Reactive peptide labeling

Abstract

Provided herein are sulfo n-hydroxysuccimidyl ester (sulfo-SE) linked peptides, methods of synthesis thereof, and methods of using such peptides for labeling of biomolecules. In particular, peptides comprising non-alkyl group such as serine, threonine, cysteine, tyrosine, glutamic acid, and aspartic acid are stably modified (e.g., without autoreactivity) with a sulfo-SE group and used to label or otherwise modify biomolecules.

1 . A composition comprising a peptide linked to a sulfo n-hydroxysuccimidyl ester (sulfo-SE) group, wherein the peptide does not comprise a cysteine or lysine residue. 
     
     
         2 . The composition of  claim 1 , wherein the sulfo-SE is linked to the N-terminus of the peptide. 
     
     
         3 . The composition of  claim 1 , wherein the sulfo-SE is linked to the C-terminus of the peptide. 
     
     
         4 . The composition of  claim 1 , wherein the sulfo-SE is linked to an amino acid sidechain of the peptide. 
     
     
         5 . The composition of  claim 1 , wherein the peptide comprises at least one non-alkyl amino acid selected from serine, threonine, tyrosine, glutamic acid, arginine, histidine, tryptophan and aspartic acid. 
     
     
         6 . The composition of  claim 5 , wherein the at least one reactive non-alkyl amino acid is a or tyrosine. 
     
     
         7 . The composition of  claim 5 , wherein the at least one reactive nucleophilic amino acid is an arginine. 
     
     
         8 . The composition of  claim 1 , wherein the sulfo-SE group is linked to the peptide by a non-peptide linker group. 
     
     
         9 . The composition of  claim 8 , wherein the linker group comprises and alkyl or heteroalkyl chain. 
     
     
         10 . The composition of  claim 8 , wherein the linker comprises one or more sidechain substituents. 
     
     
         11 . The composition of  claim 1 , wherein the peptide is 4-50 amino acids in length. 
     
     
         12 . The composition if  claim 11 , wherein the peptide is 8-20 amino acids in length. 
     
     
         13 . The composition of  claim 1 , wherein the sulfo-SE is attached to the N-terminus of the peptide. 
     
     
         14 . The composition of  claim 13 , wherein the sulfo-SE is attached to the N-terminus of the peptide via a linker group. 
     
     
         15 . The composition of  claim 1 , wherein the peptides comprise a fluorophore or chromophore conjugate. 
     
     
         16 . The composition of  claim 1 , wherein the peptide is a component of a biomolecular complex. 
     
     
         17 . The composition of  claim 14 , wherein the peptide is a component of a biomolecular complex. 
     
     
         18 . The composition of  claim 17 , wherein the peptide comprises 5 or fewer substitutions relative to SEQ ID NO: 10 (SmBiT). 
     
     
         19 . The composition of  claim 17 , wherein one or more lysine of SEQ ID NO: 1 are replaced with arginine. 
     
     
         20 . The composition of  claim 19 , wherein the peptide comprises Pep691 (SEQ ID NO: 23). 
     
     
         21 . The composition of  claim 19 , wherein the peptide comprises SmBiT (SEQ ID NO: 10). 
     
     
         22 . The composition of  claim 18 , wherein the peptide is conjugated to a fluorophore. 
     
     
         23 . The composition of  22 , wherein the peptide comprises fluorophore conjugated to an arginine. 
     
     
         24 . The composition of  23 , wherein the peptide comprises fluorophore conjugated to SEQ ID NO: 23. 
     
     
         25 . The composition of  23 , wherein the peptide comprises fluorophore conjugated to SEQ ID NO: 10. 
     
     
         26 . A method of labeling a biomolecule with a peptide comprising contacting the biomolecule with a composition of one of  claims 1 - 25 , under conditions such that the sulfo-SE group reacts with an amine on the biomolecule. 
     
     
         27 . The method of  claim 24 , wherein a peptide composition of  claim 14  contacts the biomolecule under conditions such that the sulfo-SE group reacts with amine on the biomolecule. 
     
     
         28 . The method of  claim 26  or  27 , wherein the amine is a primary amine. 
     
     
         29 . The method of  claim 14 , wherein the biomolecule is selected from the group consisting of an antigen, an antibody, an antibody fragment, a nanobody, a darpin, a non-antibody protein, a receptor, a ligand, a toxin, a cytokine, a nucleic acid, a nucleoprotein complex, a peptide, an amino acid, a sugar, a drug, and streptavidin. 
     
