Method of determining a protein in a sample
View Patent ↗The present invention relates to a method of determining a protein in a sample and the use of the method for the identification of aptamer target proteins.
1. A method of labeling a protein in a sample, comprising the steps of:
(a) providing an aptamer, which is functionalized with a labeling reagent connected to the 5′-end of the aptamer via a linker moiety, wherein the labeling reagent comprises: i) a photo-activatable crosslinking moiety and ii) a labeling moiety selected from the group consisting of biotin and desthiobiotin;
(b) incubating the aptamer of step (a) with a sample comprising a target protein of the aptamer; and
(c) irradiating the sample with ultraviolet light for crosslinking the aptamer via the photo-activatable crosslinking moiety to the protein, wherein the aptamer is not cleaved by the irradiation; thereby labeling the protein in the sample.
2. The method of claim 1 , wherein the photo-activatable crosslinking moiety is selected from the group comprising aryl azides (phenyl azide), diazirine derivatives, and benzophenone derivatives.
3. The method of claim 1 , wherein the linker moiety provides a spacer arm length of about 6 Å to 45 Å between the 5′-end of the aptamer and the crosslinking moiety.
4. The method of claim 1 , wherein the labeling reagent is Sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate.
5. The method of claim 1 , further comprising incubating the protein labeled in step (c) with avidin-, neutravidin-, or streptavidin-coated magnetic beads.
6. The method of claim 1 , wherein the protein labeled in step (c) is a purified protein, or a protein in a cell, or an exosome, or a circulating microvesicle.
7. The method of claim 1 , wherein the protein labeled in step (c) is a protein on the surface of a cell, a tissue, an exosome, or a circulating microvesicle.
8. The method of claim 1 , wherein the protein labeled in step (c) is expressed in the proteome of a cell, a tissue or an organism or is localized on exosomes or circulating microvesicles.
9. The method of claim 1 , further comprising detecting a cell, a tissue, an exosome or a circulating microvesicle comprising the protein labeled in step (c).
10. The method of claim 5 , further comprising sorting of the magnetic beads.
11. The method of claim 1 , wherein the aptamer is functionalized at the 5′-end with the following structure:
which is attached to the 5′-end of the aptamer via the alkane chain linker.
12. The method of claim 3 , wherein the spacer arm length is about 20 Å to 40 Å.
13. The method of claim 1 , wherein the method comprises incubating the protein labeled in step (c) with a construct comprising avidin-, neutravidin-, or streptavidin.
14. The method of claim 8 , wherein the cell, the tissue, or the organism comprise a disease state, or wherein the exosome or circulating microvesicle are shed from a cell or tissue comprising a disease state.
15. The method of claim 1 , wherein the protein is a biomarker of a disease state.
16. The method of claim 2 , wherein the diazirine derivative comprises 3-trifluoromethyl-3-phenyl-diazirine (TPD).
17. The method of claim 1 , further comprising detecting the protein labeled in step (c).
18. The method of claim 1 , further comprising determining an amount of the protein labeled in step (c).
19. The method of claim 1 , wherein the target protein of the aptamer is known.
20. The method of claim 1 , wherein the target protein of the aptamer is unknown.
21. The method of claim 1 , wherein the sample comprises a biological material, a cell, an organelle, a tissue, a biological fluid, a biological molecule, an extracts of any of the foregoing, or any combination thereof.