METHODS AND KITS FOR DETERMINING PROTEIN-BOUND BIOMARKERS

摘要

The invention relates to the field of diagnostic methods, in particular to detection of biological molecules using mass spectroscopy (MS). Provided is an automated high-throughput method for quantitating a low molecular weight protein-bound biomarker directly in an isolated biological sample, comprising the steps of:
(a) automated prepurification of the sample using an effective amount of an acid protease under conditions that allow for digestion of one or more binding proteins; (b) applying said protease-digested sample onto an on-line SPE column to capture at least part of said biomarker, followed by sequential washing of the solid phase; (c) eluting a fraction comprising said biomarker directly onto a liquid chromatography (LC) column comprising an apolar stationary phase and subjecting it to LC-MS or LC-MS-MS measurements to determine the amount of at least one biomarker; and (d) quantitating the biomarker(s). Also provided are kits for use in such method, for instance in the determination of vitamin D derivatives, testosterone and/or or melatonin.

主权项

1. An automated high-throughput method for quantitating at least one low molecular weight protein-bound biomarker directly in an isolated biological sample, comprising the steps of:
(a) automated prepurification of the isolated biological sample using an effective amount of an acid protease having a pH optimum in the range of pH 1.0-5.0 under conditions that allow for digestion of one or more binding proteins until they can no longer bind the at least biomarker of interest; (b) applying said protease-digested sample onto an on-line immunoaffinity solid phase extraction (SPE) column to capture at least part of said biomarker, wherein the immunoaffinity SPE column comprises an immunoaffinity sorbent comprising an antibody capable of binding the at least one biomarker of interest; and wherein the protease-digested sample is applied to the immunoaffinity SPE column without prior protein precipitation and/or centrifugation, followed by sequential washing of the solid phase; (c) eluting from the immunoaffinity SPE column a fraction comprising said at least one biomarker directly onto a liquid chromatography (LC) column comprising an apolar stationary phase and subjecting it to LC-MS or LC-MS-MS measurements to determine the amount of at least one biomarker; and (d) quantitating the biomarker(s) in the sample by correlation with standard samples.