Development of a highly sensitive liquid chromatography/tandem mass spectrometry method to quantify total and free levels of a target protein, interferon-gamma-inducible protein-10, at picomolar levels in human serum.
Rapid Commun Mass Spectrom
; 28(13): 1535-43, 2014 Jul 15.
Article
em En
| MEDLINE
| ID: mdl-24861605
ABSTRACT
RATIONALE Liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays are increasingly being used for absolute quantitation of proteins due to high specificity and low cost. However, the major challenge for the LC/MS method is insufficient sensitivity. This paper details the strategies developed to maximize the sensitivity from aspects of chromatography, mass spectrometry, and sample preparation to achieve a highly sensitive LC/MS method. METHODS:
The method is based on the LC/MS/MS measurement of a surrogate peptide generated from trypsin digestion of interferon-gamma-inducible protein-10 (IP-10). The sample preparation strategy involved selectively extracting IP-10 and removing high-abundance serum proteins through acidified protein precipitation (PPT). It was revealed in this work that these high-abundance serum proteins, if not separated from the protein of interest, could cause significant ionization saturation and high background noise in selected reaction monitoring (SRM), leading to a 100-fold higher lower limit of quantification (LLOQ).RESULTS:
Our method demonstrated that the acidified PPT could be optimized to selectively extract the protein of interest with full recovery of 97% to 103%, while the high-abundance serum proteins could be effectively removed with minimal matrix effect of 90% to 93%. For the first time, a highly sensitive LC/MS method with a LLOQ of 31.62 pM for the quantitation of IP-10 has been achieved, which is a 100-fold improvement over the generic method.CONCLUSIONS:
The described method offers excellent sensitivity with advantages of being antibody reagent independent and leads to significant cost and time savings. It has been successfully employed to determine both total and free IP-10 levels in human serum samples. This method development strategy may also be applied to other small proteins.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cromatografia Líquida
/
Espectrometria de Massas em Tandem
/
Quimiocina CXCL10
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Rapid Commun Mass Spectrom
Ano de publicação:
2014
Tipo de documento:
Article
País de afiliação:
Estados Unidos