Proteomics evaluation of chemically cleavable activity-based probes.
Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system.
Fonović
Marko
M
Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA.
Verhelst
Steven H L
SH
Sorum
Mark T
MT
Bogyo
Matthew
M
D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry; D051379:Mice; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D015335:Molecular Probes; D010455:Peptides; D002403:Cathepsins; D012697:Serine Endopeptidases