title: Challenging the limits of detection of sialylated Thomsen-Friedenreich antigens by in-gel deglycosylation and nano-LC-MALDI-TOF-MS.
abstract: The identification of sialylated Thomsen-Friedenreich antigens in proteins poses much interest in the context of cancer research. MALDI-TOF-MS is a powerful technique for this purpose; still it shows considerable low sensitivity for sialylated molecules due to in-source and metastable decomposition. Herein, we report a target-driven strategy to identify these antigens in minute amounts of glycoproteins isolated in polyacrylamide gels. The glycans were recovered from gel spots by reductive β-elimination, permethylated and analyzed by nano-LC-MALDI-TOF-MS. A computational algorithm was developed to filter spectral noise and enhance/isolate the signals of interest. Sialylated antigens were identified in minute amounts of fetuin (0.1 μg) and plasminogen (1.0 μg) by this approach.MS assignments were further validated by enzymatic methods. This methodology allowed a fivefold decrease in the current LOD of fetuin sialylated O-glycans by MALDI-TOF-MS.
authors.affiliation: Department of Chemistry, Mass Spectrometry Centre, QOPNA, University of Aveiro, Campus de Santiago, Aveiro, Portugal.
authors.forename: Andreia
authors.initials: A
authors.lastname: Almeida
authors.forename: José A
authors.initials: JA
authors.lastname: Ferreira
authors.forename: Filipe
authors.initials: F
authors.lastname: Teixeira
authors.forename: Catarina
authors.initials: C
authors.lastname: Gomes
authors.forename: M Natália D S
authors.initials: MN
authors.lastname: Cordeiro
authors.forename: Hugo
authors.initials: H
authors.lastname: Osório
authors.forename: Lúcio Lara
authors.initials: LL
authors.lastname: Santos
authors.forename: Celso A
authors.initials: CA
authors.lastname: Reis
authors.forename: Rui
authors.initials: R
authors.lastname: Vitorino
authors.forename: Francisco
authors.initials: F
authors.lastname: Amado
mesh_terms: D000465:Algorithms; D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000097:blood* / Q000737:chemistry; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D060748:Fetuins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D057230:Limit of Detection; D008745:Methylation; D009077:Mucins / Q000097:blood / Q000737:chemistry; D036103:Nanotechnology; D012794:Sialic Acids / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; D060748:Fetuins; D009077:Mucins; D012794:Sialic Acids; C016467:Thomsen-Friedenreich antigen
keywords: Nano-LC-MALDI-TOF-MS; Permethylation; Sialic acids; Simple mucin-type antigens; Thomsen-Friedenreich antigens