Patent Application
20230192761
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 13, 2022, is named R070870091US03-SEQ-RJP and is 4,860 bytes in size.
The present disclosure relates to compounds and methods for selective C-terminal functionalization of peptides. In certain embodiments, the compounds have improved water-solubility, and are suitable for use in connection with peptide sequencing methodologies.
Array-based platforms are widely used in massively parallel technologies for detecting and analyzing biomolecules, such as proteins and peptides. Examples of these platforms include microarrays used in biosensing and pixel arrays used in single molecule sequencing. Arrays generally include a collection of exceptionally small regions on the surface of a single device, with each region capable of independently assaying a sample. These regions can include selectively functionalized surface portions that are reactive toward a complementary functional moiety of a biomolecule, which permits immobilization of the biomolecule to the surface.
In one aspect, the present disclosure provides a method of selective C-terminal functionalization of a peptide, comprising:
a. reacting a plurality of peptides of Formula (I):
P—R(CO2H)n (I)
or salts thereof;
with a compound of Formula (II):
HX-L1-R1 (II)
to obtain a plurality of compounds of Formula (III):
P—R—CO—X-L1-R1]n (III)
or salts thereof; and
b. reacting the plurality of compounds of Formula (III), or salts thereof, with a compound of Formula (IV):
R2-L2-Z (IV)
to obtain a plurality of compounds of Formula (V):
P—R—CO—X-L1-Y-L2-Z]n (V)
or salts thereof; wherein m, n, P, R(CO2H)n, HX, X, L1, L2, R1, R2, Y and Z are defined herein.
In another aspect, the present disclosure provides a method of selective C-terminal amine functionalization of a peptide, comprising:
a. reacting a plurality of peptides of Formula (VI):
or salts thereof, with a compound of Formula (VII):
to obtain a plurality of compounds of Formula (VIII):
or salts thereof; and
b. reacting the plurality of compounds of Formula (VIII), or salts thereof, with a compound of Formula (IX):
R5-L4-Z1; (IX)
to afford a plurality of compounds of Formula (X):
or salts thereof; wherein P, L3, L4, R3, R4, Y1, and Z1 are as defined herein.
In another aspect, the present disclosure provides a method of selective N-functionalization of a peptide, comprising reacting a plurality of peptides of Formula (XI):
or salts thereof, wherein each P independently is a peptide having an N-terminal amine, with a compound of Formula (XII):
under conditions (a), comprising Cu2+ and a buffer having a pH of about 7-8.5; to obtain a plurality of N-terminal azido compounds of Formula (XIIIa):
or salts thereof; or
under conditions (b), comprising Cu2+ and a buffer having a pH of about 10-11; to obtain a plurality of ε-azido compounds of the Formula (XIIIb):
or salts thereof, wherein P is as defined herein.
In some embodiments, the method further comprises reacting the plurality of compounds of Formula (XIIIb) or salts thereof with a compound of Formula (XIV):
R6-L5-Z2 (XIV)
wherein R6 is a moiety comprising an alkyne or a strained alkene; L5 is a linker or is absent; and Z2 is a water-soluble moiety;
to obtain a plurality of compounds of Formula (XV), or salts thereof:
wherein Y2 is a moiety resulting from a click reaction with the azide moiety of Formula (XIIIb) and R6.
In some aspects, the present disclosure provides a method of selective functionalization in accordance with any one of the methods described herein, wherein the method comprises reacting a single peptide molecule in place of a plurality of peptides. Accordingly, in some embodiments, the method comprises reacting a single polypeptide of any one of Formulae (I), (VI), and (XI) in accordance with the methods described herein.
Aspects of the disclosure relate to methods and compositions for functionalizing a peptide, e.g., a single peptide molecule or a plurality of peptide molecules. In some embodiments, the disclosure relates to the functionalization of a terminal end or an amino acid side-chain of a peptide. In some embodiments, as described herein, functionalization can impart an attachment functionality to the peptide. For example, in some embodiments, a functionalized peptide in accordance with the application comprises a functional moiety (e.g., biotin or bis-biotin) configured for attachment to a complementary functional moiety (e.g., an avidin protein). In some embodiments, the complementary functional moiety is attached to a surface.
Accordingly, in some aspects, the disclosure relates to preparing a peptide for immobilization to a surface. In some aspects, the disclosure provides methods of immobilizing peptides to a surface of a solid support. In some embodiments, the solid support comprises a plurality of sample wells formed at the surface of the solid support. In some embodiments, each sample well of the plurality is configured to immobilize a single peptide molecule therein. In some embodiments, a plurality of peptides is contacted to the solid support to prepare an array of sample wells, where each sample well contains a single peptide molecule attached to a surface of the sample well.
In some embodiments, methods and compositions provided herein can be used to modify a terminal end or other functional group of a peptide with one or more water-soluble moieties that enhance solubility of the peptide in an aqueous solution. In some embodiments, a water-soluble moiety is useful for short peptides that result from fragmentation of a protein (e.g., via enzymatic digestion, as described herein), as short peptides are relatively insoluble in an aqueous solution. For example, in some embodiments, functionalization of a peptide (or a plurality of peptides) can comprise conjugating a water-soluble moiety to the polypeptides. Examples of water-soluble moieties include, without limitation, oligonucleotides, single- or double-stranded nucleic acids (e.g., comprising DNA, RNA, and variants thereof), polyol and polyether compounds (e.g., PEG), sugar polymers, and other charged polymers.
In one aspect, the present disclosure provides a method of selective C-terminal functionalization of a peptide, comprising:
a. reacting a plurality of peptides of Formula (I):
P—R(CO2H)n (I)
or salts thereof;
with a compound of Formula (II):
HX-L1-R1 (II)
to obtain a plurality of compounds of Formula (III):
P—RCO—X-L1-R1]n (III)
or salts thereof; and
b. reacting the plurality of compounds of Formula (III), or salts thereof, with a compound of Formula (IV):
R2-L2-Z (IV)
to obtain a plurality of compounds of Formula (V):
P—RCO—X-L1-Y-L2-Z]n (V)
or salts thereof; wherein m, n, P, R(CO2H)n, HX, X, L1, L2, R1, R2, Y and Z are defined as follows.
m is an integer of 1-25, inclusive. In certain embodiments, m is 1-10, inclusive. In certain embodiments, m is 5-10, inclusive. In certain embodiments, m is 1-5, inclusive. In certain embodiments, m is 1, 2, 3, 4, 5, 6, 7 8 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.
n is 1 or 2. In certain embodiments, n is 1. In certain embodiments, n is 2.
Each P independently is a peptide. In certain embodiments, P has 2-100 amino acid residues. In certain embodiments, P has 2-30 amino acid residues.
