Photocleavable peptide hydrogel arrays for MALDI-TOF analysis of kinase activity.
We have developed an acrylamide copolymerization strategy to immobilize acrylamide labeled peptides and proteins into a hydrogel surface and detect their modifications using MALDI-TOF mass spectrometry. Copolymerization into hydrogels is robust, compatible with "off-the-shelf" chemistry, and yields materials and surfaces that are stable to aqueous or organic solvents, drying, high or low temperature, high or low pH, oxidizing agents, sonication, mechanical contact, etc. The use of acrylamide hydrogels allows immobilization of substrates in a hydrated environment that can be used both as a biological reaction matrix and as a MALDI target. In our strategy, a substrate peptide was designed in a modular fashion to include both modification site and affinity domains. It was labeled with an acrylamide functionality using a generalized chemistry and covalently attached to the surface with a photocleavable linker, allowing for aggressive washing to remove any fouling, followed by selective release for MALDI-TOF analysis. Using this system we were able to analyze and compare v-Abl (truncated) and c-Abl (full-length) kinase activity on a peptide substrate with an affinity domain specific for the full-length kinase, observing excellent overall reproducibility in the extent of phosphorylation detected. This work serves as proof of principle for modular substrate design strategies for mass spectrometry-readable biosensors.
Parker
Laurie L
LL
Department of Biochemistry and Molecular Biology, University of Chicago, CIS 201, 929 E. 57th Street, Chicago, IL 60637, USA. lparker@uchicago.edu
Brueggemeier
Shawn B
SB
Rhee
Won Jun
WJ
Wu
Ding
D
Kent
Stephen B H
SB
Kron
Stephen J
SJ
Palecek
Sean P
SP
D000178:Acrylamides / Q000737:chemistry; D020100:Hydrogels / Q000737:chemistry*; D008027:Light; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000178:Acrylamides; D020100:Hydrogels; D010455:Peptides; D010770:Phosphotransferases