Serum Protein Fractionation and Identification

The serum proteins in each sample were separated into 12 fractions through pH 3–10 isoelectric points, using the OFFGEL fractionator (3100 OFFGEL Low Res Kit, pH 3–10; Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. Twelve fractions were loaded onto an Eksigent nanoLC 400 system and the cHiPLC® (AB Sciex, Concord, ON, Canada) and analyzed, and the proteins were identified using a TripleTOF 5600 mass spectrometer (AB Sciex). Thereafter, for relative analysis, SWATH acquisition was conducted. In each run, 100 μg/mL of samples was injected onto an Eksigent ChromXP nanoLC trap column (350 μm i.d. × 0.5 mm, ChromXP C18 3 μm) at a flow rate of 5000 nL/min. Samples were eluted from the Eksigent ChromXP nanoLC column (75 μm i.d. × 15 cm) at a flow rate of 300 nL/min for 120 min, and mobile phase B buffer was added gradually into the column (5–90%) over a 120-min total run time. The gradient of mobile phase B buffer was (time and % B) 0 min/mobile phase B 5%, 10.5 min/40%, 105.5 min/90%, 111.5 min/90%, 112 min/5%, and 120 min/5%. Mobile phase B and A buffer, and the search parameters are as described [12 (link)].