title: Increased throughput for low-abundance protein biomarker verification by liquid chromatography/tandem mass spectrometry.
abstract: Low-abundance protein quantification has historically been performed using ligand binding techniques. However, due to the time and cost associated with developing enzyme-linked immunosorbent assay (ELISA), mass spectrometric approaches are playing an increasingly important role. Protein quantification at or below the nanogram per milliliter level using liquid chromatography/tandem mass spectrometry (LC/MS/MS) typically utilizes an immunoaffinity (IA) enrichment step such as immunoprecipitation. In order to maximize mass spectrometry (MS) sensitivity, protein enrichment is followed by a proteolytic cleavage step used to generate a surrogate peptide with better mass spectrometric properties. Unlike ELISA, IA-LC/MS/MS is a serial technique that can require up to 3 days for a single batch analysis due to lengthy incubation and digestion steps. This report describes the use of immunoprecipitation in 96-well ELISA format (IPE) and microwave-assisted protein digestion to reduce the time required to perform LC/MS/MS protein analyses to within a single day. The utility of this approach was investigated through its application to previously published LC/MS/MS protein assays from our laboratory for two cardiotoxicity biomarkers, Myl3 and NTproBNP. Using commercially available antibodies, IPE and microwave-assisted digestion were used to repeat intraday validations for these markers, and intraday precision (%CV) and accuracy (%RE) did not exceed 11% or 3% for either assay, respectively. Additionally, lower limits of quantification of 100 pg/mL (NTproBNP) and 0.95 ng/mL (Myl3) were achieved.
authors.affiliation: Eli Lilly and Company, Drug Disposition Biomarker Group, Lilly Corporate Center, Indianapolis, Indiana 46285, USA. mberna@lilly.com
authors.forename: Michael
authors.initials: M
authors.lastname: Berna
authors.forename: Bradley
authors.initials: B
authors.lastname: Ackermann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D015415:Biomarkers / Q000032:analysis; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation; D008872:Microwaves; D018994:Myosin Light Chains / Q000032:analysis*; D020097:Natriuretic Peptide, Brain / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis*; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000906:Antibodies; D015415:Biomarkers; D018994:Myosin Light Chains; D010446:Peptide Fragments; C109794:pro-brain natriuretic peptide (1-76); D020097:Natriuretic Peptide, Brain