Each R(CO2H)n independently is an amino acid residue having n carboxylate moieties. n is 1 or 2. In certain embodiments, n is 1. When n is 1, R(CO2H)n is lysine or arginine. In a particular embodiment, R(CO2H)n is lysine. In another particular embodiment, R(CO2H)n is arginine. In certain embodiments, n is 2. When n is 2, R(CO2H)n is glutamic acid or aspartic acid. In a particular embodiment, R(CO2H)n is glutamic acid. In another particular embodiment, R(CO2H)n is aspartic acid.
HX is nucleophilic moiety that is capable of being acylated, wherein H is a proton. X is one or more heteroatoms. In certain embodiments, X is O, S, or NH, or NO.
L1 is a linker. In certain embodiments, L1 is a substituted or unsubstituted aliphatic chain, wherein one or more carbon atoms are optionally, independently replaced by a heteroatom, an aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety. In certain embodiments, L1 is polyethylene glycol (PEG). In other embodiments, L1 is a peptide, or an oligonucleotide. In certain embodiments, L1 is less than 5 nm. In certain embodiments L1 is less than 1 nm.
L2 is a linker, or is absent. In certain embodiments, L2 is absent. In certain embodiments, L2 is a substituted or unsubstituted aliphatic chain, wherein one or more carbon atoms are optionally, independently replaced by a heteroatom, an aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety. In certain embodiments, L2 is polyethylene glycol (PEG). In other embodiments, L2 is a peptide, or an oligonucleotide. In certain embodiments L2 is between 5-20 nm, inclusive.
R1 is a moiety comprising a click chemistry handle. In certain embodiments, R1 is a moiety comprising an azide, tetrazine, nitrile oxide, alkyne or strained alkene. In certain embodiments, the alkyne is a primary alkyne. In certain embodiments, the alkyne is a cyclic (e.g., mono- or polycyclic) alkyne (e.g., diarylcyclooctyne, or bicycle[6.1.0]nonyne). In certain embodiments, the strained alkene is trans-cyclooctene. In certain embodiments, R1 is a moiety comprising an azide. In certain embodiments, the tetrazine comprises the structure:
R2 is a moiety comprising a click chemistry handle that is complementary to R1. The click chemistry handle of R2 is capable of undergoing a click reaction (i.e., an electrocyclic reaction to form a 5-membered heterocyclic ring) with R1. For example, when R1 comprises an azide, nitrile oxide, or a tetrazine, then R2 may comprise an alkyne or a strained alkene. Conversely, when R1 comprises an alkyne or a strained alkene, then R2 may comprise an azide, nitrile oxide, or tetrazine. In certain embodiments, R2 is a moiety comprising an azide, tetrazine, nitrile oxide, alkyne or strained alkene. In certain embodiments, the alkyne is a primary alkyne. In certain embodiments, the alkyne is a cyclic (e.g., mono- or polycyclic) alkyne (e.g., diarylcyclooctyne, or bicycle[6.1.0]nonyne). In certain particular embodiments, R2 comprises BCN. In other particular embodiments, R2 comprises DBCO. In certain embodiments, the strained alkene is trans-cyclooctene. In certain embodiments, the tetrazine comprises the structure:
Y is a moiety resulting from the click reaction of R1 and R2. Y is a 5-membered heterocyclic ring resulting from an electrocyclic reaction (e.g., 3+2 cycloaddition, or 4+2 cycloaddition) between the reactive click chemistry handles of R1 and R2. In certain embodiments, Y is a diradical comprising a 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, or 1,4-dihydropyridazyl moiety.
Z is a water-soluble moiety. In certain embodiments, Z imparts water-solubility to the compound to which it is attached. In certain embodiments, Z comprises polyethylene glycol (PEG). In certain embodiments, Z comprises single-stranded DNA. In certain particular embodiments, Z comprises Q24. In certain embodiments, Z comprises double-stranded DNA. In certain embodiments (e.g., compounds of Formula (V)), Z further comprises biotin (e.g., bisbiotin). When Z comprises biotin (e.g., bisbiotin), Z may further comprise streptavidin. In certain embodiments, Z comprises double-stranded DNA. In some embodiments, the moieties of Z are capable of intermolecularly binding another molecule or surface, e.g., to anchor a compound comprising Z to the molecule or surface.
In certain embodiments, the compound of Formula (II) is of Formula (IIa):
In certain embodiments, Formula (III) is of Formula (IIIa):
In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, m is 1. In certain embodiments, m is 5.
In certain embodiments, Formula (IV) comprises TCO, and single-stranded DNA. In certain embodiments, Formula (IV) further comprises biotin (e.g., bisbiotin). In certain embodiments, Formula (IV) is Q24-BisBt-BCN. In certain embodiments, Formula (IV) is Q24-BisBt-DBCO. In certain embodiments, Formula (IV) is Q24-BisBt-TCO. Generally, Formula (IV) may comprise a branching moiety (e.g., a 1, 3, 5-tricarboxylate moiety), wherein two branches are direct or indirect attachments to biotin moieties, and the third branch is an attachment to the water soluble moiety (e.g., a polynucleotide such as Q24). As shown in
In certain embodiments, Formula (V) is of Formula (Va):
wherein m, n is 1 or 2; and L2, Y, and Z are as defined above. In certain particular embodiments, n is 1. In certain particular embodiments, n is 2. In certain particular embodiments, m is 1. In certain particular embodiments, m is 5. In certain particular embodiments, L2 is absent. In certain embodiments, Y comprises a moiety selected from 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, and 1,4-dihydropyridazyl. In certain embodiments, Z comprises single-stranded DNA. In certain embodiments, Z comprises double-stranded DNA. In certain embodiments, Z comprises biotin (e.g., bisbiotin). In certain embodiments, Z further comprises streptavidin.
In certain embodiments, the reaction of step (a) is performed in the presence of a carbodiimide reagent. In certain embodiments, the carbodiimide reagent is water soluble. In a particular embodiment, the carbodiimide reagent is 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). In certain embodiments, the reaction of step (a) is performed at a pH in the range of 3-5. In certain embodiments (e.g., when to total peptide concentration below 1 mM), the concentration of EDC is about 10 mM and the concentration of the compound of Formula (II) is about 20 mM. In certain embodiments (e.g., in connection with Trypsin/LysC digestion, as described below) the concentration of the compound of Formula (II) is about may be about 50 mM and the concentration of EDC may be about 25 mM to suppress C-terminal intramolecular cyclization.
In certain embodiments of step (a), the plurality of compounds of Formula (III) is enriched prior to step (b), for example, by passing the compounds through a G10 sephadex column and/or passing the compounds through a C18 resin column. The use of C18 resin-based enrichment is particularly useful when the compound of Formula (II) is greater than about 200 g/mol. When G-10 sephadex is used in the enrichment, the elution buffer may be 0.5×PBS (pH 7.0). When C18 resin is used in the enrichment, the elution buffer may be 0.1% formic acid with 80% acetonitrile in water. The C18 eluent may be dried and the residue resuspended in 0.5×PBS prior to step (b).
