REACTIVE PEPTIDE LABELING
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.