A novel HPLC–UV–MS method for quantitative analysis of protein glycosylation

Monoclonal antibody samples derived from transgenic plants (plantibodies) may often contain significant amounts of aglycosylated variants. Because glycosylated and non-/de-glycosylated proteins exhibit different functional and pharmacokinetic properties, accurate measurement of non- and de-glycosylated glycoprotein abundances is important. Glycosylation of plant-derived glycoproteins presents specific challenges. Here we describe a novel method to accurately measure relative and absolute amounts of non-glycosylated, de-glycosylated, and total glycosylated protein using an HPLC–UV–MS methodology. Additionally, these results were compared with glycopeptide profiling by MALDI MS. Our studies demonstrated that the quantitative aspect of HPLC–UV method was superior to MALDI MS profiling, which significantly overestimated the relative amounts of aglycosylated species in the isolated glycopeptide fractions.