2.6.1. Protein Extraction, Gel Electrophoresis Separation and Immunoblot Analysis

Biscuits were first ground coarsely using a mortar and pestle, and then milled mechanically for 30 s (three times, 10 s) in a blender (IKA Werke GmbH, Staufen im Breisgau, Germany). The biscuits' powder was weighted and 1 g of sample was combined with 20 mL of extraction buffer (50 mM Sodium carbonate/bicarbonate pH 9.6), then it was left shaking for 2 h at 60 ◦C. The extract was sonicated for 5 min, 4 s pulse and 4 s pause, 60% amplitude (VibraCell Ultrasonic Liquid Processor, Sonics and Materials Inc, Newtown, CT, USA) and centrifuged for 10 min at 3000 g at room temperature (r.t.). In this case, 10 mL of supernatant were filtered through first an Acrodisc 25 mm syringe filter by a 1.2 µm Versapor membrane (Pall Corporation, Ann Arbor, MI, USA) and then by a 0.45 µm acetate cellulose membranes (Minisart Syringe Filter, Sartorius Stedim Biotech GMbh, Goettingen, Germany). Four mL of the filtrate were loaded on centrifugation filter devices [Amicon Ultra, 3000 Da molecular weight cut-off (MWCO); Merck Millipore, Billerica, MA, USA)], and concentrated 10 times. Protein concentration of the filtered and concentrate sample was determined with the colorimetric Bicinchoninic Acid Assay (Thermo Fisher Scientific). One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of samples was performed loading 40 µg of extracted proteins onto 12% Bis-Tris Criterion XT precast gels (11 cm, Bio-Rad Laboratories, Hercules, CA, USA); separation was per-

formed in running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS) on a Criterion Cell apparatus (Bio-Rad) at 120 V. *Nutrients* **2021**, *13*, x FOR PEER REVIEW 5 of 14

**Figure 1.** Sketch of the proteomic experimental approaches. (1) Immunoblot experiments determine patient serum binding to egg and milk allergens; (2) LC-ESI-MS/MS analyses, following two different workflows and different Triple Quadrupole MS platforms (2.A and 2.B), quantify egg and milk allergens in biscuits. **Figure 1.** Sketch of the proteomic experimental approaches. (1) Immunoblot experiments determine patient serum binding to egg and milk allergens; (2) LC-ESI-MS/MS analyses, following two different workflows and different Triple Quadrupole MS platforms (2.A and 2.B), quantify egg and milk allergens in biscuits.

2.6.1. Protein Extraction, Gel Electrophoresis Separation and Immunoblot Analysis Biscuits were first ground coarsely using a mortar and pestle, and then milled mechanically for 30 s (three times, 10 s) in a blender (IKA Werke GmbH, Staufen im Breisgau, Germany). The biscuits' powder was weighted and 1 g of sample was combined with 20 mL of extraction buffer (50 mM Sodium carbonate/bicarbonate pH 9.6), then it was left shaking for 2 h at 60 °C. The extract was sonicated for 5 min, 4 s pulse and 4 s pause, 60% amplitude (VibraCell Ultrasonic Liquid Processor, Sonics and Materials Inc, Newtown, CT, USA) and centrifuged for 10 min at 3000 g at room temperature (r.t.). In this case, 10 mL of supernatant were filtered through first an Acrodisc 25 mm syringe filter by a 1.2 µm Versapor membrane (Pall Corporation, Ann Arbor, MI, USA) and then by a 0.45 µm acetate cellulose membranes (Minisart Syringe Filter, Sartorius Stedim Biotech GMbh, Goettingen, Germany). Four mL of the filtrate were loaded on centrifugation filter devices Immunoblotting was performed by transferring proteins from gel to polyvinylidene fluoride membrane (Bio-Rad) at 350 mA for 2 h, in a cold room at 4 ◦C, on Criterion™ Blotter with wire electrodes (Bio-Rad) in presence of transfer buffer (25 mM Tris, 192 mM Glycine, 20% Methanol). The membranes were left in contact with a solution of Pierce™ Protein-Free T20 Blocking Buffer (Thermo Fisher Scientific) and then incubated overnight at 4 ◦C in serum of each study subject, diluted 1:10 in Blocking Buffer. After several rinses with phosphate buffer (200 mg/L KCl, 200 mg/L KH2PO4, 8000 mg/L NaCl, 1150 mg/L Na2HPO4)- 0.1%Tween), the membrane was incubated for two hours at r.t. with a secondary antihuman IgE antibody conjugated to the enzyme alkaline phosphatase (Mouse Anti-Human IgE Fc-AP, clone B3102E8; Southern Biotech, Birmingham, AL, USA) diluted 1:500 in Blocking Buffer. The membrane was washed again in PBS-T and the immunoreactive signals were detected by a colorimetric reaction in presence of 5-bromo-4-chloroindolyl phos-

[Amicon Ultra, 3000 Da molecular weight cut-off (MWCO); Merck Millipore, Billerica,

phate/Nitroblue Tetrazolium and 0.1 M Tris, 0.5 mM MgCl2 (pH 9.5) (Alkaline Phosphate Conjugate Substrate Kit, Bio-Rad). Stained membranes were scanned with a ChemiDocTM XRS<sup>+</sup> Molecular Imager (Bio-Rad). SDS-PAGE protein molecular weight standards, to monitor the run, and proteins from biscuits containing egg and milk ingredients ("BuoniCosì", Galbusera), as positive control, were loaded. Analyses were performed on two different portions of Magretti biscuits previously administered to enrolled patients. (Table S2). Unspecific signals due to the secondary antibody were identified performing immunoblots without patients' sera or with pooled sera of non-allergic pediatric patients. A Relative Volume Quantity analysis was performed by a densitometric analysis of blots using Image Lab Software (version 6, Bio-rad). Lane of positive control (biscuits containing egg and milk) was set as reference, and for all other lanes of the blot numerical values relative to the reference were calculated as ratio of the background-adjusted lane volume divided by the background-adjusted reference volume.
