title: Robust, quantitative analysis of proteins using peptide immunoreagents, in vitro translation, and an ultrasensitive acoustic resonant sensor.
abstract: A major benefit of proteomic and genomic data is the potential for developing thousands of novel diagnostic and analytical tests of cells, tissues, and clinical samples. Monoclonal antibody technologies, phage display and mRNA display, are methods that could be used to generate affinity ligands against each member of the proteome. Increasingly, the challenge is not ligand generation, rather the analysis and affinity rank-ordering of the many ligands generated by these methods. Here, we developed a quantitative method to analyze protein interactions using in vitro translated ligands. In this assay, in vitro translated ligands generate a signal by simultaneously binding to a target immobilized on a magnetic bead and to a sensor surface in a commercial acoustic sensing device. We then normalize the binding of each ligand with its relative translation efficiency in order to rank-order the different ligands. We demonstrate the method with peptides directed against the cancer marker Bcl-xL. Our method has 4- to 10-fold higher sensitivity, using 100-fold less protein and 5-fold less antibody per sample, as compared directly with ELISA. Additionally, all analysis can be conducted in complex mixtures at physiological ionic strength. Lastly, we demonstrate the ability to use peptides as ultrahigh affinity reagents that function in complex matrices, as would be needed in diagnostic applications.
authors.affiliation: 3710 McClintock Avenue, RTH 507, Los Angeles, California 90089-2905, United States.
authors.forename: Farzad
authors.initials: F
authors.lastname: Jalali-Yazdi
authors.forename: Jasmine M
authors.initials: JM
authors.lastname: Corbin
authors.forename: Terry T
authors.initials: TT
authors.lastname: Takahashi
authors.forename: Richard W
authors.initials: RW
authors.lastname: Roberts
mesh_terms: D014408:Biomarkers, Tumor / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D018189:Immunomagnetic Separation; D007202:Indicators and Reagents; D008024:Ligands; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012313:RNA / Q000737:chemistry; D051020:bcl-X Protein / Q000737:chemistry
chemical_list: D014408:Biomarkers, Tumor; D007202:Indicators and Reagents; D008024:Ligands; D010455:Peptides; D011506:Proteins; D051020:bcl-X Protein; D012313:RNA
references.citation: Science. 2000 Sep 8;289(5485):1760-3
references.pmid: 10976071
references.citation: Protein Sci. 2007 Mar;16(3):476-84
references.pmid: 17322532
references.citation: RNA. 2002 Jul;8(7):890-903
references.pmid: 12166644
references.citation: Anal Chem. 2009 Nov 15;81(22):9499-506
references.pmid: 19848413
references.citation: Nature. 2002 Jan 10;415(6868):180-3
references.pmid: 11805837
references.citation: Acta Endocrinol (Copenh). 1990 Feb;122(2):227-32
references.pmid: 2180243
references.citation: Methods Mol Biol. 2009;535:293-314
references.pmid: 19377989
references.citation: Methods Enzymol. 1983;96:50-74
references.pmid: 6656641
references.citation: Angew Chem Int Ed Engl. 2012 Dec 7;51(50):12449-53
references.pmid: 23125174
references.citation: Science. 1990 Jul 27;249(4967):386-90
references.pmid: 1696028
references.citation: Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12297-302
references.pmid: 9356443
references.citation: Nature. 1990 Dec 6;348(6301):552-4
references.pmid: 2247164
references.citation: Clin Biochem. 2013 Aug;46(12):1079-1083
references.pmid: 23707222
references.citation: Nature. 1975 Aug 7;256(5517):495-7
references.pmid: 1172191
references.citation: Antimicrob Agents Chemother. 2009 Feb;53(2):593-602
references.pmid: 19029324
references.citation: Regul Pept. 1998 Oct 16;77(1-3):83-7
references.pmid: 9809800
references.citation: Protein Eng Des Sel. 2010 Apr;23(4):279-88
references.pmid: 20164216
references.citation: Nat Methods. 2011 May 15;8(7):551-8
references.pmid: 21572409
references.citation: Methods Enzymol. 2000;318:268-93
references.pmid: 10889994
references.citation: BMC Clin Pathol. 2008 Sep 19;8:9
references.pmid: 18803814
references.citation: Bioorg Med Chem Lett. 2001 Jul 23;11(14):1897-902
references.pmid: 11459656
references.citation: Anal Chem. 2006 Feb 1;78(3):793-800
references.pmid: 16448053
references.citation: Science. 1995 Oct 20;270(5235):467-70
references.pmid: 7569999
references.citation: Curr Protein Pept Sci. 2003 Aug;4(4):253-60
references.pmid: 14529532
references.citation: Nature. 2002 Jan 10;415(6868):141-7
references.pmid: 11805826
references.citation: Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9022-6
references.pmid: 7522328
references.citation: J Immunol Methods. 1992 Jun 24;150(1-2):5-21
references.pmid: 1613258
references.citation: Life Sci. 2007 Mar 20;80(15):1439-48
references.pmid: 17316701
references.citation: Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4665-70
references.pmid: 19255450
references.citation: J Immunol. 2007 Jun 1;178(11):7467-72
references.pmid: 17513798
references.citation: ACS Nano. 2010 Nov 23;4(11):6914-22
references.pmid: 21028792
references.citation: J Vis Exp. 2011 Feb 03;(48):
references.pmid: 21339717