title: Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers.
abstract: A major bottleneck for validation of new clinical diagnostics is the development of highly sensitive and specific assays for quantifying proteins. We previously described a method, stable isotope standards with capture by antipeptide antibodies, wherein a specific tryptic peptide is selected as a stoichiometric representative of the protein from which it is cleaved, is enriched from biological samples using immobilized antibodies, and is quantitated using mass spectrometry against a spiked internal standard to yield a measure of protein concentration. In this study, we optimized a magnetic-bead-based platform amenable to high-throughput peptide capture and demonstrated that antibody capture followed by mass spectrometry can achieve ion signal enhancements on the order of 10(3), with precision (CVs <10%) and accuracy (relative error approximately 20%) sufficient for quantifying biomarkers in the physiologically relevant ng/mL range. These methods are generally applicable to any protein or biological fluid of interest and hold great potential for providing a desperately needed bridging technology between biomarker discovery and clinical application.
authors.affiliation: Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., PO Box 19024, Seattle, WA 98109-1024, USA.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Whiteaker
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.forename: Heidi Y
authors.initials: HY
authors.lastname: Zhang
authors.forename: Li-Chia
authors.initials: LC
authors.lastname: Feng
authors.forename: Brian D
authors.initials: BD
authors.lastname: Piening
authors.forename: Leigh
authors.initials: L
authors.lastname: Anderson
authors.forename: Amanda G
authors.initials: AG
authors.lastname: Paulovich
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D015415:Biomarkers / Q000032:analysis* / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008863:Microspheres; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D000906:Antibodies; D015415:Biomarkers; D010455:Peptides