A peptide aldehyde microarray for high-throughput profiling of cellular events.
Microarrays provide exciting opportunities in the field of large-scale proteomics. With the aim to elucidate enzymatic activity and profiles within native biological samples, we developed a microarray comprising a focused positional-scanning library of enzyme inhibitors. The library was diversified across P(1)-P(4) positions, creating 270 different inhibitor sublibraries which were immobilized onto avidin slides. The peptide aldehyde-based small-molecule microarray (SMM) specifically targeted cysteine proteases, thereby enabling large-scale functional assessment of this subgroup of proteases, within fluorescently labeled samples, including pure proteins, cellular lysates, and infected samples. The arrays were shown to elicit binding fingerprints consistent with those of model proteins, specifically caspases and purified cysteine proteases from parasites (rhodesein and cruzain). When tested against lysates from apoptotic Hela and red blood cells infected with Plasmodium falciparum, clear signatures were obtained that were readily attributable to the activity of constituent proteases within these samples. Characteristic binding profiles were further able to distinguish various stages of the parasite infection in erythrocyte lysates. By converting one of our brightest microarray hits into a probe, putative protein markers were identified and pulled down from within apoptotic Hela lysates, demonstrating the potential of target validation and discovery. Taken together, these results demonstrate the utility of targeted SMMs in dissecting cellular biology in complex proteomic samples.
Department of Chemistry, 3 Science Drive 3, National University of Singapore, Singapore 117543.
Hao
H
Wu
Jingyan
J
Ge
Peng-Yu
PY
Yang
Jigang
J
Wang
Mahesh
M
Uttamchandani
Shao Q
SQ
Yao
D000447:Aldehydes / Q000737:chemistry*; D002457:Cell Extracts; D057056:Cysteine Proteases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism / Q000469:parasitology; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D010963:Plasmodium falciparum / Q000502:physiology; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
D000447:Aldehydes; D002457:Cell Extracts; D015853:Cysteine Proteinase Inhibitors; D010455:Peptides; D057056:Cysteine Proteases