N-Glycosylation profiling of intact target proteins by high-resolution mass spectrometry (MS) and glycan analysis using ion mobility-MS/MS.
Analyst
; 145(5): 1737-1748, 2020 Mar 02.
Article
em En
| MEDLINE
| ID: mdl-31913371
ABSTRACT
Glycosylation influences the structure and functionality of glycoproteins, and is regulated by genetic and environmental factors. The types and abundance of glycans on glycoproteins can vary due to diseases such as cancer, inflammation, autoimmune and neurodegenerative disorders. Due to the crucial role glycans play in modulating protein function, glycosylation analysis could lead to the discovery of novel biomarkers and is of prime importance in controlling the quality of glycoprotein biopharmaceuticals. Here, we present a method for the identification and quantification of glycoforms directly on intact proteins, after immunoaffinity purification from biological fluids. The method was validated and applied to serum transferrin and the biopharmaceutical trastuzumab. The accuracy of the method, expressed as the relative error (RE), ranged from 2.1 (at high concentrations) to 7.9% (at low concentrations), and intra- and inter-day precision, expressed as relative standard deviation (RSD), was 3.2 and 8.2%, respectively. The sensitivity and linearity of the method were suitable for serum analysis and the LOQ was calculated to be 3.1 and 4.4 µg mL-1 for transferrin (TFN) and trastuzumab (TRA), respectively. Its application to transferrin from five healthy human serum samples yielded concentrations between 1.61 and 3.17 mg mL-1, which are in agreement with blood reference levels. In parallel, the structure of the identified glycans was determined by ion mobility spectrometry coupled with tandem mass spectrometry. No chromatographic separation was required and sample preparation was performed in a semi-automatic manner, facilitating the handling of up to 12 samples at a time. This method should be useful for clinical laboratories and for the quality control of large batches of biopharmaceuticals.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polissacarídeos
/
Transferrina
/
Espectrometria de Massas em Tandem
/
Trastuzumab
/
Espectrometria de Mobilidade Iônica
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article