Xcalibur software v2

Proteomic analyses were performed as previously described29 (link) in biological replicates (n = 4) and technical duplicates (n = 2). Cell/tumor lysates and secreted sample preparations (10 μg protein) were lysed in SDS sample buffer(2% (w/v), 125 mM Tris-HCl, pH 6.8, 12.5% (v/v) glycerol, 0.02% (w/v) bromphenol blue), electrophoresed by short-range SDS-PAGE (10 × 6 mm), and visualized by Imperial Protein Stain (Invitrogen). Individual samples were excised, destained, reduced, alkylated, and trypsinized as described26 (link). A nanoflow Ultra Performance Liquid Chromatography (UPLC) instrument (Ultimate 3000 RSLCnano, Thermo Fisher Scientific) was coupled on-line to a Linear Trap Quadropole (LTQ) Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) with a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were loaded (Acclaim PepMap100, 5 mm × 300 μm i.d., μ-Precolumn packed with 5 μm C18 beads, Thermo Fisher Scientific) and separated (Acquity UPLC M-Class Peptide BEH130, C18, 1.7 μm, 75 μm × 250 mm, Waters). Data was acquired using Xcalibur software v2.1 (Thermo Fisher Scientific).

Both the unfractionated and fractionated peptide samples were separated by RPLC using a DIONEX UltiMateTM 3000LC chromatography system and MSMS analysis performed on an LTQ Orbitrap Velos using Xcalibur software v2.1 (Thermo Scientific, UK). Peptides (10 μl = ~500 ng) were injected onto the analytical column (Dionex Acclaim® PepMap RSLC C18, 2 μm, 100 Å, 75 μm i.d. × 15 cm, nanoViper.), which was maintained at 35°C and at a nanoflow rate of 0.3 μlmin^-1. Peptides were separated over linear chromatographic gradients composed of buffer A (2.5% acetonitrile [ACN]: 0.1% formic acid [FA]) and buffer B (90% ACN: 0.1% FA). Two gradients, 60 (3-50% buffer B in 40 min) and 180 min (3-60% buffer B in 140 min), were employed for analysis. Full scan MS spectra were acquired over the m/z range of 350-2000 in positive polarity mode by the Orbitrap at a resolution of 30,000. A data-dependent Top20 collision induced dissociation (CID) data acquisition method was used. The ion-trap operated with CID MSMS on the 20 most intense ions (above the minimum MS signal threshold of 500 counts).

MOMP was purified under non-denaturing conditions and analysed by Nanoflow LC-MS/MS FT/ICR following in-gel protein digestion as previously described [34 (link)]. Briefly, the gel pieces were destained by washing three times in 25 mM NH₄HCO₃ in 50% CH₃CN and once in 25 mM NH₄HCO₃ in 50% CH₃OH. Gel pieces were dried in a vacuum centrifuge and incubated with digestion buffer (50 mM NH₄HCO₃, 10 ng µl⁻¹ trypsin) at 37°C overnight. Peptides were extracted in 50% CH₃CN/1% CH₃COOH and the supernatant was evaporated to dryness in a vacuum centrifuge. Prior to MS analysis, the peptides were reconstituted in 0.2% HCOOH. After digestion, samples were subjected to LC-MS/MS analysis using a hybrid linear ion trap-FTICR mass spectrometer operated in data-dependent mode, automatically switching to MS/MS mode. MS-spectra were acquired in the FTICR, while MS/MS-spectra were acquired in the LTQ-trap. For each FTICR scan, the six most intense, double-, triple- and quadruple-charged, ions were sequentially fragmented in the linear trap by collision-induced dissociation. All tandem mass spectra were searched by Mascot (Matrix Science, London, UK) against the SwissProt database (release 54.0). Glycan structure and position were determined by the manual interpretation of the CID fragmentation spectra, using the Xcalibur software v. 2.0.7 (Thermo Scientific).

Amount of Fel d 1 and Asp f 1 was quantified using a Fel d 1- or Asp f 1-specific enzyme-linked immunosorbent assay (ELISA) kit (INDOOR Biotechnologies) following the manufacturer’s instructions. Prior to quantification of Fel d 1 by ELISA, CFE and Fel d 1 solution after PC-ions or sham treatment were 1,250-fold concentrated by lyophilization. Fel d 1 that was collected after PC-ions or sham treatment was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition and proteins were visualized by silver staining following the manufacturer’s instructions (Cosmo Bio, Tokyo, Japan). Proteins separated on SDS-PAGE were identified by liquid chromatography (LC) connected to a mass spectrometer (MS, LTQ Orbitrap XL, Thermo Fisher Scientific, Rockford, IL, USA) using a HPLC column (Develosil ODS-HG-5, Nomura Chemical, Aichi, Japan) and Xcalibur software v2.0.7 (Thermo Fisher Scientific), as described elsewhere [16 (link)].

The SCX fractions were purified through C-18 OMIX Pipette Tips (Agilent Technologies, Germany), to remove impurities and salts, and eluted in 5 μL of 65% MS buffer B (90% ACN, 0.1% FA, 10% water, 0.02% Trifluoroacetc Acid (TFA)) and 35% MS buffer A (5% ACN, 0.1% FA, 95% water, 0.02% TFA). The samples were diluted to 85 μL in MS buffer A, and injected into a nano-LC system (Proxeon Biosystems, FL) connected online to an LTQ-Orbitrap (Thermo Fisher Scientific, USA). A 90 minute linear gradient reversed phase chromatography using MS buffer A and MS buffer B, was performed at a flow rate of 400 nL/min to resolve petides on a C-18 column (75 uM × 5 cm; Proxeon Biosystems, FL). The MS parameters were: 300 Da minimum mass, 4000 Da maximum mass, automatic precursor charge selection, 10 minimum peaks per MS/MS scan; and 1 minimum scan per group. XCalibur software v.2.0.7 (Thermo Fisher Scientific, USA) was used for data acquisition.

MPM were identified by comparing retention times with those of available standards. A semi-targeted screening method was established to identify phase II metabolites (glucuronides and sulfates) when reference standards were not available. The molecular formula of each compound was generated with an accurate mass and error of 5 ppm using the Xcalibur software v2.0.7 (Thermo Fisher Scientific, San Jose, CA, USA). Data acquisition techniques, including Fourier transform mass spectrometry (FTMS) mode (scan range from m/z 100–1000) in combination with product ion scan experiments (MS2) (Orbitrap resolution from 15,000 to 30,000 FWHM), were performed to obtain information about the m/z of precursor and fragment ions, retention time, and isotope pattern. Finally, analytes were confirmed by comparing MS/MS spectra with fragments found in the literature and The Human Metabolome Database 4.0 [25 (link)].

To identify the PCs present in each type of beer, all beer fractions obtained in Section 4.2.1 were analyzed with LC-MS using an Orbitrap Exploris Mass Spectrometer, in a negative and positive mode, for accurate mass measurements. The column, the solvents, and the method (gradient, detection wavelength, and flow rate) used for the LC-MS analysis were the same as those referred to previously for the HPLC analysis (Section High-Performance Liquid Chromatography (HPLC) Analysis). The mass spectra range was from m/z 100 to 2000, and the data analysis was achieved using Xcalibur software v2.0.7 (Thermo Fisher Scientific, Waltham, MA, USA).