A highly scalable peptide-based assay system for proteomics.
We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.
Prognosys Biosciences Inc., La Jolla, California, United States of America. ikozlov@prognosysbio.com
Igor A
IA
Kozlov
Elliot R
ER
Thomsen
Sarah E
SE
Munchel
Patricia
P
Villegas
Petr
P
Capek
Austin J
AJ
Gower
Stephanie J K
SJ
Pond
Eugene
E
Chudin
Mark S
MS
Chee
D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D016174:Hepacivirus / Q000235:genetics*; D046228:Microarray Analysis / Q000379:methods; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D018076:DNA, Complementary; D010447:Peptide Hydrolases; D010455:Peptides; D010770:Phosphotransferases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases