Method of determining a protein in a sample

Abstract

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 determining a protein in a sample, comprising the steps of:
 (a) providing an aptamer, which is functionalised at the 5′-end with a labeling reagent comprising a photo-activatable crosslinking moiety, a labeling moiety, and a linker moiety connecting the labeling reagent to the 5′-end of the aptamer;   (b) incubating a sample comprising an aptamer target protein with the functionalised aptamer of step (a);   (c) irradiating the sample with ultraviolet light for crosslinking the aptamer via the photoactivatable crosslinking moiety to the protein; and   (d) determining the protein via the labeling moiety.   
     
     
         2 . The method according to  claim 1 , wherein the photo-activatable crosslinking moiety is selected from the group comprising aryl azides (phenyl azide), diazirine derivatives such as 3-trifluoromethyl-3-phenyl-diazirine (TPD), and benzophenone derivatives. 
     
     
         3 . The method according to  claim 1 , wherein the labeling moiety is selected from the group comprising biotin and desthiobiotin. 
     
     
         4 . The method according to  claim 1 , wherein the linker moiety provides a spacer arm length of about 6 Å to 45 Å, preferably about 20 Å to 40 Å, between the 5′-end of the aptamer and the crosslinking moiety. 
     
     
         5 . The method according to  claim 1 , wherein the labeling reagent is Sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)hexanoamido]ethyl-1,3′-dithiopropionate. 
     
     
         6 . The method according to  claim 1 , wherein the method comprises incubating a biotin-labeled cross-linked aptamer-protein complex formed in step (c) with avidin-, neutravidin-, or streptavidin-coated magnetic beads. 
     
     
         7 . The method according to  claim 1 , wherein step (d) includes determining a protein in a cell, or in an exosome, or in a circulating microvesicle. 
     
     
         8 . The method according to  claim 1 , wherein step (d) includes determining a protein on the surface of a cell, or an exosome, or a circulating microvesicle. 
     
     
         9 . Use of the method according to  claim 1 , for the identification of aptamer target proteins, particularly of aptamer target proteins in the proteome of cells, tissue or organisms, particularly for proteins of exosomes or circulating microvesicles. 
     
     
         10 . Use of the method according to  claim 1 , for cell surface labelling. 
     
     
         11 . Use of the method according to  claim 6  for magnetic cell sorting.