In certain embodiments of step (b), the click reaction between the plurality of compounds of Formula (III) and the compound of Formula (IV) is uncatalyzed. In certain embodiments, the click reaction is catalyzed, for example, using a copper salt (e.g., a Cu+ salt, or a Cu2+ salt that is reduced in situ to a Cu+ salt). Suitable Cu2+ salts include CuSO4. In certain embodiments, the reaction of step (b) comprises heating the reaction mixture.
In certain embodiments, the compound of Formula (IV) is added to the plurality of compounds of Formula (III). In certain embodiments, the total concentration of the compound of Formula (IV) and the plurality of compounds of Formula (III) is maintained in the range between 10 μM to 1 mM.
In certain embodiments of step (b), when Z comprises single-stranded DNA, the method further comprises hybridizing a complementary DNA strand to the single-stranded DNA to obtain a compound wherein Z comprises double-stranded DNA. In certain embodiments, the single-stranded DNA is Q24 and the complementary DNA strand is a Cy3B-labeled Q24 complementary strand.
In certain embodiments of step (b), when Z comprises biotin (e.g., bisbiotin), the method further comprises contacting the biotin (e.g., bisbiotin) with streptavidin to obtain a compound wherein Z comprises biotin (e.g., bisbiotin) and streptavidin.
In certain embodiments, the plurality of peptides of Formula (I), or salts thereof, is obtained by subjecting a protein to enzymatic digestion to obtain a digestive mixture comprising the plurality of peptides of Formula (I), or salts thereof. In certain embodiments, the enzymatic digestion comprises cleaving the C-terminal bonds of aspartic acid and/or glutamic acid residues of the protein. In certain specific embodiments, the enzymatic digestion is Glu-C digestion.
In certain embodiments, the total concentration of the plurality of peptides of Formula (I), or salts thereof, after digestion of 20 μg protein is below 100 μM.
In certain embodiments, the enzymatic digestion is performed in phosphate buffer (pH 7.8) or ammonium bicarbonate buffer (pH 4.0).
In certain embodiments, the enzymatic digestion comprises cleaving the C-terminal bonds of lysine and/or arginine residues of the protein. In certain specific embodiments, the enzymatic digestion is Trypsin+Lys-C digestion.
In certain embodiments, the carboxylic acid moieties of the protein, if present, are protected prior to the enzymatic digestion. For example, the carboxylic acid moieties of the protein, if present, may be esterified prior to enzymatic digestion. In certain specific embodiments, the esterified carboxylic acids are methyl esters.
In certain embodiments, the sulfide moieties of the protein are protected prior to enzymatic digestion. In certain specific embodiments, the sulfide moieties are protected by exposing the protein to tris(carboxyethyl)phosphine (TCEP) and iodoacetamide (ICM), or maleimide.
In certain particular embodiments, TCEP is present in the form of a hydrochloride salt, i.e., TCEP·HCl.
In certain embodiments, the method further comprises the step of enriching the digestive mixture prior to step (a).
In another aspect, the present disclosure provides a method of selective C-terminal amine functionalization of a peptide, comprising:
a. reacting a plurality of peptides of Formula (VI):
or salts thereof, with a compound of Formula (VII):
to obtain a plurality of compounds of Formula (VIII):
or salts thereof; and
b. reacting the plurality of compounds of Formula (VIII), or salts thereof, with a compound of Formula (IX):
R5-L4-Z1; (IX)
to afford a plurality of compounds of Formula (X):
or salts thereof; wherein P, L3, L4, R3, R4, Y1, and Z1 are as defined below.
Each P independently is a peptide. In certain embodiments, P has 2-100 amino acid residues. In certain embodiments, P has 2-30 amino acid residues.
L3 is a linker. In certain embodiments, L3 is a substituted or unsubstituted aliphatic chain, wherein one or more carbon atoms are optionally, independently replaced by a heteroatom, an aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety. In certain embodiments, L3 is polyethylene glycol (PEG). In other embodiments, L3 is a peptide, or an oligonucleotide.
L4 is a linker, or is absent. In certain embodiments, L4 is absent. In certain embodiments, L4 is a substituted or unsubstituted aliphatic chain, wherein one or more carbon atoms are optionally, independently replaced by a heteroatom, an aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety. In certain embodiments, L4 is polyethylene glycol (PEG). In other embodiments, L4 is a peptide, or an oligonucleotide.
R3 is a moiety comprising a click chemistry handle. In certain embodiments, R3 is a moiety comprising an azide, tetrazine, nitrile oxide, alkyne or strained alkene. In certain embodiments, the alkyne is a primary alkyne. In certain embodiments, the alkyne is a cyclic (e.g., mono- or polycyclic) alkyne (e.g., diarylcyclooctyne, or bicycle[6.1.0]nonyne). In certain embodiments, the strained alkene is trans-cyclooctene. In certain embodiments, R1 is a moiety comprising an azide. In certain embodiments, the tetrazine comprises the structure:
R4 is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In certain embodiments, R4 is substituted or unsubstituted phenyl. In certain particular embodiments, R4 is phenyl. In certain particular embodiments, R4 is 4-nitrophenyl.
R5 is a moiety comprising a click chemistry handle that is complementary to R3. The click chemistry handle of R5 is capable of undergoing a click reaction (i.e., an electrocyclic reaction to form a 5-membered heterocyclic ring) with R3. For example, when R3 comprises an azide, nitrile oxide, or a tetrazine, then R5 may comprise an alkyne or a strained alkene. Conversely, when R3 comprises an alkyne or a strained alkene, then R5 may comprise an azide, nitrile oxide, or tetrazine. In certain embodiments, R5 is a moiety comprising an azide, tetrazine, nitrile oxide, alkyne or strained alkene. In certain embodiments, the alkyne is a primary alkyne. In certain embodiments, the alkyne is a cyclic (e.g., mono- or polycyclic) alkyne (e.g., diarylcyclooctyne, or bicycle[6.1.0]nonyne). In certain particular embodiments, R5 comprises BCN. In other particular embodiments, R5 comprises DBCO. In certain embodiments, the strained alkene is trans-cyclooctene. In certain embodiments, the tetrazine comprises the structure:
Y1 is a moiety resulting from the click reaction of R3 and R5. Y1 is a 5-membered heterocyclic ring resulting from an electrocyclic reaction (e.g., 3+2 cycloaddition, or 4+2 cycloaddition) between the reactive click chemistry handles of R3 and R5. In certain embodiments, Y1 is a diradical comprising a 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, or 1,4-dihydropyridazyl moiety.
Z1 is a water-soluble moiety. In certain embodiments, Z1 imparts water-solubility to the compound to which it is attached. In certain embodiments, Z1 comprises polyethylene glycol (PEG). In certain embodiments, Z1 comprises single-stranded DNA. In certain particular embodiments, Z1 comprises Q24. In certain embodiments, Z1 comprises single-stranded DNA. In certain embodiments (e.g., compounds of Formula (V)), Z1 further comprises biotin (e.g., bisbiotin). When Z1 comprises biotin (e.g., bisbiotin), Z1 may further comprise streptavidin. In certain embodiments, Z1 comprises double-stranded DNA. In some embodiments, the moieties of Z1 are capable of intermolecularly binding another molecule or surface, e.g., to anchor a compound comprising Z1 to the molecule or surface.
