The Fine Art of Destruction: A Guide to In-Depth Glycoproteomic Analyses-Exploiting the Diagnostic Potential of Fragment Ions.
Proteomics
; 18(24): e1800282, 2018 12.
Article
en En
| MEDLINE
| ID: mdl-30427586
ABSTRACT
The unambiguous mass spectrometric identification and characterization of glycopeptides is crucial to elucidate the micro- and macroheterogeneity of glycoproteins. Here, combining lower and stepped collisional energy fragmentation for the in-depth and site-specific analysis of N- and O-glycopeptides is proposed. Using a set of four representative and biopharmaceutically relevant glycoproteins (IgG, fibrinogen, lactotransferrin, and ribonuclease B), the benefits and limitations of the developed workflow are highlighted and a state-of-the-art blueprint for conducting high-quality in-depth N- and O-glycoproteomic analyses is provided. Further, a modified and improved version of cotton hydrophilic interaction liquid chromatography-based solid phase extraction for glycopeptide enrichment is described. For the unambiguous identification of N-glycopeptides, the use of a conserved yet, rarely employed-fragmentation signature [Mpeptide +H+0,2 X GlcNAc]+ is proposed. It is shown for the first time that this fragmentation signature can consistently be found across all N-glycopeptides, but not on O-glycopeptides. Moreover, the use of the relative abundance of oxonium ions to retrieve glycan structure information, for example, differentiation of hybrid- and high-mannose-type N-glycans or differentiation between antenna GlcNAc and bisecting GlcNAc, is systematically and comprehensively evaluated. The findings may increase confidence and comprehensiveness in manual and software-assisted glycoproteomics.
Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Polisacáridos
/
Ribonucleasas
/
Fibrinógeno
/
Inmunoglobulina G
/
Glicopéptidos
/
Glicoproteínas
/
Lactoferrina
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Proteomics
Asunto de la revista:
BIOQUIMICA
Año:
2018
Tipo del documento:
Article
País de afiliación:
Alemania