Systems and methods for biomolecule quantitation
Inventors
Mallick, Parag • EGERTSON, Jarrett D. • KAPP, Gregory
Assignees
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Publication Number
US-12092642-B2
Publication Date
2024-09-17
Expiration Date
Abstract
Systems and methods for obtaining qualitative or quantitative measurements of proteoforms of polypeptides are described. The described methods include measurements of affinity reagent binding on single-molecule polypeptide arrays to distinguish between polypeptide isoforms. The described methods may provide high resolution quantitative comparisons of proteoforms with very low copy numbers.
Core Innovation
The invention characterizes proteoforms of a polypeptide using an array in which a plurality of polypeptides includes a first proteoform and a second proteoform, and each proteoform is present at an individually observable address with each individual address comprising a single polypeptide. The method contacts the array with a first affinity reagent and a second affinity reagent, and at least one of the affinity reagents binds promiscuously to the first and second proteoforms.
After sequential contact, the method detects the presence or absence of binding of the affinity reagents at the individually observable addresses to characterize proteoforms, including observing the first proteoform and the second proteoform at single molecule resolution at corresponding addresses. The disclosure further describes proteoform quantitation and characterization using sequential or multiplex affinity-reagent binding and signal detection at each address, followed by decoding to infer which proteoforms are present.
The disclosure further describes binding reporter signals, including labeled affinity formats such as fluorescent, FRET, isotopic, and nucleic-acid tags, together with probabilistic decoding, confidence-threshold decoding, and multi-cycle affinity reagent workflows using addressable resolvable sites. Proteoform characterization may be refined for post-translational modification and splicing isoforms, including phosphorylation and deamidation (Gln/Asn), and may include anti-pan and proteoform-specific antibodies for EGFR.
Claims Coverage
The independent claim is directed to a method that characterizes proteoforms using an individually observable address array, sequential affinity reagent binding with promiscuous binding, and single-molecule-resolution detection of binding presence or absence at specific addresses. The independent claim includes three inventive features.
Individually observable address array with single-polypeptide addresses
An array comprising a plurality of polypeptides where the plurality includes a first proteoform and a second proteoform, each proteoform present at an individually observable address, with each individual address comprising a single polypeptide.
Sequential affinity reagent binding with promiscuous cross-binding
Contacting the array with a first affinity reagent and a second affinity reagent, where the first affinity reagent binds the first proteoform and the second affinity reagent binds the second proteoform, the first affinity reagent is removed prior to contacting with the second affinity reagent, and at least one of the first and second affinity reagents binds promiscuously to the first and second proteoforms.
Single-molecule-resolution detection of binding presence/absence at addresses
Detecting the presence or absence of binding of the affinity reagents at the individually observable addresses to characterize at least one proteoform, including observing the first proteoform and the second proteoform at single molecule resolution at corresponding addresses.
Across the independent claim, proteoform characterization is achieved by addressing single polypeptides on an array, applying sequential affinity reagents with promiscuous binding, and detecting binding presence or absence at individually observable addresses with single-molecule-resolution observations for both proteoforms.
Stated Advantages
Allows characterization of proteoforms of a polypeptide by observing binding presence or absence at individually observable addresses with single molecule resolution.
Enables proteoform characterization even when at least one affinity reagent binds promiscuously to multiple proteoforms.
Documented Applications
Proteoform quantitation/characterization using individually addressable single-molecule polypeptide arrays with sequential or multiplex affinity-reagent binding and signal detection at each address, followed by decoding to infer which proteoforms are present.
Proteoform characterization refined for post-translational modification and splicing isoforms, including phosphorylation and deamidation (Gln/Asn), using affinity binding context for the relevant proteoforms.
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