Plasma PCSK9 measurement by liquid chromatography-Tandem mass spectrometry and comparison with conventional ELISA.
J Chromatogr B Analyt Technol Biomed Life Sci
; 1044-1045: 24-29, 2017 Feb 15.
Article
em En
| MEDLINE
| ID: mdl-28064066
ABSTRACT
The combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and trypsin proteolysis is an effective tool for accurate quantitation of multiple proteins in a single run. However, expensive samples pre-treatment as immunoenrichment are often required to analyze low abundant proteins. Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a circulating regulator of low-density lipoprotein metabolism, was studied as an example of a low abundant plasma protein. We investigated post-proteolysis solid-phase extraction (SPE) as an alternative strategy to improve its detection. After optimization of pretreatment, including denaturation, reduction, alkylation, tryptic digestion and selective SPE concentration, 91±7% of PCSK9 was recovered from human plasma samples and coefficients of variation were less than 13.2% with a lower limit of quantification of 37.5ng/ml. This LC-MS/MS method was compared with standard enzyme-linked immunosorbent assay in 30 human plasma samples with a broad range of PCSK9 concentrations. Both methods were significantly correlated (r=0.936, p<0.001) with less than 7% of the values out of the 95% confidence interval and similar concentrations were measured using either LC-MS/MS or ELISA methods (514.2±217.2 vs. 504.2±231.0ng/ml, respectively- p=NS). This method involving SPE is an effective measurement tool for low abundant plasma protein analysis that could be easily included in multiplexed assays.
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MEDLINE
Assunto principal:
Ensaio de Imunoadsorção Enzimática
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Cromatografia Líquida
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Espectrometria de Massas em Tandem
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Pró-Proteína Convertase 9
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article