Speed vacuum concentrator

In-gel digestion was determined according to the method of Losuwannarak et al. [41 (link)]. To reduce the disulfide bonds of total protein isolated from rat brains, dithiothreitol (10 mM) in ammonium bicarbonate (10 mM) was added. Reformation of disulfide bonds in proteins was blocked by alkylation with iodoacetamide (30 mM) in ammonium bicarbonate (10 mM). Protein samples were digested with sequencing-grade porcine trypsin (ratio of 1:20; Promega, Mannheim, Germany) and incubated overnight at 37 °C. Tryptic peptides were dried using a speed vacuum concentrator (Thermo Fisher Scientific, Waltham, MA, USA) and resuspended in 0.1% formic acid for nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS).

Culture supernatant generated from the shake flask was purified by Protein-A affinity chromatography and further used for glycan and charge variant analysis. An Acquity UPLC HILIC-BEH Glycan chromatography column (1.7 μm, 2.1 mm × 150 mm) was used for glycan analysis in Acquity UPLC H class (Waters, United States) system. The eluent mobile phase-A (50 mM ammonium formate, pH 4.5) and mobile phase-B (Acetonitrile) were used to run the HPLC for glycan analysis. The peaks obtained were detected using Fluorescence detector (λ ex = 330 nm and λ e = 420 nm). For analysis, Protein A purified samples (20 μl) were denatured and subsequently deglycosylated using PNGaseF enzyme (NEB, United States) and incubated at 37°C for 3 h. The deglycosylated samples were dried by speed vacuum concentrator (Thermo Fisher Scientific, United States) and then released N-Linked Glycans were labeled with 2-AB reagent (Waters, United States) by incubating in a heating block at 65°C for around 2.5 h. The dried sample was reconstituted in 50 μl of Mobile Phase A and B buffers and injected 10 μl on HILIC-UPLC BEH Glycan column for the separation of N-linked glycans. The peaks obtained were analyzed using Empower 3.0 software.

The ethanol extract was dried by spinning in a speed vacuum concentrator (Thermo Fisher Scientific Inc., Waltham, MA) and washed with a mixture of 1 mL of chloroform/methanol (2:1) and 200 μL of water. The organic layer was transferred to a new tube and dried. The extract was then re-suspended in a chloroform/methanol (2:1) solution at the concentration of 5 mg/mL and separated by thin layer chromatography on an aluminum-backed Silica gel plate (TLC Silica gel 60, Merck KGaA, Darmstadt, Germany) with a development solution (chloroform/methanol/water [90:10:1]). The Silica gel plates were then stained using a polymolybdate solution.

The Acquity UPLC H class (Waters, USA) system was used for antibody Glycan analysis for the culture supernatant generated from the shake flask experiment. An Acquity UPLC BEH Glycan chromatography column (1.7um, 2.1mm x 150mm) was used with a flow rate 0.5 to 0.25 mL/min, column temperature 60°C, and sample storage temperature 2–8°C. The eluent mobile phase-A (50mM ammonium formate, pH 4.5) and mobile phase-B (Acetonitrile) were used to run the HPLC for glycan analysis. Detection was done using Fluorescence detector (λ ex = 330nm & λ em = 420nm). For sample preparation, Protein A purified sample 20μl each were denatured followed by deglycosylated using PNGaseF enzyme (NEB,USA) and incubated at 37°C for 3 hours. The deglycosylated samples were dried by speed vacuum concentrator (Thermo Fisher Scientific, USA) and then released N-Linked Glycans were labeled with 2-AB reagent (Waters, USA) by incubating in a heating block at 65°C for around 2.5 hours. Free 2-AB dye was removed by acetone clean-up followed by evaporation by dry heat. The dried sample was reconstituted with 20μL of Mobile Phase A and 30μL of Mobile Phase B buffers and injected 10uL on HILIC-UPLC BEH Glycan column for the separation of N- linked glycans. The peaks were identified by comparing with control samples and integrated for area % determination using Waters Empower 3.0 software.

Proteins were extracted from both A. flavus-infected and uninfected samples of T₃ generation HIGS lines (F-4 & F-5) and the WT control ICGV 91114, as previously described [41 (link)]. The protein concentration was determined and normalized by loading an equal amount of each sample in the polyacrylamide gel electrophoresis (PAGE). The proteins were then subjected to reduction, alkylation, and overnight trypsin digestion using sequencing-grade porcine trypsin (Promega, Madison, WI, USA). Peptides from each fraction were extracted separately in 60% (v/v) acetonitrile (ACN) containing 0.1% (v/v) formic acid, sonicated in ice for 30 min, followed by concentrating in a speed vacuum concentrator (Thermo Scientific, Waltham, MA, USA) and purification using C18 spin columns (Thermo Scientific, Waltham, MA, USA). These samples were either immediately used for proteomics analysis or stored at −80 °C for further use.