In certain embodiments, the compound of Formula (VII) is selected from:
In certain embodiments, Formula (VIII) is of Formula (VIIIa) or Formula (VIIIb):
In certain embodiments, Formula (IX) comprises TCO, single-stranded DNA, and biotin (e.g., bisbiotin). In certain embodiments, Formula (IX) is Q24-BisBt-BCN. In certain embodiments, Formula (IX) is Q24-BisBt-DBCO. In certain embodiments, Formula (IX) is Q24-BisBt-TCO. Generally, Formula (IX) may comprise a branching moiety (e.g., a 1, 3, 5-tricarboxylate moiety), wherein two branches are direct or indirect attachments to biotin moieties, and the third branch is an attachment to the water soluble moiety (e.g., a polynucleotide such as Q24). As shown in
In certain embodiments, the reaction of step (a) is performed in the presence of a buffer having a concentration in the range of about 20 mM-500 mM and a pH in the range of about 9-11, and acetonitrile in the range of about 20-70% of total volume. In certain embodiments, the reaction of step (a) is performed in pH 9.5 buffer/acetonitrile (1:3 v/v) at approximately 37° C. In certain embodiments, the reaction of step (a) is performed using a concentration of the compound of Formula (VII) of about 500 μM-50 mM.
In certain embodiments, the plurality of compounds of Formula (VIII) is enriched prior to step (b). In certain embodiments, the enrichment comprises ethyl acetate/hexane extraction. Suitable ranges for ethyl acetate/hexane include, but are not limited to, 20 to 100 volume % ethyl acetate in hexanes. In certain embodiments, the volume of organic solvent used in the extraction is about 10× the volume of aqueous layer. Other water immiscible organic solvents can be used in the extraction, e.g., diethyl ether, dichloromethane, chloroform, benzene, toluene, and n-1-butanol.
In certain embodiments, the reaction of step (b) comprises reacting the compounds of Formula (VIII) with about one equivalent of the compound of Formula (IX). In certain embodiments, the reaction of step (b) comprises heating the reaction mixture.
In certain embodiments of step (b), when Z1 comprises single-stranded DNA, the method further comprises hybridizing a complementary DNA strand to the single-stranded DNA to obtain a compound wherein Z1 comprises double-stranded DNA. In certain embodiments, the single-stranded DNA is Q24 and the complementary DNA strand is a Cy3B-labeled Q24 complementary strand.
In certain embodiments of step (b), when Z1 comprises biotin (e.g., bisbiotin), the method further comprises contacting the biotin (e.g., bisbiotin) with streptavidin to obtain a compound wherein Z1 comprises biotin (e.g., bisbiotin) and streptavidin.
In certain embodiments, the plurality of peptides of Formula (VI), or salts thereof, is obtained by subjecting a protein to enzymatic digestion to obtain a digestive mixture comprising the plurality of peptides of Formula (VI), or salts thereof. The enzymatic digestion comprises cleaving the C-terminal bonds of lysine and/or arginine residues of the protein. In certain embodiments, the enzymatic digestion is performed using Trypsin, Lys-C, or a combination thereof. In certain embodiments, the enzymatic digestion comprises reacting the protein with Trypsin and Lys-C in Tris-HCl buffer (pH 8.5). In certain embodiments, the total concentration of the plurality of peptides of Formula (VI), or salts thereof, after digestion of 20 μg protein is below 100 μM.
In certain embodiments, the enzymatic digestion comprises reacting the protein with Trypsin, wherein the molar ratio of Trypsin:protein is in the range of 1:50 to 1:200, inclusive.
In certain embodiments, the sulfide moieties of the protein are protected prior to enzymatic digestion. In certain specific embodiments, the sulfide moieties are protected by exposing the protein to tris(carboxyethyl)phosphine (TCEP) and iodoacetamide (ICM), or maleimide.
In certain embodiments, the method further comprises the step of enriching the digestive mixture prior to step (a). In certain embodiments, the digestive mixture is used in the method of selective C-terminal amine functionalization of a peptide without enrichment or purification.
In another aspect, the present disclosure provides a method of selective N-functionalization of a peptide, comprising reacting a plurality of peptides of Formula (XI):
or salts thereof, wherein each P independently is a peptide having an N-terminal amine, with a compound of Formula (XII):
under conditions (a), comprising Cu2+, or a precursor thereof, and a buffer having a pH of about 7-8.5; to obtain a plurality of N-terminal azido compounds of Formula (XIIIa):
or salts thereof; or
under conditions (b), comprising Cu2+, or a precursor thereof, and a buffer having a pH of about 10-11; to obtain a plurality of ε-azido compounds of the Formula (XIIIb):
or salts thereof.
In some embodiments, the compound of Formula (XII) is present in the form of a salt. In certain particular embodiments, the compound of Formula (XII) is imidazole-1-sulfonyl azide tetrafluoroborate. In some embodiments, the compound of Formula (XII), or salt thereof, is present in the form of a solution. In certain embodiments, the solution comprises a base (e.g., potassium hydroxide).
Each P independently is a peptide having an N-terminal amine. In certain embodiments, P has 2-100 amino acid residues. In certain embodiments, P has 2-30 amino acid residues.
In certain embodiments, conditions (a) comprise suitable Cu2+ salts, for example CuSO4. In certain embodiments, conditions (a) comprise reaction at about 25° C. for about 30-60 minutes. In a particular embodiment, conditions (a) comprise reaction at ambient temperature (e.g., about 25° C.) for about 60 minutes.
In certain embodiments, the compound of Formula (XII) is replaced by an aryl/heteroaryl sulfonyl azide compound that is not larger than 500 Da. For example, the compound of Formula (XII) may be replaced with a compound of Formula (XIIa):
wherein RA is substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
In certain embodiments, conditions (b) include comprise phosphate or bicarbonate buffer at pH 10.5. In certain embodiments, conditions (b) comprise suitable Cu2+ salts, for example CuSO4. In certain embodiments, conditions (b) comprise about 2.4 molar equivalents of CuSO4 relative to the compound of Formula (XI). In certain conditions (b) comprise about 20 molar equivalents of the compound of Formula (XII) relative to the compound of Formula (XI). In certain embodiments, conditions (b) comprise reaction at about 25° C. In certain embodiments, conditions (b) comprise reaction for about 30-60 minutes. In a particular embodiment, conditions (b) comprise reaction at ambient temperature (e.g., about 25° C.) for about 60 minutes.
In some embodiments, the compound of Formula (XIIa) is present in the form of a solution. In certain embodiments, the solution comprises a base (e.g., potassium hydroxide).
