Affi prep

Isolation of N-linked glycopeptides (N-glycopeptides) from plasma was performed as previously described^{21 (link), 27 (link), 32 (link), 33 (link)}. Briefly, starting with aliquots of 25 μl individual or pooled plasma, samples were diluted 10 fold with ammonium bicarbonate (100 mM), then denatured with 50% TFE (2, 2, 2-Trifluoroethanol, J.T. Baker, Philipsburg, NJ) and digested with mass spectrometry-grade trypsin (Promega, Madison, WI). The glycopeptides were desalted using C18 cartridges (Waters, Milford, MA, USA) and were then oxidized with 10 mM NaIO₄. The resulting oxidized glycopeptides were coupled to hydrazide resin (Affi-prep, Bio-Rad, Hercules, CA) by incubation in coupling buffer (100 mM sodium acetate and 1.5 M sodium chloride, pH 4.5) overnight at room temperature with bottom-over-head rotation. The unbound non-glycosylated peptides were removed by several washes of sodium chloride (1.5 M), 80% ACN and ammonium bicarbonate (100 mM), respectively. N-glycopeptides were finally eluted from the resin by addition of PNGase F in 50 mM ammonium bicarbonate, pH 7.5 and incubation overnight at 37°C. A MCX (mixed-mode cation exchange) desalting step using an Oasis μElusion plate (Waters, Milford, MA) was performed before LC-MS analysis.