Protein isoform discrimination and quantitative measurements thereof
View Patent ↗The invention relates to methods, reagents and apparatus for detecting protein isoforms (e.g., those due to alternative splicing, or different disease protein isoforms or degradation products) in a sample, including using combinations of capture agents to identify the isoforms to be detected/measured.
1. A method for detecting unambiguously the presence of one or more of a plurality of target protein isoforms in a sample, the method comprising:
a. fragmenting the target protein isoforms using a mixture of preselected proteases to produce a plurality of target peptides each comprising a first epitope and a second epitope;
b. contacting the plurality of target peptides with immobilized first binding agents that bind to respective first epitopes, wherein at least a portion of the respective first epitopes are present on peptides other than the target peptides;
c. contacting the plurality of target peptides with detectably labeled second binding agents that bind to respective second epitopes; and
d. detecting target peptides bound by combinations of said first and second binding agents to indicate unambiguously the presence of the target protein isoforms in the sample.
2. The method of claim 1 , wherein at least a portion of the respective second epitopes each are present on more than one peptide.
3. The method of claim 2 , wherein at least a portion of the combinations of first and second epitopes each are present on more than one peptide.
4. The method of claim 3 , wherein step d. comprises deconvoluting relative signals from target peptides bound by respective combinations of said first and second binding agents.
5. The method of 1 , wherein the mixture of preselected proteases comprises trypsin or Lys-C.
6. The method of claim 1 , wherein the first binding agents are immobilized on a solid support at known positions.
7. The method of claim 6 , wherein the solid support comprises an array.
8. The method of claim 6 , wherein the solid support comprises a plurality of beads.
9. The method of claim 1 , wherein the sample is pre-treated by purification and/or denaturation prior to step b.
10. The method of claim 1 , wherein the first and/or the second binding agents are antibodies or functional antibody fragments thereof.
11. The method of claim 1 , further comprising quantitating the binding of said first or second binding agents to said target peptides to determine the amount and/or concentration of said target protein isoforms in the sample.
12. The method of claim 1 , wherein one of the second binding agents is detectably labeled with a fluorescent label.
13. The method of claim 1 , wherein the target protein isoforms comprise expression products of alternatively spliced RNAs.
14. The method of claim 13 , wherein the expression products of alternatively spliced RNAs are the only isoforms present in the sample.
15. The method of claim 1 , wherein the first and second epitopes do not span junctions between expression products of different exons.
16. The method of claim 1 , wherein the target peptides comprise a first epitope comprising at least a portion of an expression product of a first exon and a second epitope comprising at least a portion of an expression product of a second exon.
17. The method of claim 1 , further comprising quantitating the binding of said binding agents to determine at least the relative quantity of at least two different target protein isoforms in the sample.
18. The method of claim 1 , wherein detection of binding is effected by detecting an optical signal generated by an optical label on the second binding agents bound to a said target peptide captured at a selected position on a solid support.
19. The method of claim 1 , wherein step d. comprises detecting multiple target peptides bound by multiple combinations of said first and second binding agents to determine unambiguously whether a said target protein isoform is present in the sample.
20. A method for detecting a target protein in a sample comprising a mixture of proteins, the method comprising:
a. fragmenting the target protein using preselected proteases to produce peptides each comprising a first linear epitope and a second linear epitope, both of which potentially are present in other proteins in the sample;
b. contacting said peptides with a pair of first and second binding agents which bind to said first and second linear epitopes, respectively, the binding of the combination of the binding agents being unambiguously indicative of the presence of the target protein in the sample; and,
c. detecting the binding of said first and second binding agents to said peptides as an indication of the presence of the target protein in the sample.