In certain embodiments, the N-terminal:ε selectivity under conditions (b) is at least about 90%.
In certain embodiments, methods utilizing diazo transfer chemistry as described herein further comprise the step of quenching (i.e., neutralizing) unreacted sulfonyl azide reagent by addition of a material which neutralizes the sulfonyl azide reagent. In certain embodiments, the material is a resin or bead, e.g., a polystyrene polyamine bead. Advantageously, a resin or bead may be removed by filtration.
In some embodiments, the plurality of peptides of Formula (XI), or salts thereof, is obtained by subjecting a protein to enzymatic digestion to obtain a digestive mixture comprising the plurality of peptides of Formula (XI), or salts thereof. The enzymatic digestion comprises cleaving the C-terminal bonds of aspartic acid and/or glutamic acid residues of the protein.
In some embodiments, the enzymatic digestion is Trypsin+Lys-C digestion. In some embodiments, the Trypsin+Lys-C digestion comprises reacting the protein with Trypsin and Lys-C at room temperature in pH 9.5 buffer.
In some embodiments, the method further comprises enriching the plurality of compounds of Formula (XIIIb), or salts thereof.
In some embodiments, the method further comprises reacting the plurality of compounds of Formula (XIIIb) or salts thereof with a compound of Formula (XIV):
R6-L5-Z2 (XIV)
wherein R6 is a moiety comprising an alkyne or a strained alkene; L5 is a linker or is absent; and Z2 is a water-soluble moiety;
to obtain a plurality of compounds of Formula (XV), or salts thereof:
wherein Y2 is a moiety resulting from a click reaction with the azide moiety of Formula (XIIIb) and R6.
R6 is a moiety comprising a click chemistry handle that is complementary to the azide moiety of Formula (XIIIb). The click chemistry handle of R6 is capable of undergoing a click reaction (i.e., an electrocyclic reaction to form a 5-membered heterocyclic ring) with the azide moiety of Formula (XIIIb). In certain embodiments, R6 comprises an alkyne or a strained alkene. In certain embodiments, the alkyne is a primary alkyne. In certain embodiments, the alkyne is a cyclic (e.g., mono- or polycyclic) alkyne (e.g., diarylcyclooctyne, or bicycle[6.1.0]nonyne). In certain particular embodiments, R6 comprises BCN. In other particular embodiments, R6 comprises DBCO. In certain embodiments, the strained alkene is trans-cyclooctene.
In certain embodiments, L5 is absent. In certain embodiments, L5 is a substituted or unsubstituted aliphatic chain, wherein one or more carbon atoms are optionally replaced by a heteroatom, an aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety. In certain embodiments, L5 is polyethylene glycol (PEG). In other embodiments, L5 is a peptide, or an oligonucleotide.
In certain embodiments, Z2 comprises PEG. In certain embodiments, Z2 comprises single-stranded DNA. In certain embodiments, Z2 comprises double-stranded DNA. In certain embodiments, Z2 further comprises biotin (e.g., bisbiotin). In certain embodiments, when Z2 comprises single-stranded DNA, the method further comprises hybridizing a complementary DNA strand to the single-stranded DNA to obtain a compound wherein Z2 comprises double-stranded DNA. In certain embodiments, the single-stranded DNA is Q24 and the complementary DNA strand is Cy3B.
In certain embodiments, the compound of Formula (XIV) comprises TCO, single-stranded DNA, and biotin (e.g., bisbiotin). In certain embodiments, Formula (XIV) is Q24-BisBt-BCN. In certain embodiments, Formula (XIV) is Q24-BisBt-DBCO. In certain embodiments, Formula (XIV) is Q24-BisBt-TCO. Generally, Formula (XIV) may comprise a branching moiety (e.g., a 1, 3, 5-tricarboxylate moiety), wherein two branches are direct or indirect attachments to biotin moieties, and the third branch is an attachment to the water soluble moiety (e.g., a polynucleotide such as Q24). As shown in
In certain embodiments, the compound of Formula (XIV) comprises DBCO, single-stranded DNA, and streptavidin (SV). In certain particular embodiments, the compound of Formula (XIV) is DBCO-Q24-SV.
In certain embodiments, when Z2 comprises biotin (e.g., bisbiotin), the method further comprises contacting the biotin (e.g., bisbiotin) with streptavidin to obtain a compound wherein Z2 comprises biotin (e.g., bisbiotin) and streptavidin. In other embodiments, when Z2 comprises streptavidin, the method further comprises contacting the streptavidin with biotin (e.g., bisbiotin) to obtain a compound wherein Z2 comprises streptavidin and biotin (e.g., bisbiotin).
In certain embodiments, the reaction used to conjugate the host to the tag is a “click chemistry” reaction (e.g., the Huisgen alkyne-azide cycloaddition). It is to be understood that any “click chemistry” reaction known in the art can be used to this end. Click chemistry is a chemical approach introduced by Sharpless in 2001 and describes chemistry tailored to generate substances quickly and reliably by joining small units together. See, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004-2021; Evans, Australian Journal of Chemistry (2007) 60: 384-395). Exemplary coupling reactions (some of which may be classified as “click chemistry”) include, but are not limited to, formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems); azide-alkyne Huisgen cycloaddition; thiol-yne addition; imine formation; Michael additions (e.g., maleimide addition); and Diels-Alder reactions (e.g., tetrazine [4+2] cyclo addition).
The term “click chemistry” refers to a chemical synthesis technique introduced by K. Barry Sharpless of The Scripps Research Institute, describing chemistry tailored to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together. See, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004-2021; Evans, Australian Journal of Chemistry (2007) 60: 384-395). Exemplary reactions include, but are not limited to, azide-alkyne Huisgen cycloaddition; and Diels-Alder reactions (e.g., tetrazine [4+2] cycloaddition). In some embodiments, click chemistry reactions are modular, wide in scope, give high chemical yields, generate inoffensive byproducts, are stereospecific, exhibit a large thermodynamic driving force >84 kJ/mol to favor a reaction with a single reaction product, and/or can be carried out under physiological conditions. In some embodiments, a click chemistry reaction exhibits high atom economy, can be carried out under simple reaction conditions, use readily available starting materials and reagents, uses no toxic solvents or use a solvent that is benign or easily removed (preferably water), and/or provides simple product isolation by non-chromatographic methods (crystallization or distillation).
The term “click chemistry handle,” as used herein, refers to a reactant, or a reactive group, that can partake in a click chemistry reaction. For example, a strained alkyne, e.g., a cyclooctyne, is a click chemistry handle, since it can partake in a strain-promoted cycloaddition (see, e.g., Table 1). In general, click chemistry reactions require at least two molecules comprising click chemistry handles that can react with each other. Such click chemistry handle pairs that are reactive with each other are sometimes referred to herein as partner click chemistry handles. For example, an azide is a partner click chemistry handle to a cyclooctyne or any other alkyne. Exemplary click chemistry handles suitable for use according to some aspects of this invention are described herein, for example, in Tables 1 and 2. Other suitable click chemistry handles are known to those of skill in the art.
