title: Automation of High-Throughput Mass Spectrometry-Based Plasma N-Glycome Analysis with Linkage-Specific Sialic Acid Esterification.
abstract: Glycosylation is a post-translational modification of key importance with heterogeneous structural characteristics. Previously, we have developed a robust, high-throughput MALDI-TOF-MS method for the comprehensive profiling of human plasma N-glycans. In this approach, sialic acid residues are derivatized with linkage-specificity, namely the ethylation of α2,6-linked sialic acid residues with parallel lactone formation of α2,3-linked sialic acids. In the current study, this procedure was used as a starting point for the automation of all steps on a liquid-handling robot system. This resulted in a time-efficient and fully standardized procedure with throughput times of 2.5 h for a first set of 96 samples and approximately 1 h extra for each additional sample plate. The mass analysis of the thus-obtained glycans was highly reproducible in terms of relative quantification, with improved interday repeatability as compared to that of manual processing.
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC) , PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Marco R
authors.initials: MR
authors.lastname: Bladergroen
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC) , PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Karli R
authors.initials: KR
authors.lastname: Reiding
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC) , PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Agnes L
authors.initials: AL
authors.lastname: Hipgrave Ederveen
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC) , PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Gerda C M
authors.initials: GC
authors.lastname: Vreeker
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC) , PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Florent
authors.initials: F
authors.lastname: Clerc
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC) , PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Holst
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC) , PO Box 9600, 2300 RC Leiden, The Netherlands.|Department of Rheumatology, Erasmus University Medical Center , 3000 CA Rotterdam, The Netherlands.
authors.forename: Albert
authors.initials: A
authors.lastname: Bondt
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC) , PO Box 9600, 2300 RC Leiden, The Netherlands.|Division of BioAnalytical Chemistry, VU University Amsterdam , 1081 HV Amsterdam, The Netherlands.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC) , PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Yuri E M
authors.initials: YE
authors.lastname: van der Burgt
mesh_terms: D001331:Automation / Q000379:methods*; D004951:Esterification; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D057166:High-Throughput Screening Assays / Q000379:methods*; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D006023:Glycoproteins; D019158:N-Acetylneuraminic Acid
keywords: MALDI-TOF−MS; ethyl esterification; glycan analysis; glycomics; high-throughput strategies; proteomics; robotization; sample preparation