SYSTEMS AND METHODS FOR BIOMOLECULE QUANTITATION
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.
1 . (canceled)
2 . An array composition, comprising:
(a) a solid support comprising a plurality of sites, wherein each site is optically resolvable at single-analyte resolution from any other site of the plurality of sites, wherein a plurality of polypeptides is attached to sites of the plurality of sites, wherein each of the sites of the plurality of sites is attached to a single polypeptide of the plurality of polypeptides; and
(b) affinity reagents of a plurality of affinity reagents bound to polypeptides of the plurality of polypeptides, wherein each affinity reagent of the plurality of affinity reagents recognizes a characteristic of a proteoform of a species of polypeptide,
wherein polypeptides of the plurality of polypeptides are of the species of polypeptide, and wherein the plurality of polypeptides comprises two or more proteoforms of the species of polypeptide.
3 . The array composition of claim 2 , wherein a polypeptide of the polypeptides of the plurality of polypeptides has a full-length primary structure of the species of polypeptide.
4 . The array composition of claim 2 , wherein a polypeptide of the polypeptides of the plurality of polypeptides is a splicing isoform of the species of polypeptide.
5 . The array composition of claim 2 , wherein each site of the plurality of sites is attached to an anchoring group, wherein the single polypeptide of the plurality of polypeptides is attached to the anchoring group.
6 . The array composition of claim 5 , wherein the anchoring group comprises a biomolecule, polymer, or particle.
7 . The array composition of claim 6 , wherein the particle comprises a structured nucleic acid particle.
8 . The array composition of claim 2 , wherein the plurality of polypeptides comprises a second species of polypeptides.
9 . The array composition of claim 8 , wherein the second species of polypeptide comprises two or more proteoforms of the second species of polypeptide.
10 . The array composition of claim 9 , wherein each affinity reagent of the plurality of affinity reagents further recognizes a proteoform of the second species of polypeptide.
11 . The array composition of claim 2 , wherein a proteoform of the two or more proteoforms of the species of polypeptide comprises a post-translational modification selected from the group consisting of: myristoylation, palmitoylation, isoprenylation, prenylation, farnesylation, geranylgeranylation, lipoylation, flavin moiety attachment, Heme C attachment, phosphopantetheinylation, retinylidene Schiff base formation, dipthamide formation, ethanolamine phosphoglycerol attachment, hypusine, beta-Lysine addition, acylation, acetylation, deacetylation, formylation, alkylation, methylation, C-terminal amidation, arginylation, polyglutamylation, polyglyclyation, butyrylation, gammacarboxylation, glycosylation, glycation, polysialylation, malonylation, hydroxylation, iodination, nucleotide addition, phosphoate ester formation, phosphoramidate formation, phosphorylation, adenylylation, uridylylation, propionylation, pyrolglutamate formation, S-glutathionylation, nitrosylation, S-sulfenylation, S-sulfinylation, S-sulfonylation, succinylation, sulfation, glycation, carbamylation, carbonylation, isopeptide bond formation, biotinylation, oxidation, reduction, pegylation, ISGylation, SUMOylation, ubiquitination, neddylation, pupylation, citrullination, deamidation, elminylation, disulfide bridge formation, proteolytic cleavage, isoaspartate formation, and racemization.
12 . The array composition of claim 2 , wherein the characteristic of the proteoform recognized by each affinity reagent is an epitope containing a post-translational modification of the proteoform of the species of polypeptide.
13 . The array composition of claim 2 , wherein the characteristic of the proteoform recognized by each affinity reagent is a functional group of a post-translational modification of the proteoform of the species of polypeptide.
14 . The array composition of claim 2 , wherein each site is separated from any other site of the plurality of sites by at least 0.5 microns.
15 . The array composition of claim 14 , wherein each site is separated from any other site of the plurality of sites by at least 1 micron.
16 . The array composition of claim 2 , wherein the solid support comprises silicon, silicon dioxide, borosilicate glass, fused silica, or quartz.
17 . The array composition of claim 2 , wherein each affinity reagent of the plurality of affinity reagents further comprises a detectable label.
18 . A system for detecting the presence of a polypeptide proteoform, comprising:
(a) a solid support comprising a plurality of sites, wherein each site is optically resolvable from any other site of the plurality of sites, wherein a plurality of polypeptides is attached to sites of the plurality of sites, wherein each of the sites of the plurality of sites is attached to a single polypeptide of the plurality of polypeptides;
(b) a plurality of detectable affinity reagents, wherein each detectable affinity reagent of the plurality of detectable affinity reagents recognizes a proteoform of a species of polypeptide; and
(c) a detector that is configured to detect presence or absence of an optical signal from a detectable affinity reagent at each site of the plurality of sites,
wherein polypeptides of the plurality of polypeptides are of the species of polypeptide, and wherein the plurality of polypeptides comprises two or more proteoforms of the species of polypeptide.
19 . The system of claim 18 , further comprising a processor that is configured to: i) receive from the detector a presence or absence of the optical signal at each site of the plurality of sites, and ii) determine for each site of the plurality of sites a presence or absence of the proteoform of the species of polypeptide.
20 . The system of claim 19 , wherein the processor is further configured to quantify an abundance of the proteoform of the species of polypeptide in the plurality of polypeptides.
21 . A method for detecting a polypeptide proteoform, comprising:
(a) providing a solid support comprising a plurality of sites, wherein each site is optically resolvable from any other site of the plurality of sites, wherein a plurality of polypeptides is attached to sites of the plurality of sites, wherein each of the sites of the plurality of sites is attached to a single polypeptide of the plurality of polypeptides;
(b) contacting to the solid support a plurality of detectable affinity reagents, wherein each detectable affinity reagent of the plurality of detectable affinity reagents recognizes a proteoform of a species of polypeptide; and
(c) detecting at single-analyte resolution for each site of the plurality of sites a presence or absence of an optical signal from a detectable affinity reagent at each site of the plurality of sites, thereby detecting a presence or absence of the proteoform of the species of polypeptide at each site of the plurality of sites.