Trypsin lys c

A slightly modified SCAD method^{50 (link)} was used to prepare all pancreas samples. Each sample was dissolved in 150 μL of extraction buffer solution (4% SDS, 50 mM Tris buffer) and sonicated using a probe sonicator (Thermo Fisher Scientific). Protein extracts were reduced with 10 mM dithiothreitol for 30 min at room temperature and alkylated with 50 mM iodoacetamide for another 30 min in dark before quenched with dithiothreitol. Proteins were then precipitated with 80% (v/v) cold acetone (−20 °C) overnight. Samples were centrifuged at 14,000 × g for 15 min, after which supernatant containing SDS (in the extraction buffer) was discarded. Pellets were rinsed with cold acetone again and air-dried at room temperature. Eight moles of urea was added to dissolve the pellets and 50 mM Tris buffer was used to dilute the samples to a urea concentration <1 M. On-pellet digestion was performed with LysC/trypsin (Promega) in a 50:1 ratio (protein:enzyme, w/w) at 37 °C overnight. The digestion was quenched with 1% trifluoroacetic acid and samples were desalted with Sep-Pak C18 cartridges (Waters). Concentrations of peptide mixture were measured by peptide assay (Thermo Fisher Scientific). One hundred micrograms of peptide was aliquoted for each sample, dried in vacuo, and reconstituted in 0.5 M triethylammonium bicarbonate prior to DiLeu labeling.

Samples were fully resuspended in 50 µL of 10-mM DTT in 8-M urea and incubated in a thermomixer for 1 h at 37 °C. A total 50 µL of alkylation reagent mixture (97.5% acetonitile (ACN), 0.5% triethylphosphine, 2% iodoethanol) was added to each sample, and again incubated for 1 h in a thermomixer at 37 °C. After alkylation, samples were dried in a vacuum centrifuge and resuspended in 200 µL of 0.05 µg/uL Lys-C/Trypsin (Promega) dissolved in 25-mM ABC. Samples were transferred to a barocycler (50 °C, 60 cycles; 50 s at 20 kPSI and 10 s at 1 ATM), in which proteolysis was carried out. Peptides were desalted with the Pierce Peptide Desalting Spin Columns (Thermo Fisher Scientific, USA). A total of 20 μg of peptides from each sample were saved for global analysis. The remainder was used for phosphopeptide enrichment, performed with PolyMac spin tips (Tymora Analytical, West Lafayette, IN, USA), following manufacturer’s recommendations.

Protein concentration was determined using a NanoPhotometer (IMPLEN). For each sample, 100 μl were digested. Reduction occurred by 5 mM DTT in ABC buffer (50 mM ammonium bicarbonate/NH₃, pH 8.5) for 30 min at 37 °C. For alkylation, 15 mM iodoacetamide in ABC buffer were added to samples and incubated for 30 min at RT in the dark. For digestion, 10 μg protein was digested by 0.3 μg LysC/Trypsin (Promega) for 3 h at 37 °C. ABC buffer was added to a final volume of 79.2 μl and further incubated o/n at 37 °C. The reaction was stopped by adding 100% (v/v) formic acid to a final concentration of 1% (v/v) formic acid. Remaining particles were removed by centrifugation and the peptide-containing supernatant was transferred to HPLC vials and subjected to mass spectrometry (MS) measuring 1 μg digested protein.

Phytanic acid, urea, thiourea, iodoacetamide, 1,4-Dithioerythritol, Tris base, non-ionic detergent octylphenoxy poly(ethyleneoxy)ethanol (IGEPAL^® CA-630), and zwitterionic detergent Amidosulfobetaine-14 (ASB-14) were purchased from Sigma-Aldrich. LysC/Trypsin was purchased from Promega (UK). MTBSTFA + 1%TBDMS (N-Methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide, Tertbutyldimetheylchlorosilane) was purchased from Thermofisher Scientific. Heptadecanoic-17,17,17-d3 acid was purchased from Qmx laboratories Ltd. (Essex, UK). 2-hydroxyPhytanic acid was purchased from LGC (Middlesex, UK). All proteomic solvents were of UPLC grade. All lipid analysis solvents were of HPLC grade. All other reagents were of analytical grade.

A total of 300 μg of protein from each diluting pool sample, which was made up of five individual samples, was taken for protein digestion according to the FASP procedure (Wiśniewski et al., 2009 (link)). Briefly, the detergent (i.e., SDS), DTT and other low-molecular-weight components were removed using 200 μL of urea (UA) buffer (8 M UA, 150 mM Tris-HCl, pH 8.0) by repeated ultrafiltration (Microcon units, 30 kDa) facilitated by centrifugation. Then, iodoacetamide in UA buffer (final concentration 50 mM) was added to block reduced cysteine residues, followed by incubation of the samples for 20 min in darkness. The filter was washed with 100 μL of UA buffer three times and then with 100 μL of 40 mM NH₄HCO₃ twice. Finally, the protein suspension was digested with 4 μg of LysC/trypsin (Promega) in 40 μL of 40 mM NH₄HCO₃ overnight at 37°C for 18 h, followed by termination of the digestion procedure with an appropriate amount of formic acid (FA). The resulting peptides were collected by centrifugation. Then, the peptides were desalted using a C18 cartridge (Sigma-Aldrich) and resolved with OD280 peptide quantification.

An overnight culture of MRSA USA300 cells were treated with 10 × MIC of auranofin (1.25 μg/ml), linezolid (20 μg/ml) and vancomycin (10 μg/ml) for one hour at 37 °C. Bacterial cells were centrifuged and sequence grade Lys-C/Trypsin (Promega) was used to enzymatically digest samples. Samples were reduced and alkylated prior to digestion. All trypsin digestions were carried out in a Barocycler NEP2320 (PBI) at 50 °C under 20 kpsi for two hours. After digestion, samples were cleaned using MicroSpin C18 columns (Nest Group, Inc.) and the resulting pellets were re-suspended in 97% H₂O/3% ACN/0.1% FA. A small aliquot (5 μL) of sample was analyzed via nanoLC-MS/MS49 (link).

Proteins eluted from columns during the specific-binding pH 2.9 elution step were submitted to the University of California, Davis Proteomics Core and subjected to LC-MS/MS analysis. Following proteolytic digestion at a 1:25 ratio of Lys-C/trypsin (Promega) and protein, 550 μL of ammonium bicarbonate was added to dilute urea and activate trypsin overnight at 37 °C. Samples were then subject to liquid chromatography-mass spectrometry on a Exactive Plus Orbitrap Mass Spectrometer (Thermo Scientific) in conjunction with an EASY-nLC II nano UHPLC, a 75 micron, 150 mm silica column filled with Magic C18 200A 3U, and Proxeon nanospray source. Tandem mass spectra were extracted and charge state deconvoluted with IDPicker 2.0, which included a cRAP database of common laboratory contaminants and an equal number of reverse protein sequences (n = 6). Distribution of subcellular locations and biological processes of identified antigenic proteins were investigated using Uniprot database^{17 (link)}.