In some embodiments, click chemistry handles are used that can react to form covalent bonds in the presence of a metal catalyst, e.g., copper (II). In some embodiments, click chemistry handles are used that can react to form covalent bonds in the absence of a metal catalyst. Such click chemistry handles are well known to those of skill in the art and include the click chemistry handles described in Becer, Hoogenboom, and Schubert, Click Chemistry beyond Metal-Catalyzed Cycloaddition, Angewandte Chemie International Edition (2009) 48: 4900-4908.
[a]RT = room temperature, DMF = N,N-dimethylformamide, NMP = N-methylpyrolidone, THF = tetrahydrofuran, CH3CN = acetonitrile.
Additional click chemistry handles suitable for use in methods of conjugation described herein are well known to those of skill in the art, and such click chemistry handles include, but are not limited to, the click chemistry reaction partners, groups, and handles described in PCT/US2012/044584 and references therein, which references are incorporated herein by reference for click chemistry handles and methodology.
In certain aspects, the present disclosure provides compounds of Formulae (II), (IIa), (III), (IIIa), (IV), (V), (Va), (VII), (VIII), (VIIIa), (VIIIb), (XIV), (X), (XI), (XII), (XIIIa), (XIIIb), (XV), and salts thereof, as described herein in various embodiments.
In certain embodiments, the compounds are water soluble.
In certain embodiments, the compounds are useful for applications relating to the analysis of proteins and peptides, such as peptide sequencing. For example, in certain embodiments, compounds of Formulae (V), (X), (XV), and salts thereof, may be covalently or non-covalently attached to a surface.
In certain single molecule analytical methods, a molecule to be analyzed is immobilized onto surfaces such that the molecule may be monitored without interference from other reaction components in solution. In some embodiments, surface immobilization of the molecule allows the molecule to be confined to a desired region of a surface for real-time monitoring of a reaction involving the molecule.
Accordingly, in some aspects, the application provides methods of immobilizing a peptide to a surface by attaching any one of the compounds described herein to a surface of a solid support. In some embodiments, the methods comprise contacting a compound of Formula (V), (X), (XV), or a salt thereof, to a surface of a solid support. In some embodiments, the surface is functionalized with a complementary functional moiety configured for attachment (e.g., covalent or non-covalent attachment) to a functionalized terminal end of a peptide. In some embodiments, the solid support comprises a plurality of sample wells formed at the surface of the solid support. In some embodiments, the methods comprise immobilizing a single peptide to a surface of each of a plurality of sample wells. In some embodiments, confining a single peptide per sample well is advantageous for single molecule detection methods, e.g., single molecule peptide sequencing.
As used herein, in some embodiments, a surface refers to a surface of a substrate or solid support. In some embodiments, a solid support refers to a material, layer, or other structure having a surface, such as a receiving surface, that is capable of supporting a deposited material, such as a functionalized peptide described herein. In some embodiments, a receiving surface of a substrate may optionally have one or more features, including nanoscale or microscale recessed features such as an array of sample wells. In some embodiments, an array is a planar arrangement of elements such as sensors or sample wells. An array may be one or two dimensional. A one dimensional array is an array having one column or row of elements in the first dimension and a plurality of columns or rows in the second dimension. The number of columns or rows in the first and second dimensions may or may not be the same. In some embodiments, the array may include, for example, 102, 103, 104, 105, 106, or 107 sample wells.
An example scheme of peptide surface immobilization is depicted in
In some embodiments, peptide 900 comprising functionalized terminal end 902 is contacted with complementary functional moiety 904 of the solid support to form a covalent or non-covalent linkage group. In some embodiments, functionalized terminal end 902 and complementary functional moiety 904 comprise partner click chemistry handles, e.g., which form a covalent linkage group between peptide 900 and the solid support. Suitable click chemistry handles are described elsewhere herein. In some embodiments, functionalized terminal end 902 and complementary functional moiety 904 comprise non-covalent binding partners, e.g., which form a non-covalent linkage group between peptide 900 and the solid support. Examples of non-covalent binding partners include complementary oligonucleotide strands (e.g., complementary nucleic acid strands, including DNA, RNA, and variants thereof), protein-protein binding partners (e.g., barnase and barstar), and protein-ligand binding partners (e.g., biotin and streptavidin).
In panel (II), peptide 900 is shown immobilized to the bottom surface through a linkage group formed by contacting functionalized terminal end 902 and complementary functional moiety 904. In this example, peptide 900 is attached through a non-covalent linkage group, which is depicted in the zoomed region of panel (III). As shown, in some embodiments, the non-covalent linkage group comprises an avidin protein 920. Avidin proteins are biotin-binding proteins, generally having a biotin binding site at each of four subunits of the avidin protein. Avidin proteins include, for example, avidin, streptavidin, traptavidin, tamavidin, bradavidin, xenavidin, and homologs and variants thereof. In some embodiments, avidin protein 920 is streptavidin. The multivalency of avidin protein 920 can allow for various linkage configurations, as each of the four binding sites are independently capable of binding a biotin molecule (shown as white circles).
As shown in panel (III), in some embodiments, the non-covalent linkage is formed by avidin protein 920 bound to a first bis-biotin moiety 922 and a second bis-biotin moiety 924. In some embodiments, functionalized terminal end 902 comprises first bis-biotin moiety 922, and complementary functional moiety 904 comprises second bis-biotin moiety 924. In some embodiments, functionalized terminal end 902 comprises avidin protein 920 prior to being contacted with complementary functional moiety 904. In some embodiments, complementary functional moiety 904 comprises avidin protein 920 prior to being contacted with functionalized terminal end 902.
In some embodiments, functionalized terminal end 902 comprises first bis-biotin moiety 922 and a water-soluble moiety, where the water-soluble moiety forms a linkage between first bis-biotin moiety 922 and an amino acid (e.g., a terminal amino acid) of peptide 900. Water-soluble moieties are described in detail elsewhere herein.
In some embodiments, the compounds described herein may be subjected to peptide sequencing (also referred to as “polypeptide sequencing”) by detecting single molecule binding interactions during a peptide degradation process. In some embodiments, the peptide being degraded is a compound of Formula (V), (X), (XV), or a salt thereof, that is covalently or non-covalently attached to a surface.
As used herein, sequencing a polypeptide refers to determining sequence information for a polypeptide. In some embodiments, this can involve determining the identity of each sequential amino acid for a portion (or all) of the polypeptide. However, in some embodiments, this can involve assessing the identity of a subset of amino acids within the polypeptide (e.g., and determining the relative position of one or more amino acid types without determining the identity of each amino acid in the polypeptide). In some embodiments, amino acid content information can be obtained from a polypeptide without directly determining the relative position of different types of amino acids in the polypeptide. The amino acid content alone may be used to infer the identity of the polypeptide that is present (e.g., by comparing the amino acid content to a database of polypeptide information and determining which polypeptide(s) have the same amino acid content).