     
         30 . A method of labeling a peptide with a sulfo-SE moiety comprising contacting the peptide with a sulfo-NHS compound under conditions such that the hydroxy of the sulfo-NHS compound reacts with the terminal amine of the peptide, wherein the peptide does not comprise a cysteine or lysine residue. 
     
     
         31 . The method of  claim 20 , wherein the peptide comprises at least one reactive nucleophilic amino acid. 
     
     
         32 . A composition comprising a biomolecule labeled with a peptide of one of  claims 1 - 25 . 
     
     
         33 . A method comprising contacting the composition of  claim 32  with an analyte. 
     
     
         34 . The method of  claim 33 , wherein the analyte is selected from the group consisting of an antigen, an antibody, an antibody fragment, a nanobody, a darpin, a non-antibody protein, a receptor, a ligand, a toxin, a cytokine, a nucleic acid, a nucleoprotein complex, a peptide, an amino acid, a sugar, a drug, and streptavidin. 
     
     
         35 . The method of  claim 35 , wherein the analyte is linked to a complementary polypeptide capable of forming a bioluminescent complex with the peptide on the biomolecule. 
     
     
         36 . The method of  claim 35 , further comprising contacting the bioluminescent complex with a substrate for the bioluminescent complex and detecting luminescence. 
     
     
         37 . A composition comprising an analyte labeled with a peptide of one of  claims 1 - 25 . 
     
     
         38 . A method comprising contacting the composition of  claim 37  with a biomolecule. 
     
     
         39 . The method of  claim 38 , wherein the biomolecule is linked to a complementary polypeptide capable of forming a bioluminescent complex with the peptide on the analyte. 
     
     
         40 . The method of  claim 39 , further comprising contacting the bioluminescent complex with a substrate for the bioluminescent complex and detecting luminescence, fluorescence, and/or BRET. 
     
     
         41 . A composition comprising an analyte labeled with a first peptide of one of  claims 1 - 25  and a biomolecule labelled with a second peptide of one of  claims 1 - 25 , wherein the first and second peptides are capable of forming a bioluminescent complex in the presence of a complementary polypeptide. 
     
     
         42 . A method comprising contacting the analyte and biomolecule of  claim 41  with the complementary polypeptide and forming the bioluminescent complex. 
     
     
         43 . The method of  claim 42 , further comprising contacting the bioluminescent complex with a substrate for the bioluminescent complex and detecting luminescence. 
     
     
         44 . The method of one of  claims 26 - 31 ,  33 - 36 ,  38 - 40 , and  42 - 43 , wherein one or more of the peptides is a fluorophore or chromophore-conjugated peptide. 
     
     
         45 . The method of  claim 44 , further comprising detecting fluorescence/light and/or BRET from the bioluminescent complex to the fluorophore or chromophore. 
     
     
         46 . The method of  claim 45 , wherein the number of labeling per biomolecule is calculated by the number of fluorophore or chromophore molecules per biomolecule. 
     
     
         47 . The method of  claim 44 , the fluorophore molecule is a FAM, TAMRA, ROX, silo-rhodamine, BODIPY, TOM, Dyomics dye, or a carbon-rhodamine, but not limited to those fluorophores. 
     
     
         48 . A method comprising
 (a) forming a bioluminescent complex of a SEQ ID NO: 1 (SmBiT) labeled-analyte biomolecule and the LgBiT-labeled analyte biomolecule specific antibody;   (b) contacting the bioluminescent complex with the analyte;   (c) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and   (d) detecting light output from the bioluminescent complex.   
     
     
         49 . A method comprising
 (a) contacting an analyte with a SEQ ID NO: 1 (SmBiT) labeled-analyte specific antibody and a LgBiT-labeled analyte-specific antibody, and forming a bioluminescent complex;   (b) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and   (c) detecting light output from the bioluminescent complex.   
     
     
         50 . A method comprising:
 (a) contacting an analyte with a SEQ ID NO: 1 (SmBiT) labeled-analyte specific antibody, a SEQ ID NO: 11 (HiBiT)-labeled analyte-specific antibody, and a polypeptide copable for forming a bioluminescent complex with HiBiT and SmBiT;   (b) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and   (c) detecting light output from the bioluminescent complex.   
     
     
         51 . A method comprising contacting a biomolecule labeled with a composition of one of  claims 1 - 25  with an analyte. 
     
