Sample Preparation Scale-Up for Deep N-glycomic Analysis of Human Serum by Capillary Electrophoresis and CE-ESI-MS.
Mol Cell Proteomics
; 18(12): 2524-2531, 2019 12.
Article
em En
| MEDLINE
| ID: mdl-31628258
ABSTRACT
We introduce an efficient sample preparation workflow to facilitate deep N-glycomics analysis of the human serum by capillary electrophoresis with laser induced fluorescence (CE-LIF) detection and to accommodate the higher sample concentration requirement of electrospray ionization mass spectrometry connected to capillary electrophoresis (CE-ESI-MS). A novel, temperature gradient denaturing protocol was applied on amine functionalized magnetic bead partitioned glycoproteins to circumvent the otherwise prevalent precipitation issue. During this process, the free sugar content of the serum was significantly decreased as well, accommodating enhanced PNGase F mediated release of the N-linked carbohydrates. The liberated oligosaccharides were tagged with aminopyrene-trisulfonate, utilizing a modified evaporative labeling protocol. Processing the samples with this new workflow enabled deep CE-LIF analysis of the human serum N-glycome and provided the appropriate amount of material for CE-ESI-MS analysis in negative ionization mode.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Glicoproteínas
/
Eletroforese Capilar
/
Espectrometria de Massas por Ionização por Electrospray
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Glicômica
Limite:
Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article