In some aspects, peptide sequencing of the compounds of the disclosure may be performed by identifying one or more types of amino acids of a polypeptide. In some embodiments, one or more amino acids (e.g., terminal amino acids and/or internal amino acids) of the polypeptide are labeled (e.g., directly or indirectly, for example using a binding agent such as an amino acid recognition molecule) and the relative positions of the labeled amino acids in the polypeptide are determined. In some embodiments, the relative positions of amino acids in a polypeptide are determined using a series of amino acid labeling and cleavage steps. However, in some embodiments, the relative position of labeled amino acids in a polypeptide can be determined without removing amino acids from the polypeptide but by translocating a labeled polypeptide through a pore (e.g., a protein channel) and detecting a signal (e.g., a FRET signal) from the labeled amino acid(s) during translocation through the pore in order to determine the relative position of the labeled amino acids in the polypeptide molecule.
In some embodiments, the identity of a terminal amino acid (e.g., an N-terminal or a C-terminal amino acid) is assessed after which the terminal amino acid is removed and the identity of the next amino acid at the terminus is assessed, and this process is repeated until a plurality of successive amino acids in the polypeptide are assessed. In some embodiments, assessing the identity of an amino acid comprises determining the type of amino acid that is present. In some embodiments, determining the type of amino acid comprises determining the actual amino acid identity, for example by determining which of the naturally-occurring 20 amino acids is the terminal amino acid is (e.g., using a binding agent that is specific for an individual terminal amino acid). In some embodiments, the type of amino acid is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine, and valine.
However, in some embodiments assessing the identity of a terminal amino acid type can comprise determining a subset of potential amino acids that can be present at the terminus of the polypeptide. In some embodiments, this can be accomplished by determining that an amino acid is not one or more specific amino acids (and therefore could be any of the other amino acids). In some embodiments, this can be accomplished by determining which of a specified subset of amino acids (e.g., based on size, charge, hydrophobicity, post-translational modification, binding properties) could be at the terminus of the polypeptide (e.g., using a binding agent that binds to a specified subset of two or more terminal amino acids). In some embodiments, assessing the identity of a terminal amino acid type comprises determining that an amino acid comprises a post-translational modification.
In some embodiments, a protein or polypeptide can be digested into a plurality of smaller polypeptides and sequence information can be obtained from one or more of these smaller polypeptides (e.g., using a method that involves sequentially assessing a terminal amino acid of a polypeptide and removing that amino acid to expose the next amino acid at the terminus). In some embodiments, methods of peptide sequencing may involve subjecting a polypeptide terminus to repeated cycles of terminal amino acid detection and terminal amino acid cleavage.
A non-limiting example of polypeptide sequencing by iterative terminal amino acid detection and cleavage is depicted in
In some embodiments, polypeptide 1000 is immobilized to surface 1004 through a functionalization moiety at one terminal end such that the other terminal end is free for detecting and cleaving of a terminal amino acid in a sequencing reaction. Accordingly, in some embodiments, the reagents used in certain polypeptide sequencing reactions preferentially interact with terminal amino acids at the non-immobilized (e.g., free) terminus of polypeptide 1000. In this way, polypeptide 1000 remains immobilized over repeated cycles of detecting and cleaving. To this end, in some embodiments, linker 1002 may be designed according to a desired set of conditions used for detecting and cleaving, e.g., to limit detachment of polypeptide 1000 from surface 1004. Suitable linker compositions and techniques for functionalizing polypeptides (e.g., which may be used for immobilizing a polypeptide to a surface) are described in detail elsewhere herein.
In some embodiments, as shown in
In some embodiments, the method further comprises identifying the amino acid (terminal or internal amino acid) of polypeptide 1000 by detecting labeled amino acid recognition molecule 1006. In some embodiments, detecting comprises detecting a luminescence from labeled amino acid recognition molecule 1006. In some embodiments, the luminescence is uniquely associated with labeled amino acid recognition molecule 1006, and the luminescence is thereby associated with the type of amino acid to which labeled amino acid recognition molecule 1006 selectively binds. As such, in some embodiments, the type of amino acid is identified by determining one or more luminescence properties of labeled amino acid recognition molecule 1006.
In some embodiments, polypeptide sequencing proceeds by (2) removing the terminal amino acid by contacting polypeptide 1000 with an exopeptidase 1008 that binds and cleaves the terminal amino acid of polypeptide 1000. Upon removal of the terminal amino acid by exopeptidase 1008, polypeptide sequencing proceeds by (3) subjecting polypeptide 1000 (having n−1 amino acids) to additional cycles of terminal amino acid recognition and cleavage. In some embodiments, steps (1) through (3) occur in the same reaction mixture, e.g., as in a dynamic peptide sequencing reaction. In some embodiments, steps (1) through (3) may be carried out using other methods known in the art, such as peptide sequencing by Edman degradation.
Edman degradation involves repeated cycles of modifying and cleaving the terminal amino acid of a polypeptide, wherein each successively cleaved amino acid is identified to determine an amino acid sequence of the polypeptide. Referring to
In some embodiments, Edman degradation proceeds by (2) removing the terminal amino acid by contacting polypeptide 1000 with an exopeptidase 1008 that specifically binds and cleaves the isothiocyanate-modified terminal amino acid. In some embodiments, exopeptidase 1008 comprises a modified cysteine protease. In some embodiments, exopeptidase 1008 comprises a modified cysteine protease, such as a cysteine protease from Trypanosoma cruzi (see, e.g., Borgo, et al. (2015) Protein Science 24:571-579). In yet other embodiments, step (2) comprises removing the terminal amino acid by subjecting polypeptide 1000 to chemical (e.g., acidic, basic) conditions sufficient to cleave the isothiocyanate-modified terminal amino acid. In some embodiments, Edman degradation proceeds by (3) washing polypeptide 1000 following terminal amino acid cleavage. In some embodiments, washing comprises removing exopeptidase 1008. In some embodiments, washing comprises restoring polypeptide 1000 to neutral pH conditions (e.g., following chemical cleavage by acidic or basic conditions). In some embodiments, sequencing by Edman degradation comprises repeating steps (1) through (3) for a plurality of cycles.
In some embodiments, peptide sequencing can be carried out in a dynamic peptide sequencing reaction. In some embodiments, referring again to
In some embodiments, dynamic polypeptide sequencing is carried out in real-time by evaluating binding interactions of terminal amino acids with labeled amino acid recognition molecules and a cleaving reagent (e.g., an exopeptidase).
