl of porcine trypsin (0.4 μg/μl, sequencing grade modified; Promega, Madison, WI, catalog number V511C) at 37 °C overnight. The digest was dried and resuspended in 20 μl of 0.1% formic acid in preparation for MS. The digests were stored at −20 °C. Unlabeled, control porcine tubulin was treated similarly and analyzed by LC-MS/MS for diethylphosphate labeling and cross-linked peptides.