4.2. Mass Spectrometry Analyses

For the determination of the intact mass of Bet v 2, 1 μg of protein either reduced or untreated (disulfide-bridged) protein was desalted with C18 ZipTips (EMD Millipore, Billerica, MA, USA). Using direct infusion at a flow rate of 1 μL/min, mass determination was done with a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) at maximum resolution. Background peaks of the solvent were used for lock mass calibration, resulting in an accuracy of less than 2 ppm. For deconvolution of the raw data, the Xtract modul of Xcalibur (Thermo Fisher Scientific) was used. To determine intramolecular disulfide bridges, Bet v 2 were digested with the ProteoExtract All-in-One Trypsin Digestion Kit (EMD Millipore, Billerica, MA, USA) omitting the reduction/alkylation step. After the digest, samples were desalted using C18 ZipTips. Resulting peptides were separated by reverse-phase nano-HPLC (Dionex Ultimate 3000, Thermo Fisher Scientific, column: PepSwift Monolithic Nano Column, 100 μm × 25 cm, Dionex). The column was developed with an acetonitrile gradient (Solvent A: 0.1% (v/v) FA/0.01% (v/v) TFA; solvent B: 0.1% (v/v) FA/0.01% (v/v) TFA/90% (v/v) ACN; 5–45% B in 60 min) at a flow rate of 1 μL/min at 55 °C). The HPLC was directly coupled via nano electrospray to the Q Exactive mass spectrometer (Thermo Fisher Scientific). After deconvolution with Xtract, the mass list of uncharged signals was exported to xQuest [23] and cross-linked peptides were matched to the Bet v 2 sequence with xBobcat. For sequence determination, tryptic peptides were HPLC-separated as described above and fragmented in the mass spectrometer with a top 12 method using a normalized fragmentation energy of 27%. Sequence analyses were performed with Peaks Studio 8 (Bioinformatics Solutions, Waterloo, ON, Canada).