     
         52 . The method of  claim 51 , wherein the analyte is selected from the group consisting of an antigen, an antibody, a non-antibody protein, a receptor, a ligand, a toxin, a cytokine, a nucleic acid, a peptide, an amino acid, a sugar, a drug, a nucleoprotein complex, biotin, and streptavidin. 
     
     
         53 . The method of  claim 51 , wherein the analyte biomolecule is labeled with SEQ ID NO: 1 (SmBiT). 
     
     
         54 . The method of  claim 51 , wherein
 (a) forming a bioluminescent complex from a SEQ ID NO: 1 (SmBiT) labeled-analyte biomolecule and a LgBiT-labeled analyte biomolecule specific antibody;   (b) contacting the bioluminescent complex with the analyte;   (c) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and   (d) detecting light output from the bioluminescent complex.   
     
     
         55 . The method of  claim 51 , wherein
 (a) forming a bioluminescent complex from SEQ ID NO: 1 (SmBiT) labeled- and LgBiT-labeled analyte specific antibodies;   (b) contacting the bioluminescent complex with the analyte;   (c) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and   (d) detecting light output from the bioluminescent complex.   
     
     
         56 . The method of  claim 51 , wherein
 (a) contacting an analyte with SEQ ID NO: 10 (SmBiT)-labeled antibodies or receptors and SEQ ID NO: 11 (HiBiT)-labeled antibodies or receptors;   (b) contacting the analyte with LgBiT to form a bioluminescent complex;   (c) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and   (d) detecting light output from the bioluminescent complex.   
     
     
         57 . The method of  claim 51 , wherein
 a) contacting SmBiT- or HiBiT-labeled-analyte biomolecule with a HiBiT- or SmBiT-labeled analyte biomolecule specific antibody;   (b) contacting with LgBiT to form a bioluminescent complex;   (c) contacting with the analyte   (d) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and   (e) detecting light output from the bioluminescent complex.   
     
     
         58 . The method of  claim 57 , wherein the peptide is a fluorophore or chromophore-conjugated peptide. 
     
     
         59 . The method of  claim 58 , wherein the number of labeling per biomolecule is calculated by the number of fluorophore or chromophore molecules per biomolecule. 
     
     
         60 . The method of  claim 58 , the fluorophore molecule is a FAM, TAMRA, ROX, silo-rhodamine, BODIPY, TOM, Dyomics dye, or a carbon-rhodamine, but not limited to those fluorophores. 
     
     
         61 . The method of  claim 51 , wherein
 (a) forming a bioluminescent complex from a fluorophore-conjugated SEQ ID NO: 1 (SmBiT) labeled-analyte biomolecule and a LgBiT-labeled analyte biomolecule specific antibody;   (b) contacting the bioluminescent complex with the analyte;   (c) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and   (d) detecting light output from the bioluminescent complex.   
     
     
         62 . The method of  claim 51 , wherein
 (a) contacting an analyte with both a fluorophore-conjugated SEQ ID NO: 1 (SmBiT) labeled- and LgBiT-labeled analyte specific antibodies, forming a bioluminescent complex   (b) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and   (c) detecting light output from the bioluminescent complex.   
     
     
         63 . The method of  claim 51 , wherein
 (a) one of SEQ ID NO: 10 (SmBiT) and SEQ ID NO: 11 (HiBiT)-peptides is the fluorophore-conjugated peptide;   (b) the analyte contacts SEQ ID NO: 10 (SmBiT) and SEQ ID NO: 11 (HiBiT)-labeled antibodies, or receptors, or combination, wherein one of the peptides is a fluorophore-conjugated peptide;   (b) contacting with LgBiT to form a bioluminescent complex;   (c) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and   (d) detecting light output from the bioluminescent complex.   
     
     
         64 . A method wherein the analyte biomolecule or analyte specific antibody is labeled with fluorophore-conjugated SEQ ID NO: 10 (SmBiT) or SEQ ID NO: 11 (HiBiT), wherein
 (a) SEQ ID NO: 10 (SmBiT) or SEQ ID NO: 11 (HiBiT)-labeled-analyte biomolecule contacts the SEQ ID NO: 11 (HiBiT) or SEQ ID NO: 10 (SmBiT)-labeled analyte biomolecule specific antibody, wherein one of the peptides is the fluorophore-conjugated peptide;   (b) contacting with LgBiT to form a bioluminescent complex;   (c) contacting with the analyte   (d) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and   (e) detecting light output from the bioluminescent complex.