Global proteomic screening of random bead arrays using mass spectrometry imaging
Inventors
Lim, Mark J • Bergo, Vladislav B • Rothschild, Kenneth J
Assignees
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Publication Number
US-12013392-B2
Publication Date
2024-06-18
Expiration Date
Abstract
Methods for proteomic screening on random protein-bead arrays by mass spec is described. Photocleavable mass tags are utilized to code a protein library (bait molecules) displayed on beads randomly arrayed in an array substrate. A library of probes (prey) can be mixed with the protein-bead array to query the array. Because mass spec can detect multiple mass tags, it is possible to rapidly identify all of the interactions resulting from this mixing.
Core Innovation
The document describes bead-based global proteomic screening using random protein-bead arrays (Bead-GPS/Bead-GPS™) in which bait-prey interactions are detected by decoding photocleavable mass tags. Photocleavable mass tags (PC-mass tags) encode bait identity on individual beads and can also encode probes/prey, and mass spectrometry imaging detects the released tags or fragments to identify bait-prey interactions.
A key element is the use of photocleavable mass tags having a general structure in which B is a spacer and Y is a reacted react-able moiety, together with detecting the photocleavable mass tags or fragments by mass spectrometry. The approach is stated to enable detection and quantification, including expression profiling using isotope-coded PC tags and functional screening including protein modifications.
The document further describes compositions and methods in which a bait molecule attached to a surface binds an analyte at a first binding site, and a probe bound to the analyte at a different second binding site carries a photocleavable mass tag. Electromagnetic radiation photo-releases the photocleavable mass tag, and mass spectrometry detects the released mass tag or fragment, including attachment strategies such as affinity-based attachment via (stre)avidin/biotin and direct covalent strategies with photocleavable linkers.
Claims Coverage
The provided independent claims are clm-00001, clm-00007, and clm-00014. Together they cover three main inventive features: multiplexed analyte interaction detection on beads using photocleavable mass tags and mass spectrometry detection of released tags or fragments; analyte detection and quantification by binding to bait and probe with photo-release of photocleavable mass tags; and a composition format using spatially distinct binding sites for bait and probe.
Two-bead analyte interaction detection using photocleavable mass-tag probes on beads
A method comprising providing first and second beads with different bait molecules, contacting the beads with a solution comprising analyte molecules to form reacted beads, contacting reacted beads with one or more probes each comprising a photocleavable mass tag having a structure where B is a spacer and Y is a reacted react-able moiety, and detecting the photocleavable mass tags or fragments thereof from the reacted beads by mass spectrometry.
Photo-release detection and quantification via photocleavable mass-tagged probes detected by mass spectrometry
A method for detection and quantification comprising providing a bait molecule attached to a surface and a probe comprising a photocleavable mass tag with the structure where B is a spacer and Y is a reacted react-able moiety, contacting the bait with a sample comprising an analyte to form a complex, contacting the complex with the probe to create a reacted probe, treating the reacted probe with electromagnetic radiation to photo-release the photocleavable mass tag, and detecting the photo-released mass tag or fragments thereof by mass spectrometry.
Composition with bait and probe bound at different analyte binding sites carrying a photocleavable mass tag
A composition for detection and quantification comprising a bait molecule attached to a surface bound to an analyte at a first binding site, and a probe bound to the analyte at a second binding site different from the first binding site, where the probe comprises a photocleavable mass tag having the general structure where B is a spacer and Y is a reacted react-able moiety.
Across the independent claims, the document covers methods and compositions in which analyte-bait interactions are identified through probes that carry photocleavable mass tags, where electromagnetic radiation photo-releases the tags or tags are associated with reacted beads, and mass spectrometry detects the released mass tags or fragments for detection and, in the later claims, quantification. The bead-based multi-bait format and the spatially distinct bait/probe binding-site composition are explicitly encompassed.
Stated Advantages
Enables multiplexed proteomics by decoding bait-prey interactions using mass spectrometry detection of released photocleavable mass tags or fragments.
Supports biomarker discovery, drug/library screening, and expression profiling using isotope-coded PC mass tags.
Enables functional screening including detection of protein modifications.
Provides detection and quantification of analytes by photo-release of photocleavable mass tags followed by mass spectrometry detection.
Documented Applications
Biomarker discovery using autoantibodies, including detection of autoantibodies and protein abundance changes.
Drug/library screening.
Expression profiling using isotope-coded PC mass tags.
Functional screening, including protein modifications such as phosphorylation, ubiquitination, and methylation.
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