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.

[1]  L L Needham,et al.  Isotope dilution--mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I. , 1996, Clinical chemistry.

[2]  A. Howell,et al.  Tamoxifen ("Nolvadex"): a review. , 2002, Cancer treatment reviews.

[3]  R. Aebersold,et al.  Scoring proteomes with proteotypic peptide probes , 2005, Nature Reviews Molecular Cell Biology.

[4]  J. Ross,et al.  The HER‐2/neu Oncogene in Breast Cancer: Prognostic Factor, Predictive Factor, and Target for Therapy , 1998, The oncologist.

[5]  M. Lindsay.  Target discovery , 2003, Nature Reviews Drug Discovery.

[6]  M. Karas,et al.  Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. , 1988, Analytical chemistry.

[7]  E. Winer,et al.  Trastuzumab/chemotherapy combinations in metastatic breast cancer. , 2002, Seminars in oncology.

[8]  J. Ross,et al.  The HER‐2/neu Oncogene in Breast Cancer: Prognostic Factor, Predictive Factor, and Target for Therapy , 1998, Stem cells.

[9]  J. Brand,et al.  Depletion efficiency and recovery of trace markers from a multiparameter immunodepletion column , 2006, Proteomics.

[10]  David R Goodlett,et al.  Large‐scale evaluation of quantitative reproducibility and proteome coverage using acid cleavable isotope coded affinity tag mass spectrometry for proteomic profiling , 2005, Proteomics.

[11]  K. Duffin,et al.  Online immunoaffinity liquid chromatography/tandem mass spectrometry determination of a type II collagen peptide biomarker in rat urine: Investigation of the impact of collision-induced dissociation fluctuation on peptide quantitation. , 2006, Analytical biochemistry.

[12]  S. Gygi,et al.  Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[13]  L. Belghiti,et al.  Prognostic factors in breast cancer , 2002 .

[14]  D. Haber,et al.  Molecular targeted therapy of lung cancer: EGFR mutations and response to EGFR inhibitors. , 2005, Cold Spring Harbor symposia on quantitative biology.

[15]  Ning Zhang,et al.  High Throughput Proteome Screening for Biomarker Detection* , 2005, Molecular & Cellular Proteomics.

[16]  M. Kanaoka,et al.  18O‐labeling quantitative proteomics using an ion trap mass spectrometer , 2005, Proteomics.

[17]  Werner Zolg,et al.  Quantification of C‐reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C‐labeled peptide standards , 2004, Proteomics.

[18]  G. Siuzdak,et al.  The Expanding Role of Mass Spectrometry in Metabolite Profiling and Characterization , 2005, Chembiochem : a European journal of chemical biology.

[19]  S. Gygi,et al.  Quantitative analysis of complex protein mixtures using isotope-coded affinity tags , 1999, Nature Biotechnology.

[20]  T. Shaler,et al.  Quantification of intermediate-abundance proteins in serum by multiple reaction monitoring mass spectrometry in a single-quadrupole ion trap. , 2006, Analytical chemistry.

[21]  A. Ullrich,et al.  Smart drugs: tyrosine kinase inhibitors in cancer therapy. , 2002, Cancer cell.

[22]  William S Hancock,et al.  Targeted proteomics of low-level proteins in human plasma by LC/MSn: using human growth hormone as a model system. , 2002, Journal of proteome research.

[23]  George G Klee,et al.  Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-Ms/MS using protein cleavage and isotope dilution mass spectrometry. , 2004, Journal of proteome research.

[24]  Donald H Chace,et al.  A biochemical perspective on the use of tandem mass spectrometry for newborn screening and clinical testing. , 2005, Clinical biochemistry.

[25]  Darryl B. Hardie,et al.  Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). , 2004, Journal of proteome research.

[26]  E. Leonard,et al.  α1-Antichymotrypsin Is the Human Plasma Inhibitor of Macrophage Ectoenzymes That Cleave Pro-macrophage Stimulating Protein* , 2001, The Journal of Biological Chemistry.

[27]  M. Mann,et al.  Electrospray Ionization for Mass Spectrometry of Large Biomolecules , 1990 .

[28]  S. Edge,et al.  Prognostic factors in breast cancer , 2005 .

[29]  Steven Butcher.  Target Discovery and Validation in the Post-Genomic Era , 2003, Neurochemical Research.

[30]  Leigh Anderson,et al.  Quantitative Mass Spectrometric Multiple Reaction Monitoring Assays for Major Plasma Proteins* , 2006, Molecular & Cellular Proteomics.

[31]  N. Anderson,et al.  The Human Plasma Proteome: History, Character, and Diagnostic Prospects , 2003, Molecular & Cellular Proteomics.

[32]  Ivo Safarik,et al.  Magnetic techniques for the isolation and purification of proteins and peptides , 2004, Biomagnetic research and technology.

[33]  B. Gibbs,et al.  Mass spectrometric quantitation of C-reactive protein using labeled tryptic peptides. , 2006, Analytical biochemistry.

[34]  M. Adler.  Immuno-PCR as a clinical laboratory tool. , 2005, Advances in clinical chemistry.

[35]  K Vajda,et al.  [Prognostic factors in breast cancer]. , 1998, Orvosi hetilap.

[36]  Christof M Niemeyer,et al.  Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification. , 2005, Trends in biotechnology.

[37]  Ruedi Aebersold,et al.  Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags. , 2002, Journal of proteome research.

[38]  N. Anderson,et al.  The Human Plasma Proteome , 2002, Molecular & Cellular Proteomics.