Photocleavable Peptide-Conjugated Magnetic Beads for Protein Kinase Assays by MALDI-TOF MS

Abstract

Peptides were immobilized onto superparamagnetic beads via photocleavable linkers. This enabled simple, rapid, and label-free protein kinase assays via MALDI-TOF MS detection of substrate peptide phosphorylation. Abltide, a model substrate for the Abl protein tyrosine kinase model, was coupled onto amine-terminated beads, incubated with ATP and recombinant c-Abl kinase, and released and further detected to determine phosphorylation. Abltide phosphorylation was found to depend significantly on the length and composition of linkers to the bead surface. Inserting a diblock spacer of poly(glycine) and poly(ethylene glycol) segments markedly enhanced phosphorylation. To validate the assay, the activity of two small-molecule kinase inhibitors, imatinib and dasatinib, which target the oncogenic mutant tyrosine kinase Bcr-Abl to treat chronic myeloid leukemia (CML), was tested. Examining inhibition of the purified c-Abl or Bcr-Abl in K562 CML cell extracts, IC50 values were determined to be consistent with the literature. This simple, label-free, MALDI-based protein kinase assay can be readily adapted to allow multiplexed assays of multiple peptide substrates and/or analysis of alternative post-translational modifications as a tool for drug discovery and clinical testing.

Photocleavable peptide–oligonucleotide conjugates for protein kinase assays by MALDI-TOF MS

“…Nonetheless, optimal MALDI matrices for ionization of peptides and oligonucleotides are distinct and the ionization properties of peptide-oligonucleotide conjugates and particularly of phosphorylated conjugates are unpredictable. Toward obviating this confounding factor, we drew on prior studies 25, 29 that demonstrated that a photocleavable cross-linker could be introduced to provide reliable detection of peptide phosphorylation by MALDI-TOF MS.…”

“…We have previously demonstrated that magnetic beads are a powerful tool for hybrid and solid-phase protein kinase assays, offering separation and suitability to automated procedures as well as favorable analysis by MALDI-TOF MS. 24–25 To pull out protein kinase products from kinase reaction mixtures by magnetic beads through DNA hybridization for detecting degree of phosphorylation, we investigated the immobilization of the complementary thiol-modified oligonucleotide (5′-HS GTACTTCCTTAAACGACGCAGG-3′) on magnetic beads. First, to prevent nonspecific interactions, a poly(ethylene glycol) (PEG) spacer linker was grafted onto the surface of magnetic beads according as previously reported (Figure 4).…”

“…First, to prevent nonspecific interactions, a poly(ethylene glycol) (PEG) spacer linker was grafted onto the surface of magnetic beads according as previously reported (Figure 4). 25 Surface PEG-modified magnetic beads were further reacted with 3-maleimido-propionic acid to produce surface maleimide-functionalized magnetic beads. To immobilize the complementary oligonucleotide onto magnetic beads, the commercially available complementary thiol-modified oligonucleotide was activated by treating with 50 mM TCEP neutral solution for 30 min, and followed by purification with desalting spin columns.…”

“…However, the common use of luminescent or fluorescent probes, typically based on antibody recognition, [12][13][14][15][16][17] offers only indirect, label-dependent detection of tyrosine phosphorylation, thereby compromising dynamic range, and sensitivity and specificity and providing limited potential for multiplexing. The application of modern chemical approaches has led to several advances in detection methods for homogenous assays 8,12,[18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] but only limited progress has been reported on multiplexing of substrates and/or kinases. 31,32,[37][38][39] Notably, Imperiali and colleagues 39 reported a fluorescencebased homogeneous assay enabling multiplexed kinase detection in cell lysates.…”

“…[40][41][42] Applying this principle to homogeneous assays, Kubota et al 32 developed a single-reaction strategy to measure the differential phosphorylation in 90 peptide substrates using electrospray ionization mass spectrometry. We 24,25,[43][44][45] and others [46][47][48] have exploited matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a tool for label-free detection of peptide phosphorylation in heterogeneous assays with substrates tethered to MALDI targets, self-assembled monolayers, microarrays, microparticles and other geometries. MALDI-TOF MS-based phosphorylation detection is comparatively simple, does not require elaborate chromatography-based separation steps, is compatible with HTS and has the potential for unlimited multiplexing.…”