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Challenging the limits of detection of sialylated T homsen– F riedenreich antigens by in‐gel deglycosylation and nano‐ LC ‐ MALDI ‐ TOF ‐ MS
Author(s) -
Almeida Andreia,
Ferreira José A.,
Teixeira Filipe,
Gomes Catarina,
Cordeiro M. Natália D. S.,
Osório Hugo,
Santos Lúcio Lara,
Reis Celso A.,
Vitorino Rui,
Amado Francisco
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201300148
Subject(s) - chemistry , fetuin , glycan , glycoprotein , antigen , context (archaeology) , biochemistry , chromatography , mass spectrometry , microbiology and biotechnology , immunology , biology , paleontology
The identification of sialylated T homsen–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 .