title: Quantitative profiling of O-glycans by electrospray ionization- and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry after in-gel derivatization with isotope-coded 1-phenyl-3-methyl-5-pyrazolone.
abstract: The potential and benefits of isotope-coded labeling in the context of MS-based glycan profiling are evaluated focusing on the analysis of O-glycans. For this purpose, a derivatization strategy using d0/d5-1-phenyl-3-methyl-5-pyrazolone (PMP) is employed, allowing O-glycan release and derivatization to be achieved in one single step. The paper demonstrates that this release and derivatization reaction can be carried out also in-gel with only marginal loss in sensitivity compared to in-solution derivatization. Such an effective in-gel reaction allows one to extend this release/labeling method also to glycoprotein/glycoform samples pre-separated by gel-electrophoresis without the need of extracting the proteins/digested peptides from the gel. With highly O-glycosylated proteins (e.g. mucins) LODs in the range of 0.4 μg glycoprotein (100 fmol) loaded onto the electrophoresis gel can be attained, with minor glycosylated proteins (like IgAs, FVII, FIX) the LODs were in the range of 80-100 μg (250 pmol-1.5 nmol) glycoprotein loaded onto the gel. As second aspect, the potential of isotope coded labeling as internal standardization strategy for the reliable determination of quantitative glycan profiles via MALDI-MS is investigated. Towards this goal, a number of established and emerging MALDI matrices were tested for PMP-glycan quantitation, and their performance is compared with that of ESI-based measurements. The crystalline matrix 2,6-dihydroxyacetophenone (DHAP) and the ionic liquid matrix N,N-diisopropyl-ethyl-ammonium 2,4,6-trihydroxyacetophenone (DIEA-THAP) showed potential for MALDI-based quantitation of PMP-labeled O-glycans. We also provide a comprehensive overview on the performance of MS-based glycan quantitation approaches by comparing sensitivity, LOD, accuracy and repeatability data obtained with RP-HPLC-ESI-MS, stand-alone nano-ESI-MS with a spray-nozzle chip, and MALDI-MS. Finally, the suitability of the isotope-coded PMP labeling strategy for O-glycan profiling of biological important proteins is demonstrated by comparative analysis of IgA immunoglobulins and two coagulation factors.
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währinger Straße 38, A-1090 Vienna, Austria.
authors.forename: Siniša
authors.initials: S
authors.lastname: Sić
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währinger Straße 38, A-1090 Vienna, Austria.
authors.forename: Norbert M
authors.initials: NM
authors.lastname: Maier
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währinger Straße 38, A-1090 Vienna, Austria. Electronic address: Andreas.Rizzi@univie.ac.at.
authors.forename: Andreas M
authors.initials: AM
authors.lastname: Rizzi
mesh_terms: D000983:Antipyrine / Q000031:analogs & derivatives* / Q000737:chemistry; D000077553:Edaravone; D004586:Electrophoresis; D005782:Gels / Q000737:chemistry; D007553:Isotope Labeling; D011134:Polysaccharides / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005782:Gels; D011134:Polysaccharides; D012967:Sodium Dodecyl Sulfate; D000077553:Edaravone; D000983:Antipyrine
keywords: 1-Phenyl-3-methyl-5-pyrazolone; Ionic liquid matrix; Mass spectrometry; O-glycans; Stable isotope coded labeling