Identification and characterization of isomeric N-glycans of human alfa-acid-glycoprotein by stable isotope labelling and ZIC-HILIC-MS in combination with exoglycosidase digestion.
In this study, a ZIC-HILIC-MS methodology for the analysis of N-glycan isomers was optimized to obtain greater detection sensitivity and thus identify more glycan structures in hAGP. In a second step, this method was combined with glycan reductive isotope labelling (GRIL) through [(12)C6]/[(13)C6]-aniline and exoglycosidase digestion to characterize the different glycan isomers. The GRIL method allows the peak areas resulting from two different labelled samples to be compared, since neither retention time shifts nor variations in the ionization of glycans between these samples are obtained. First, sialic acid linkage assignations were performed for most hAGP glycan isomers with α2-3 sialidase digestion. Bi-, tri- and tetraantennary glycan isomers with different terminal sialic acid linkages to galactose (α2-3 or α2-6) were assigned, and the potential of this technique for the structural characterization of isobaric isomers was therefore demonstrated. Furthermore, fucose linkage isomers of hAGP glycans were also characterized using this isotope-labelling approach in combination with α1-3,4 fucosidase and β1-4 galactosidase digestion. α1-3 antennary fucoses and α1-6 core fucosylation were detected in hAGP fucosylated glycans. These established methodologies can be extremely useful for patho-glycomic studies to characterize glycoproteins of biomedical interest and find novel glycan isomers that could be used as biomarkers in cancer research.
Department of Analytical Chemistry, University of Barcelona, Diagonal 645, E-08028 Barcelona, Spain.
Montserrat
M
Mancera-Arteu
Department of Analytical Chemistry, University of Barcelona, Diagonal 645, E-08028 Barcelona, Spain. Electronic address: estelagimenez@ub.edu.
Estela
E
Giménez
Department of Analytical Chemistry, University of Barcelona, Diagonal 645, E-08028 Barcelona, Spain.
José
J
Barbosa
Department of Analytical Chemistry, University of Barcelona, Diagonal 645, E-08028 Barcelona, Spain.
Victòria
V
Sanz-Nebot
D002853:Chromatography, Liquid / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis*
D006023:Glycoproteins; D011134:Polysaccharides; D006026:Glycoside Hydrolases
Aniline; Enzymes; Glycan; Isomer; Mass spectrometry; ZIC-HILIC; hAGP