Advertisement

 

 

Absolute quantitation of disease protein biomarkers in a single LC-MS acquisition using apolipoprotein F as an example.

Absolute quantitation of disease protein biomarkers in a single LC-MS acquisition using apolipoprotein F as an example.
Author Information (click to view)

Kumar A, Gangadharan B, Cobbold J, Thursz M, Zitzmann N,


Kumar A, Gangadharan B, Cobbold J, Thursz M, Zitzmann N, (click to view)

Kumar A, Gangadharan B, Cobbold J, Thursz M, Zitzmann N,

Advertisement

Scientific reports 2017 09 217(1) 12072 doi 10.1038/s41598-017-12229-2
Abstract

LC-MS and immunoassay can detect protein biomarkers. Immunoassays are more commonly used but can potentially be outperformed by LC-MS. These techniques have limitations including the necessity to generate separate calibration curves for each biomarker. We present a rapid mass spectrometry-based assay utilising a universal calibration curve. For the first time we analyse clinical samples using the HeavyPeptide IGNIS kit which establishes a 6-point calibration curve and determines the biomarker concentration in a single LC-MS acquisition. IGNIS was tested using apolipoprotein F (APO-F), a potential biomarker for non-alcoholic fatty liver disease (NAFLD). Human serum and IGNIS prime peptides were digested and the IGNIS assay was used to quantify APO-F in clinical samples. Digestion of IGNIS prime peptides was optimised using trypsin and SMART Digest™. IGNIS was 9 times faster than the conventional LC-MS method for determining the concentration of APO-F in serum. APO-F decreased across NAFLD stages. Inter/intra-day variation and stability post sample preparation for one of the peptides was ≤13% coefficient of variation (CV). SMART Digest™ enabled complete digestion in 30 minutes compared to 24 hours using in-solution trypsin digestion. We have optimised the IGNIS kit to quantify APO-F as a NAFLD biomarker in serum using a single LC-MS acquisition.

Submit a Comment

Your email address will not be published. Required fields are marked *

sixteen − five =

[ HIDE/SHOW ]