Top 12 abundant protein depletion spin columns

To identify the proteins present in the 15.1 kDa and 15.9 kDa peaks, ten µL of a single patient’s serum were applied to Top 12 Abundant Protein Depletion Spin Columns (Thermo Scientific Pierce) according to the manufacturer’s protocol. Five hundred µL of the eluate were concentrated on a 3 kDa molecular weight cut-off column (Amicon) in 25 mM Tris-HCl, pH 8.0. Twenty µL of depleted serum were separated by 1D SDS-PAGE on an 18% Tris-glycine denaturing gel (TGX, Bio-Rad) at 150 V for 70 min and compared against SeeBlue Plus 2 pre-stained protein standard (Life Technologies). The gel was rinsed 5 times with deionized water and stained overnight in See Band staining solution (Gene Bio-Application Ltd.) A band corresponding to 15 to 16 kDa was excised and an in-gel trypsin digest
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Samples were analysed by nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS), using a nanoAcquity UPLC system (Waters MS Technologies, Manchester, UK). Peptide identification was performed using ProteinLynx Global SERVER v3.1 (Waters).