4.3. Quantitative Proteomics Analysis

The fractionated samples were dissolved in 10 μL of solution A (2% acetonitrile in 0.1% formic acid), and 500 ng of each fraction were loaded onto a nanoLC 1D plus system (Eksigent, Walpole, MA, USA) consisting of an in-house C12 resin (4 µm Proteo 90 Å; Phenomenex Inc., Torrance, CA, USA) and a capillary column (ID 75 µm, OD 150 µm; Molex, Lisle, IL, USA). Elution was conducted using a gradient liquid chromatography method (0–25% acetonitrile for 90 min) and was analyzed with an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) in positive ion mode at the Mass Spectrometry Convergence Research Center. The m/z data collection range was set at 300–1800 m/z, and a higher-energy collisional dissociation collision mode was used for fragmentation. The quantitative mass spectrometry analyses were technically duplicated for each of the pooled peptide samples (n = 4).

All mass spectra data were input to MaxQuant 1.5.1.0 to obtain bioinformatics information, and the human proteome database (updated 12/13/2018) was downloaded from Uniprot (https://www.uniprot.org/proteomes/UP000005640, accessed on 8 March 2021). GO, InterPro, and KEGG pathways were analyzed using DAVID Functional Annotation Bioinformatics Microarray Analysis (https://david.ncifcrf.gov/, accessed on 8 March 2021). Perseus 1.6.0.7 (Max-Planck-Institute of Biochemistry, Planegg, Germany) was used for clustering protein groups depending on the protein regulation patterns. The STRING analytical tool (https://string-db.org/, accessed on 8 March 2021) was used to search specific protein networks according to regulation differences resulting from the α-AMA treatments at different concentrations.