Correlated matrix‐assisted laser desorption/ionization mass spectrometry and fluorescent imaging of photocleavable peptide‐coded random bead‐arrays

Abstract

RATIONALERapidly performing global proteomic screens is an important goal in the post-genomic era. Correlated matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and fluorescent imaging of photocleavable peptide-coded random bead-arrays was evaluated as a critical step in a new method for proteomic screening that combines many of the advantages of MS with fluorescence-based microarrays.METHODSSmall peptide-coded model bead libraries containing up to 20 different bead species were constructed by attaching peptides to 30–34 µm diameter glass, agarose or TentaGel® beads using photocleavable biotin or a custom-designed photocleavable linker. The peptide-coded bead libraries were randomly arrayed into custom gold-coated micro-well plates with 45 µm diameter wells and subjected to fluorescence and MALDI mass spectrometric imaging (MALDI-MSI).RESULTSPhotocleavable mass-tags from individual beads in these libraries were spatially localized as ∼65 µm spots using MALDI-MSI with high sensitivity and mass resolution. Fluorescently tagged beads were identified and correlated with their matching photocleavable mass-tags by comparing the fluorescence and MALDI-MS images of the same bead-array. Post-translational modification of the peptide Kemptide was also detected on individual beads in a photocleavable peptide-coded bead-array by MALDI-MSI alone, after exposure of the beads to protein kinase A (PKA).CONCLUSIONSCorrelated MALDI-MS and fluorescent imaging of photocleavable peptide-coded random bead-arrays can provide a basis for performing global proteomic screening. © 2013 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons, Ltd.

Proteome-wide drug screening using mass spectrometric imaging of bead-arrays

“…Synchronization of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and fluorescence images as described previously 28 was used to validate the PC-Mass-Tag coding of individual kinase-beads in the library. Since all 50 kinases contained a common FLAG epitope tag, the bead library was probed with a fluorescently labeled (DyLight650®) anti-FLAG antibody.…”

“…A specific kinase (LCK) chosen in the library was also simultaneously probed with an LCK-specific antibody that was fluorescently labeled with a different fluorophore (Phycoerythrin). After antibody binding, the 50-member kinase-bead library was randomly incorporated into a micro-well plate to form the array (micro-well plates contain approximately 1 million wells in the footprint of a standard microscope slide, each well sized large enough to hold only a single 34 μm bead 28 ).…”

“…Previously, we demonstrated that photocleavable peptides can be utilized as PC-Mass-Tags to encode random bead-arrays. In combination with correlated MALDI-MSI and fluorescence readout, this provided a label-based method for detecting protein-protein molecular interaction such as between a protein library and fluorescently labeled antibodies or other proteins 28 . Here, we demonstrate a label-free method for detection of drug-protein interactions that uses PC-Mass-Tags and proteins attached to beads but does not require fluorescence detection.…”

Rapid LC-MS Based High-Throughput Screening Method, Affording No False Positives or False Negatives, Identifies a New Inhibitor for Carbonic Anhydrase

“…Various MS-based techniques have been developed over the past few decades, and these methods can be subdivided into those approaches that either do or do not require a chromatographic separation step as part of the analysis. The most commonly used MS-based methods without chromatography rely on MALDI-TOF-MS 8 – 10 . This MS platform is fast and sensitive, but matrix interferences and poor reproducibility are some shortcomings that can contribute to false positive and false negative identifications 11 , 12 .…”

HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications

“…One of the vital tasks of drug discovery is fast and effective identification of high affinity ligands from libraries containing these vast numbers of compounds. When a screening method based on fluorescent [1–2] or chemiluminescent [3] read-out is feasible, tens of thousands of compounds can be assayed per day. Many potential drug candidates, however, cannot be probed using these standard assays because some druggable interactions, such as protein binding events, cannot be readily monitored by a change in fluorescence.…”