Isolation of N-linked glycopeptides from plasma.

Proteomic analysis of blood plasma can potentially identify biomarkers that are useful for classifying the physiological or pathological status of an individual and for monitoring the effects of therapy. However, the complexity of the plasma proteome, the large number of peptides generated per protein due to dynamic protein post-translational modifications of each protein, and sequence variations among individuals pose great challenges to current proteomic technologies. To overcome these challenges, we have recently developed a method for the high-throughput analysis of glycoproteins using solid-phase extraction of N-linked glycopeptides (SPEG). Here we describe a procedure for plasma analysis using SPEG in which each step of SPEG was optimized. The performance of optimization was monitored using mouse plasma spiked with radioactive-labeled human plasma glycoproteins. Our data show that a standard procedure for plasma proteome analysis can be developed using the SPEG technique, mainly due to the relatively constant protein content in plasma.