Trifluoroacetic acid

Membrane eluted proteins were reduced with 10 mM dithiothreitol for 30 min at 56°C and alkylated with 20 mM iodoacetamide for 30 min at RT in the dark. The samples were precipitated overnight at -80°C using ethanol 99.5%. Precipitated samples were centrifuged at 14000 x g for 15 min at 4°C, the pellet was dissolved in 25mM ammonium bicarbonate and digested to peptides using trypsin (Sigma-Aldrich) for 18 h at 37°C. The reaction was stopped using 5 μl of 10% trifluoroacetic acid (Sigma-Aldrich), desalted using Ultra Microspin C18 columns (Nest Group, Southborough, MA) and dried using a SpeedVac. The dried peptides were dissolved in 25 µl of 2% acetonitrile, 0.1% trifluoroacetic acid (Sigma-Aldrich) and the concentration was measured using a DS-11 FX spectrophotometer (DENovix Inc, Wilmington, DE).

Liquid chromatography was performed to measure the rate of chemical degradation. Samples from the stability study were analysed using an Agilent 1260 infinity system equipped with an Agilent 1260 II infinity DAD UV detector, an Agilent 1260 infinity pump, an Agilent 1260 autosampler (Agilent Technologies, Santa Clara, CA, USA), and a BioResolve reversed phase column (Polyphenyl, 450 Å, 2.7 µ, 3 × 150 mm, Waters, Milford, MA, USA). A gradient was run with mobile phase A (0.08% formic acid (VWR) and 0.02% trifluoroacetic acid (Sigma-Aldrich, St. Louis, MO, USA) in Milli-Q water, and mobile phase B (0.08% formic acid (VRW) and 0.02% trifluoroacetic acid (Sigma-Aldrich) in acetonitrile (Sigma-Aldrich). Mobile phase B increased 10–31% in the 0–15 min period, 31–40% in the 15–45 min period, and 40–95% in the 45–50 min period. All samples were analysed at a concentration of 0.33 mg/mL in 10% acetonitrile (Sigma-Aldrich, St. Louis, MO, USA) and Milli-Q water. The flow was set to 1 mL/min, the injection volume to 5 µL, and the column oven temperature to 60 °C. Signal data were collected at two wavelengths, 215 and 280 nm. Samples were kept at 4 °C in the autosampler during the run. The LC chromatograms were analysed using Open LAB CDS software (Agilent Technologies, Santa Clara, CA, USA).

HLA class I molecules were isolated using standard immunoaffinity purification as described previously,^{53 (link),54 (link)}. In brief, cell pellets were lysed in 10 mM CHAPS/PBS (AppliChem/Lonza) containing 1× protease inhibitor (Complete; Roche). Mouse MHC molecules were removed using a 1 h immunoaffinity purification with H-2K-specific monoclonal antibody 20-8-4S, covalently linked to CNBr-activated sepharose (GE Healthcare). Remaining HLA molecules were purified overnight using the pan-HLA class I-specific monoclonal antibody W6/32 or a mix of the pan-HLA class II-specific monoclonal antibody Tü39 and the HLA-DR-specific monoclonal antibody L243, covalently linked to CNBr-activated Sepharose. MHC–peptide complexes were eluted by repeated addition of 0.2% trifluoroacetic acid (Merck). Elution fractions E1–E4 were pooled, and free MHC ligands were isolated by ultrafiltration using centrifugal filter units (Amicon; Merck Millipore). MHC ligands were extracted and desalted from the filtrate using ZipTip C18 pipette tips (Merck Millipore). Extracted peptides were eluted in 35 µl of acetonitrile (Merck)/0.1% trifluoroacetic acid, centrifuged to complete dryness and resuspended in 25 µl of 1% acetonitrile/0.05% trifluoroacetic acid. Samples were stored at −20 °C until analysis by LC–MS/MS.

HLA class I molecules were isolated using standard immunoaffinity purification as described previously,^{53 (link),54 (link)}. In brief, cell pellets were lysed in 10 mM CHAPS/PBS (AppliChem/Lonza) containing 1× protease inhibitor (Complete; Roche). Mouse MHC molecules were removed using a 1 h immunoaffinity purification with H-2K-specific monoclonal antibody 20-8-4S, covalently linked to CNBr-activated sepharose (GE Healthcare). Remaining HLA molecules were purified overnight using the pan-HLA class I-specific monoclonal antibody W6/32 or a mix of the pan-HLA class II-specific monoclonal antibody Tü39 and the HLA-DR-specific monoclonal antibody L243, covalently linked to CNBr-activated Sepharose. MHC–peptide complexes were eluted by repeated addition of 0.2% trifluoroacetic acid (Merck). Elution fractions E1–E4 were pooled, and free MHC ligands were isolated by ultrafiltration using centrifugal filter units (Amicon; Merck Millipore). MHC ligands were extracted and desalted from the filtrate using ZipTip C18 pipette tips (Merck Millipore). Extracted peptides were eluted in 35 µl of acetonitrile (Merck)/0.1% trifluoroacetic acid, centrifuged to complete dryness and resuspended in 25 µl of 1% acetonitrile/0.05% trifluoroacetic acid. Samples were stored at −20 °C until analysis by LC–MS/MS.

The stationary phase of liquid chromatography (LC) was a 5 µm C18 column from Thermo Fisher Scientific (15 cm × 4.6 mm). The mobile phase A was comprised of acetonitrile (Merck Millipore) and 0.1% of trifluoroacetic acid (Merck Millipore), and the mobile phase B was comprised of deionized water and 0.1% of trifluoroacetic acid. The gradient of mobile phases was set with a flow rate of 0.5 mL/min. The temperature of the autosampler was set at 4 °C, and the injection volume was 5 µL. The total run time of the LC was 20 min. Ionization and detection of the analyte were performed on a triple quadrupole mass spectrometer (API-2000, AB Sciex Pte. Ltd.), operating in positive ion mode. Quantitation was done using multiple reaction monitoring (MRM) mode to monitor protonated precursor → ion transition product of m/z 103.1 → 75.0 for DCS of this study. The concentration range of DCS for the calculation curve was from 0 to 0.3 mg/mL. The standard curve was built as y = 2E+07x + 43665, and the value of r² was 0.9986.

Dichloromethane (DCM), acetonitrile (ACN),
and HPLC grade water were supplied by Pharmco. 2-Chlorotrityl chloride
resin, dimethylformamide (DMF), bromoacetic acid, N,N-diisopropylethylamine (DIEA), diisopropylcarbodiimide
(DIC), trifluoroacetic acid (TFA), triisopropylsilane (TIPS), PyBOP,
benzylamine, 1-napthylmethylamine, and benzhydrylamine were provided
by Millipore Sigma. N-Boc-1,3-diaminopropane was
provided by Matrix Scientific.