2.4. Proteomics and Glycoproteomic Analysis Using nLC-Orbitrap LC-MS/MS

For the proteomics and glycoproteomics analyses, membrane fractions were separately prepared from the tissue samples following the procedure above. The membrane proteins were reconstituted with 60 µL of 8 M urea and sonicated for 20 minutes for denaturation. Two microliters (2 µL) dithiothreitol (DTT, 550 mM in 50 mM NH₄HCO₃) was added to the samples, and the mixture was incubated for 50 min at 55 °C. The free cysteine was alkylated with 4 µL of iodoacetamide (450 mM) for 20 minutes in the dark at ambient temperature. The reaction was quenched by the addition of 420 µL buffer (50 mM NH₄HCO₃). Trypsin (10 µL, 0.1 mg/mL) was then added to the mixture, and tryptic digestion was performed at 37 °C for 18 h. For proteomic analysis, tryptic peptides were purified using C-18 SPE cartridges, eluted with 80% ACN and 0.1% TFA, dried in vacuo and stored at −20 °C before LC-MS/MS analysis. For site-specific glycoproteomic analysis, glycopeptides were enriched from tryptic digest preparation using HILIC solid-phase extraction, eluted with H₂O containing 0.1% TFA, dried in vacuo and stored at −20 °C until LC-MS/MS analysis. Purified peptides and glycopeptides were quantified using a Pierce BCA assay kit following the manufacturer’s instructions (ThermoFisher, Waltham, MA, USA), and adjusted to concentrations of 0.5 µg/µL and 0.1 µg/µL, respectively, before injection in LC-MS/MS.

Tryptic peptides and glycopeptides samples were analyzed in an UltiMate™ WPS-3000RS nanoLC system coupled with an Orbitrap Fusion Lumos MS system (ThermoFisher Scientific). One (1) microliter of each sample was injected, and the analytes were separated using an Acclaim™ PepMap™ 100C18 LC Column (75 µm × 150 mm, particle size: 2 µm; ThermoFisher Scientific) at a flow rate of 300 nL/min. Water containing 0.08% formic acid and 80% acetonitrile containing 0.1% formic acid were used as solvents A and B, respectively. MS spectra were collected with a mass range of m/z 700–2000 for MS1 and m/z of ≥120 for MS2, at a rate of 1.5 s per spectrum in the positive ionization mode. The filtered precursor ions in each MS spectrum were subjected to fragmentation through 30% higher-energy C-trap dissociation (HCD) using nitrogen gas as a carrier.

Mass spectrometry data were analyzed using the Byos workflow (Protein Metrics, Cupertino, CA, USA). For qualitative analysis using Byonic (Protein Metrics), proteins were identified against the human proteome database [37] using a precursor mass tolerance of 20 ppm and fragment mass tolerance of 10 ppm. Data analysis parameters used included C-terminal cleavage by trypsin (K and R cleavage sites) with at most two missed cleavages and the following peptide modifications: carbamidomethyl at the cysteine, oxidation at the methionine, deamidation at asparagine and glutamine, acetylation at the protein N-terminal, glutamine to pyro-glutamate at the N-terminal, and glutamate to pyro-glutamate at the N-terminal. Protein IDs were filtered at 1% FDR. For glycopeptide/glycoprotein identification, an additional search was performed in Byonic using an in-house N-glycan database. Each protein was quantified using Byologic (Protein Metrics) by quantifying the XIC (extracted ion chromatogram) area sum of the top 3 most abundant peptides per protein. The XICs were then normalized to total ion count before statistical analysis. On the other hand, glycoform quantification was normalized to each protein’s glycosite to yield the percentage glycan distribution of a particular glycoform.

Statistical analysis (multiple t-tests with an FDR correction of 5%) was conducted using GraphPad Prism (version 9.3.1 for Windows, GraphPad Software, San Diego, CA, USA, www.graphpad.com, accessed on 5 May 2022) to identify significantly over- and under-expressed proteins, glycoproteins, and pathways. Pathway enrichment analysis were annotated using PantherGO [38] and then plotted as heatmaps in GraphPad Prism.