title: Combining ultracentrifugation and peptide termini group-specific immunoprecipitation for multiplex plasma protein analysis.
abstract: Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques.
authors.affiliation: NMI Natural and Medical Sciences Institute, University of Tübingen, 72770 Reutlingen, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Volk
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Schreiber
authors.forename: David
authors.initials: D
authors.lastname: Eisen
authors.forename: Calvin
authors.initials: C
authors.lastname: Wiese
authors.forename: Hannes
authors.initials: H
authors.lastname: Planatscher
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Pynn
authors.forename: Dieter
authors.initials: D
authors.lastname: Stoll
authors.forename: Markus F
authors.initials: MF
authors.lastname: Templin
authors.forename: Thomas O
authors.initials: TO
authors.lastname: Joos
authors.forename: Oliver
authors.initials: O
authors.lastname: Pötz
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry; D000936:Antigen-Antibody Complex / Q000737:chemistry; D015415:Biomarkers / Q000032:analysis; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D003285:Contractile Proteins / Q000032:analysis; D064448:Filamins; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008840:Microfilament Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling; D013395:Sucrose; D014357:Trypsin; D014461:Ultracentrifugation / Q000379:methods
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D015415:Biomarkers; D001798:Blood Proteins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D010455:Peptides; D013395:Sucrose; D014357:Trypsin