title: Particle-based N-linked glycan analysis of selected proteins from biological samples using nonglycosylated binders.
abstract: 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.
authors.affiliation: Department of Environmental Science and Analytical Chemistry, Stockholm University, Stockholm, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Sroka-Bartnicka
authors.affiliation: Department of Environmental Science and Analytical Chemistry, Stockholm University, Stockholm, Sweden.
authors.forename: Isabella
authors.initials: I
authors.lastname: Karlsson
authors.affiliation: Department of Environmental Science and Analytical Chemistry, Stockholm University, Stockholm, Sweden.
authors.forename: Lorena
authors.initials: L
authors.lastname: Ndreu
authors.affiliation: Department of Environmental Science and Analytical Chemistry, Stockholm University, Stockholm, Sweden.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Quaranta
authors.affiliation: Department of Environmental Science and Analytical Chemistry, Stockholm University, Stockholm, Sweden.
authors.forename: Matthijs
authors.initials: M
authors.lastname: Pijnappel
authors.affiliation: Department of Environmental Science and Analytical Chemistry, Stockholm University, Stockholm, Sweden. Electronic address: gunnar.thorsen@aces.su.se.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Thorsén
mesh_terms: D001331:Automation; D002244:Carbon / Q000737:chemistry; D019075:Electrophoresis, Capillary; D005818:Genetic Engineering; D006031:Glycosylation; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry; D008280:Magnetics; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D016062:Porosity; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012794:Sialic Acids / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin / Q000737:chemistry*; D014168:Transferrin / Q000134:cerebrospinal fluid / Q000737:chemistry; D000515:alpha 1-Antitrypsin / Q000097:blood / Q000134:cerebrospinal fluid
chemical_list: D007128:Immunoglobulin Fragments; D011134:Polysaccharides; D011506:Proteins; D012794:Sialic Acids; D014168:Transferrin; D000515:alpha 1-Antitrypsin; D002244:Carbon; D019809:Streptavidin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: Biomarkers; Cerebrospinal fluid; Glycosylation pattern; MALDI-MS; Magnetic beads; VHH antibody fragments