Mriga Dutt, Marisa N. Duong, Scott Bringans, Renée S. Richards, Richard Lipscombe & Michelle M. Hill
Abstract
Aberrant protein glycosylation is a characteristic of diverse diseases which has been explored as biomarkers. To support translational serum glycoprotein biomarker discovery and validation, we developed a semi-automated workflow using individual lectin-coupled magnetic beads to conduct lectin pulldowns in a high-throughput format. Lectins are naturally occurring glycoprotein binding proteins widely used in glycobiology. While lectin-affinity isolation has been coupled to mass spectrometry-based proteomics, the lectin magnetic bead array (LeMBA) platform allows technically robust screening and measurement of clinical cohorts. This chapter describes detailed lectin-magnetic bead coupling, serum denaturation, lectin magnetic bead pulldown, and on-bead trypsin digest. The resulting tryptic peptides are analyzed by untargeted or targeted liquid chromatography-mass spectrometry (LC-MS), for biomarker discovery, or qualification/validation, respectively. LeMBA-MS generates quantitative data for glycoforms based on lectin affinity of the glycoprotein coupled with MS measurement of one or more prototypic peptides and has successfully been used to discover and validate novel serum cancer glycoprotein biomarkers. This chapter includes detailed protocols for two different liquid handlers, along with recommendations on quality control measures for clinical biomarker studies.
