Pegylated, steptavidin-conjugated quantum dots are effective detection elements for reverse-phase protein microarrays.
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.
Geho
David
D
FDA-NCI Clinical Proteomics Program, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Lahar
Nicholas
N
Gurnani
Prem
P
Huebschman
Michael
M
Herrmann
Paul
P
Espina
Virginia
V
Shi
Alice
A
Wulfkuhle
Julia
J
Garner
Harold
H
Petricoin
Emanuel
E
Liotta
Lance A
LA
Rosenblatt
Kevin P
KP
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
D002457:Cell Extracts; D011506:Proteins; D011092:Polyethylene Glycols; D019809:Streptavidin