title: Pegylated, steptavidin-conjugated quantum dots are effective detection elements for reverse-phase protein microarrays.
abstract: Protein microarray technologies provide a means of investigating the proteomic content of clinical biopsy specimens in order to determine the relative activity of key nodes within cellular signaling pathways. A particular kind of protein microarray, the reverse-phase microarray, is being evaluated in clinical trials because of its potential to utilize limited amounts of cellular material obtained through biopsy. Using this approach, cellular lysates are arrayed in dilution curves on nitrocellulose substrates for subsequent probing with antibodies. To improve the sensitivity and utility of reverse-phase microarrays, we tested whether a new reporter technology as well as a new detection instrument could enhance microarray performance. We describe the use of an inorganic fluorescent nanoparticle conjugated to streptavidin, Qdot 655 Sav, in a reverse-phase protein microarray format for signal pathway profiling. Moreover, a pegylated form of this bioconjugate, Qdot 655 Sav, is found to have superior detection characteristics in assays performed on cellular protein extracts over the nonpegylated form of the bioconjugate. Hyperspectral imaging of the quantum dot microarray enabled unamplified detection of signaling proteins within defined cellular lysates, which indicates that this approach may be amenable to multiplexed, high-throughput reverse-phase protein microarrays in which numerous analytes are measured in parallel within a single spot.
authors.affiliation: FDA-NCI Clinical Proteomics Program, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: David
authors.initials: D
authors.lastname: Geho
authors.forename: Nicholas
authors.initials: N
authors.lastname: Lahar
authors.forename: Prem
authors.initials: P
authors.lastname: Gurnani
authors.forename: Michael
authors.initials: M
authors.lastname: Huebschman
authors.forename: Paul
authors.initials: P
authors.lastname: Herrmann
authors.forename: Virginia
authors.initials: V
authors.lastname: Espina
authors.forename: Alice
authors.initials: A
authors.lastname: Shi
authors.forename: Julia
authors.initials: J
authors.lastname: Wulfkuhle
authors.forename: Harold
authors.initials: H
authors.lastname: Garner
authors.forename: Emanuel
authors.initials: E
authors.lastname: Petricoin
authors.forename: Lance A
authors.initials: LA
authors.lastname: Liotta
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Rosenblatt
mesh_terms: D002138:Calibration; D002457:Cell Extracts / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D007834:Lasers; D011092:Polyethylene Glycols / Q000737:chemistry*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D045663:Quantum Dots; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry*; D013997:Time Factors
chemical_list: D002457:Cell Extracts; D011506:Proteins; D011092:Polyethylene Glycols; D019809:Streptavidin