Analysis of phospholipase A2 glycosylation patterns from venom of individual bees by capillary electrophoresis/electrospray ionization mass spectrometry using an ion trap mass spectrometer

A method based on tryptic digestion, ultrafiltration and capillary electrophoresis/mass spectrometry (CE/MS) has been developed for the analysis of the glycosylation pattern in the phospholipase A2 (PLA) of individual honeybees. Without reducing the disulfide bonds, PLA was digested with trypsin and filtered with a 3 kDa molecular weight (MW) cut-off membrane. With this procedure, the glycopeptides could be isolated from the nonglycosylated peptides. After tryptic digestion and ultrafiltration, the disulfide bonds were reduced before analysis by CE. To reduce the adsorption, CE separation was performed on successive multiple ionic-polymer (SMIL) polybrene (PB) coated capillary columns. The SMIL-PB columns allowed partial separation of the glycopeptides and eight glycopeptides were identified by on-line coupling of CE with electrospray ionization (ESI) mass spectrometry. The analysis of phospholipase A2 from the venom of individual bees indicated that the variation and relative abundances of different glycopeptides were similar between the younger and the older bees.