Particle-based N-linked glycan analysis of selected proteins from biological samples using nonglycosylated binders

[Display omitted] •A method capable of multiplexed analyses of N-glycan profiles of specific proteins has been developed.•Streptavidin-coated magnetic beads are used in combination with nonglycosylated VHH antibody fragments.•Enzymatic deglycosylation and desialylation are performed on the beads using PNGase F and neuraminidase.•A simple clean-up step using porous graphitic carbon spin tips is performed before MALDI-TOF-MS analysis.•The glycosylation patterns of alpha-1-antitrypsin and transferrin in human serum and cerebrospinal fluid has been measured. Glycosylation is one of the most common and important post-translational modifications, influencing both the chemical and the biological properties of proteins. Studying the glycosylation of the entire protein population of a sample can be challenging because variations in the concentrations of certain proteins can enhance or obscure changes in glycosylation. Furthermore, alterations in the glycosylation pattern of individual proteins, exhibiting larger variability in disease states, have been suggested as biomarkers for different types of cancer, as well as inflammatory and neurodegenerative diseases. In this paper, we present a rapid and efficient method for glycosylation analysis of individual proteins focusing on changes in the degree of fucosylation or other alterations to the core structure of the glycans, such as the presence of bisecting N-acetylglucosamines and a modified degree of branching. Streptavidin-coated magnetic beads are used in combination with genetically engineered immunoaffinity binders, called VHH antibody fragments. A major advantage of the VHHs is that they are nonglycosylated; thus, enzymatic release of glycans from the targeted protein can be performed directly on the beads. After deglycosylation, the glycans are analyzed by MALDI-TOF-MS. The developed method was evaluated concerning its specificity, and thereafter implemented for studying the glycosylation pattern of two different proteins, alpha-1-antitrypsin and transferrin, in human serum and cerebrospinal fluid. To our knowledge, this is the first example of a protein array-type experiment that employs bead-based immunoaffinity purification in combination with mass spectrometry analysis for fast and efficient glycan analysis of individual proteins in biological fluid.