Sensitive single-molecule protein quantification and protein complex detection in a microarray format.
Single-molecule protein analysis provides sensitive protein quantitation with a digital read-out and is promising for studying biological systems and detecting biomarkers clinically. However, current single-molecule platforms rely on the quantification of one protein at a time. Conventional antibody microarrays are scalable to detect many proteins simultaneously, but they rely on less sensitive and less quantitative quantification by the ensemble averaging of fluorescent molecules. Here, we demonstrate a single-molecule protein assay in a microarray format enabled by an ultra-low background surface and single-molecule imaging. The digital read-out provides a highly sensitive, low femtomolar limit of detection and four orders of magnitude of dynamic range through the use of hybrid digital-analog quantification. From crude cell lysate, we measured levels of p53 and MDM2 in parallel, proving the concept of a digital antibody microarray for use in proteomic profiling. We also applied the single-molecule microarray to detect the p53-MDM2 protein complex in cell lysate. Our study is promising for development and application of single-molecule protein methods because it represents a technological bridge between single-plex and highly multiplex studies.
Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University, St. Louis, MO 63108, USA.
Lee A
LA
Tessler
Robi D
RD
Mitra
D045744:Cell Line, Tumor; D006801:Humans; D046912:Multiprotein Complexes / Q000032:analysis*; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000032:analysis; D051736:Proto-Oncogene Proteins c-mdm2 / Q000032:analysis*; D016159:Tumor Suppressor Protein p53 / Q000032:analysis*
D046912:Multiprotein Complexes; D011506:Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2