title: Dephosphorylation of intact glycoprotein to greatly improve digestion efficiency coupled with matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometric analysis.
abstract: Sialylation is essential for a variety of cellular functions. Herein, we used bovine fetuin with three potential N-linked glycosylation sites containing complex-type glycan structures, four potential O-linked glycosylation sites and six potential phosphorylation sites as a model compound to develop a highly-efficient digestion strategy for sialylated glycoproteins and efficient enrichment strategy for sialylated glycopeptides using titanium dioxide. The former according to the process of alkaline phosphatase digestion followed by tryptic digestion and then proteinase K digestion could greatly improve the enzymatic efficiency on fetuin, and the latter could obviously enhance the enrichment efficiency for multisialylated glycopeptides using phosphoric acid solution as elution buffer. The mass spectra of the enriched glycopeptides derived from fetuin reveal that several series of the ion clusters with mass difference of 291 Da correspond to the presence of multisialylated glycopeptides. In addition, the approach was applied to characterize the sialylated status of α2-macroglobulin and transferrin, respectively, from the sera of healthy subjects and sex- and age-matched patients with thyroid cancer, and their spectra indicate that the change in the amount of the glycoforms containing different number of sialic acid (SA) residues from one glycosylation site may be used to differentiate between healthy subjects and cancer cases.
authors.affiliation: Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100005, PR China.
authors.forename: Fenjie
authors.initials: F
authors.lastname: Li
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Wang
authors.forename: Yujie
authors.initials: Y
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Liu
authors.forename: Zhili
authors.initials: Z
authors.lastname: Li
mesh_terms: D000818:Animals; D002417:Cattle; D017357:Cyclotrons; D005583:Fourier Analysis; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010766:Phosphorylation / Q000502:physiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D006023:Glycoproteins
keywords: 2,5-dihydroxylbenzoic acid; ACN; ATP; CBB; CDG; DHB; DTT; Dephosphorylation; IAA; IG/I(G-H(2)O); IG/I(G-SA); Intact glycoprotein; MALDI-FTICR MS; Matrix-assisted laser desorption/ionization–Fourier transform ion cyclotron resonance mass spectrometry; PTM(s); SA; Sialylated glycopeptide; TFA; TP; TPA; Titanium dioxide; acetonitrile; alkaline phosphatase digestion followed by tryptic digestion and then proteinase K digestion; an intensity ratio of sialylated glycopeptide to its dehydrated counterpart, where G represents sialylated glycopeptide; an intensity ratio of sialylated glycopeptide to its less sialylated counterpart; carbohydrate-deficient glycoproteins; coomassie brilliant blue; dithiothreitol; iodoacetamide; matrix-assisted laser desorption/ionization–Fourier transform ion cyclotron resonance mass spectrometry; post translational modification(s); sialic acid; trifluoroacetic acid; tryptic digestion followed by proteinase K digestion; tryptic digestion followed by proteinase K digestion and then alkaline phosphatase digestion