Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers.
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.
Whiteaker
Jeffrey R
JR
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., PO Box 19024, Seattle, WA 98109-1024, USA.
Zhao
Lei
L
Zhang
Heidi Y
HY
Feng
Li-Chia
LC
Piening
Brian D
BD
Anderson
Leigh
L
Paulovich
Amanda G
AG
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
D000906:Antibodies; D015415:Biomarkers; D010455:Peptides