Photocleavable peptide hydrogel arrays for MALDI-TOF analysis of kinase activity

Abstract

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.

A Bead-Based Activity Screen for Small-Molecule Inhibitors of Signal Transduction in Chronic Myelogenous Leukemia Cells

“…Similarly, substrates that included p40 were phosphorylated more rapidly than Abltide alone (Figure 3D). Mass spectrometry and fluorescent anti-phosphotyrosine antibody labeling confirmed that signal increases were not the result of phosphorylation at an additional tyrosine in p40 (17, 36). Results from these competition assays suggested that p40 recruits Bcr-Abl to the bead surface by interaction with the SH3 domain.…”

“…Previous work using immobilized Abltide to measure Bcr-Abl activity in cell lysates demonstrated that altered orientations resulted in increased signal intensities (35). It was also shown that inclusion of p40, the high-affinity peptide ligand for the Abl kinase SH3 domain, led to more efficient phosphorylation of Abltide (17, 36). To test the influence of peptide orientation on assay sensitivity we synthesized Abltide with a carboxyl-terminal cysteine.…”

Lab‐on‐a‐plate: Extending the functionality of MALDI‐MS and LDI‐MS targets

“…A similar approach was also adopted by Parker et al (2006); here, acrylamide-labeled peptides were immobilized in a hydrogel surface and the enzymatic activity of a kinase was studied by detecting the phosphorylated peptides (Fig. 15).…”

“…15). Cleavage of peptides from the plate was performed directly using the MALDI laser, or by treatment with an auxiliary UV light source, and the matrix added subsequently to enable detection by MALDI-MS (Parker et al, 2006). Yet another protocol for immobilization of substrates of an enzyme was demonstrated by Mrksich and co-workers who implemented SAMDI-MS.…”

Deciphering Enzyme Function Using Peptide Arrays

“…Regioselective immobilisation of poly(desoxythymidin)-modified kinase substrates onto differently coated glass slides was reported for PKA and c-Src [49]. Photocleavable acrylamide labelled cysteine-containing kinase substrates were incorporated into peptide-acrylamide copolymer hydrogel surfaces and v-Abl-or c-Abl-mediated phosphorylation was detected by MALDI-TOF/TOF subsequent to laser-induced cleavage at the ß-(2-Nitrophenyl)-ß-alanine residue [50]. There are several additional chemistries used for the chemoselective immobilisation of peptides onto different surfaces (comprehensively reviewed in [19]) like formation of covalent bonds by reaction of salicylhydroxamic acids with 1,3-phenyldiboronic acid derivatives [51][52][53] or semicarbazides with aldehydes [54][55][56][57][58][59][60] or using thio-ene chemistry [61][62][63][64] but no applications for enzyme profiling have been described so far.…”