The procedure for 2-dimensional gel electrophoresis (2-DE) has been described in previous studies.24 (link),28 (link) Briefly, 40 µL plasma sample from each subject was depleted of albumin and immunoglobulin G (IgG) using ProteoPrep (Sigma-Aldrich Co, St Louis, MO, USA), followed by protein concentration measurement using 2D-Quant Kit (GE Healthcare, Little Chalfont, UK). Samples were further desalted with PD-10 columns (GE Healthcare) and lyophilized prior to use in the first dimension. Lyophilized proteins were resolved in 2-DE urea sample buffer according to Gorg et al,31 (link) and 100 µg of total protein from each subject was applied in the first dimension and further run in second dimension using Ettan™ DALTsix Electrophoresis unit (Amersham, Pharmacia Biotech, Uppsala, Sweden).24 (link),28 (link)
Separated proteins were fluorescently stained with SYPRO Ruby® (Bio-Rad Laboratories, Hercules, CA, USA), and gels were visualized using a charged coupled device (CCD) camera (VersaDoc™ Imaging system 4000 MP; Bio-Rad Laboratories). 2-DE protein patterns were analyzed and quantified using software PDQuest Advanced version 8.0.1 (Bio-Rad Laboratories). Protein spots of interest were excised from the gel and, after tryptic digestion, were analyzed by mass spectrometry using ultrafleXtreme™ matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF; Bruker Daltronik GmbH, Bremen, Germany). Database search was performed in ProteinProspector MS-Fit version 5.14.4 including Swiss-Prot database version 2015.3.5 as described in previous studies.28 (link),32 (link)
Separated proteins were fluorescently stained with SYPRO Ruby® (Bio-Rad Laboratories, Hercules, CA, USA), and gels were visualized using a charged coupled device (CCD) camera (VersaDoc™ Imaging system 4000 MP; Bio-Rad Laboratories). 2-DE protein patterns were analyzed and quantified using software PDQuest Advanced version 8.0.1 (Bio-Rad Laboratories). Protein spots of interest were excised from the gel and, after tryptic digestion, were analyzed by mass spectrometry using ultrafleXtreme™ matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF; Bruker Daltronik GmbH, Bremen, Germany). Database search was performed in ProteinProspector MS-Fit version 5.14.4 including Swiss-Prot database version 2015.3.5 as described in previous studies.28 (link),32 (link)
