title: Evaluation of protein N-glycosylation in 2-DE: Erythropoietin as a study case.
abstract: The structure, function, and physico-chemical properties of many proteins are determined by PTM, being glycosylation the most complex. This study describes how a combination of typical proteomics methods (2-DE) combines with glycomics strategies (HPLC, MALDI-TOF-MS, exoglycosidases sequencing) to yield comprehensive data about single spot-microheterogeneity, providing meaningful information for the detection of disease markers, pharmaceutical industry, antidoping control, etc. Recombinant erythropoietin and its hyperglycosylated analogue darbepoetin-alpha were chosen as showcases because of their relevance in these fields and the analytical challenge they represent. The combined approach yielded good results in terms of sample complexity (mixture glycoforms), reproducibility, sensitivity ( approximately 25 pmoles of glycoprotein/spot), and identification of the underlying protein. Heterogeneity was present in all spots but with a clear tendency; spots proximal to the anode contained the highest amount of tetra-antennary tetra-sialylated glycans, whereas the opposite occurred for spots proximal to the cathode with the majority of the structures being undersialylated. Spot microheterogeneity proved a consequence of the multiple glycosylation sites as they contributed directly to the number of possibilities to account for a discrete charge in a single spot. The interest of this combined glycoproteomics method resides in the efficiency for detecting and quantifying subtle dissimilarities originated from altered ratios of identical glycans including N-acetyl-lactosamine repeats, acetylation, or antigenic epitopes, that do not significantly contribute to the electrophoretic mobility, but affect the glycan microheterogeneity and the potential underlying related functionality.
authors.affiliation: Pharmacology Research Unit - Bio-analysis group, IMIM, PRBB, Barcelona, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Llop
authors.forename: Ricardo Gutiérrez
authors.initials: RG
authors.lastname: Gallego
authors.forename: Viviana
authors.initials: V
authors.lastname: Belalcazar
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Gerwig
authors.forename: Johannis P
authors.initials: JP
authors.lastname: Kamerling
authors.forename: Jordi
authors.initials: J
authors.lastname: Segura
authors.forename: José A
authors.initials: JA
authors.lastname: Pascual
mesh_terms: D000118:Acetylglucosaminidase / Q000032:analysis / Q000737:chemistry; D000215:Acylation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D000068256:Darbepoetin alfa; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004921:Erythropoietin / Q000031:analogs & derivatives / Q000032:analysis* / Q000737:chemistry; D054794:Glycomics / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000032:analysis / Q000737:chemistry; D025942:Oligosaccharides, Branched-Chain / Q000032:analysis / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D001616:beta-Galactosidase / Q000032:analysis / Q000737:chemistry
chemical_list: D006023:Glycoproteins; D025942:Oligosaccharides, Branched-Chain; D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin; D000068256:Darbepoetin alfa; D009439:Neuraminidase; D001616:beta-Galactosidase; D000118:Acetylglucosaminidase