title: Antibody-Coupled Magnetic Beads Can Be Reused in Immuno-MRM Assays To Reduce Cost and Extend Antibody Supply.
abstract: Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring mass spectrometry (immuno-MRM) enables precise quantification of peptides. Affinity-purified polyclonal antibodies are routinely used as affinity reagents in immuno-MRM assays, but they are not renewable, limiting the number of experiments that can be performed. In this technical note, we describe a workflow to regenerate anti-peptide polyclonal antibodies coupled to magnetic beads for enrichments in multiplex immuno-MRM assays. A multiplexed panel of 44 antibodies (targeting 60 peptides) is used to show that peptide analytes can be effectively stripped off of antibodies using acid washing without compromising assay performance. The performance of the multiplexed panel (determined by correlation, agreement, and precision of reused assays) is reproducible (R(2) between 0.81 and 0.99) and consistent (median CVs 8-15%) for at least 10 times of washing and reuse. Application of this workflow to immuno-MRM studies greatly reduces per sample assay cost and increases the number of samples that can be interrogated with a limited supply of polyclonal antibody reagent. This allows more characterization for promising and desirable targets prior to committing funds and efforts to conversion to a renewable monoclonal antibody.
authors: Fred Hutchinson Cancer Research Center , 1100 Fairview Avenue North, Seattle, Washington 98109, United States.
authors: Lei
authors: L
authors: Zhao
authors: Fred Hutchinson Cancer Research Center , 1100 Fairview Avenue North, Seattle, Washington 98109, United States.
authors: Jeffrey R
authors: JR
authors: Whiteaker
authors: Fred Hutchinson Cancer Research Center , 1100 Fairview Avenue North, Seattle, Washington 98109, United States.
authors: Uliana J
authors: UJ
authors: Voytovich
authors: Fred Hutchinson Cancer Research Center , 1100 Fairview Avenue North, Seattle, Washington 98109, United States.
authors: Richard G
authors: RG
authors: Ivey
authors: Fred Hutchinson Cancer Research Center , 1100 Fairview Avenue North, Seattle, Washington 98109, United States.
authors: Amanda G
authors: AG
authors: Paulovich
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000191:economics* / Q000295:instrumentation / Q000379:methods; D002853:Chromatography, Liquid; D004847:Epithelial Cells / Q000737:chemistry / Q000378:metabolism; D017743:Equipment Reuse; D006801:Humans; D018189:Immunomagnetic Separation / Q000191:economics* / Q000295:instrumentation / Q000379:methods; D007553:Isotope Labeling; D059346:Magnets; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000032:analysis*; D059748:Proteolysis; D014357:Trypsin / Q000737:chemistry
chemical_list: D000906:Antibodies; D010455:Peptides; D014357:Trypsin
keywords: Targeted proteomics; antibody; immunoaffinity enrichment; mass spectrometry; peptide assays; quantitative proteomics