As further shown in the inset panel (left) of
In some embodiments, dynamic peptide sequencing is performed by observing different association events, e.g., association events between an amino acid recognition molecule and an amino acid at a terminal end of a peptide, wherein each association event produces a change in magnitude of a signal, e.g., a luminescence signal, that persists for a duration of time. In some embodiments, observing different association events, e.g., association events between an amino acid recognition molecule and an amino acid at a terminal end of a peptide, can be performed during a peptide degradation process. In some embodiments, a transition from one characteristic signal pattern to another is indicative of amino acid cleavage (e.g., amino acid cleavage resulting from peptide degradation). In some embodiments, amino acid cleavage refers to the removal of at least one amino acid from a terminus of a polypeptide (e.g., the removal of at least one terminal amino acid from the polypeptide). In some embodiments, amino acid cleavage is determined by inference based on a time duration between characteristic signal patterns. In some embodiments, amino acid cleavage is determined by detecting a change in signal produced by association of a labeled cleaving reagent with an amino acid at the terminus of the polypeptide. As amino acids are sequentially cleaved from the terminus of the polypeptide during degradation, a series of changes in magnitude, or a series of signal pulses, is detected.
In some embodiments, signal pulse information may be used to identify an amino acid based on a characteristic pattern in a series of signal pulses. In some embodiments, a characteristic pattern comprises a plurality of signal pulses, each signal pulse comprising a pulse duration. In some embodiments, the plurality of signal pulses may be characterized by a summary statistic (e.g., mean, median, time decay constant) of the distribution of pulse durations in a characteristic pattern. In some embodiments, the mean pulse duration of a characteristic pattern is between about 1 millisecond and about 10 seconds (e.g., between about 1 ms and about 1 s, between about 1 ms and about 100 ms, between about 1 ms and about 10 ms, between about 10 ms and about 10 s, between about 100 ms and about 10 s, between about 1 s and about 10 s, between about 10 ms and about 100 ms, or between about 100 ms and about 500 ms). In some embodiments, different characteristic patterns corresponding to different types of amino acids in a single polypeptide may be distinguished from one another based on a statistically significant difference in the summary statistic. For example, in some embodiments, one characteristic pattern may be distinguishable from another characteristic pattern based on a difference in mean pulse duration of at least 10 milliseconds (e.g., between about 10 ms and about 10 s, between about 10 ms and about 1 s, between about 10 ms and about 100 ms, between about 100 ms and about 10 s, between about 1 s and about 10 s, or between about 100 ms and about 1 s). It should be appreciated that, in some embodiments, smaller differences in mean pulse duration between different characteristic patterns may require a greater number of pulse durations within each characteristic pattern to distinguish one from another with statistical confidence.
In some embodiments, peptides to be used in accordance with the methods of the disclosure (e.g., to be modified or functionalized using any one of the compounds of the disclosure, e.g., compounds of Formulae (II)-(IV), (VI), (VII), or (XII)-(XIV)) may be derived from, purified from, or isolated from any conceivable source.
For example, in some embodiments, a peptide sample is derived from, purified from, or isolated from a cell lysate, a single-cell, a population of cells, or a tissue. In some embodiments, a peptide sample is derived from, purified from, or isolated from a biological sample. In some embodiments, a a biological sample is a blood, saliva, sputum, feces, urine or buccal swab. In some embodiments, a biological sample is from a human, a non-human primate, a rodent, a dog, a cat, a horse, or any other mammal. In some embodiments, a biological sample is from a bacterial cell culture (e.g., an E. coli bacterial cell culture). A bacterial cell culture may comprise gram positive bacterial cells and/or gram negative bacterial cells.
In some embodiments, a peptide sample has been previously extracted via user-developed methods from metagenomic samples or environmental samples. In some embodiments, a peptide sample is derived from, purified from, or isolated from a blood sample, which may be a freshly drawn blood sample from a subject (e.g., a human subject) or a dried blood sample (e.g., preserved on solid media (e.g. Guthrie cards)). A blood sample may comprise whole blood, serum, plasma, red blood cells, and/or white blood cells.
In some embodiments, a biological sample (e.g., a biological sample comprising a peptide sample for use in the disclosure), may be lysed (e.g., disrupted, degraded and/or otherwise digested). In some embodiments, a biological sample comprising a peptide sample for use in the disclosure is lysed using any one of known physical or chemical methodologies to release the peptide sample from within cells or tissues. In some embodiments, a biological sample comprising a peptide sample for use in the disclosure is lysed using an electrolytic method, an enzymatic method, a detergent-based method, and/or mechanical homogenization. In some embodiments, a biological sample comprising a peptide sample for use in the disclosure requires multiple lysis methods performed in series. In some embodiments, a lysis method further includes use of a mill to grind a sample, sonication, surface acoustic waves (SAW), freeze-thaw cycles, heating, addition of detergents, addition of protein degradants (e.g., enzymes such as hydrolases or proteases), and/or addition of cell wall digesting enzymes (e.g., lysozyme or zymolase). Exemplary detergents (e.g., non-ionic detergents) for lysis include polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, polysorbates and alkylphenol ethoxylates, preferably nonylphenol ethoxylates, alkylglucosides and/or polyoxyethylene alkyl phenyl ethers. In some embodiments, lysis methods involve heating a sample for at least 1-30 min, 1-25 min, 5-25 min, 5-20 min, 10-30 min, 5-10 min, 10-20 min, or at least 5 min at a desired temperature (e.g., at least 60° C., at least 70° C., at least 80° C., at least 90° C., or at least 95° C.).
In some embodiments, a peptide sample for use in the disclosure is purified, e.g., following lysis of a biological sample, before terminal end functionalization of the peptides of the peptide sample. In some embodiments, a peptide sample is purified following terminal end functionalization by the methods described herein.
In some embodiments, a peptide sample is purified using chromatography (e.g., affinity chromatography that selectively binds the sample) or electrophoresis. In some embodiments, a peptide sample is purified in the presence of precipitating agents. In some embodiments, after a purification step or method, a peptide sample may be washed and/or released from a purification matrix (e.g., affinity chromatography matrix) using an elution buffer. In some embodiments, a purification step or method may comprise the use of a reversibly switchable polymer, such as an electroactive polymer. In some embodiments, a sample may be purified by electrophoretic passage of a sample through a porous matrix (e.g., cellulose acetate, agarose, acrylamide).
In some embodiments, a peptide sample may be fragmented before terminal end functionalization using the methods described herein. In some embodiments, a peptide sample may be fragmented to produce peptide fragments of any length. Fragmentation of proteins may, in some embodiments, be accomplished using chemical and/or enzymatic (e.g., proteolytic enzymes such as trypsin) methods. In some embodiments, mean fragment length may be controlled by reaction time, temperature, and concentration of sample and/or enzymes (e.g., restriction enzymes, transposases).
In some embodiments, a biological sample comprising a peptide sample for use in the disclosure is enriched for a target peptide to be terminally functionalized. In some embodiments, a biological sample is enriched for a target peptide using an electropheretic method. In some embodiments, a biological sample is enriched for a target peptide using affinity SCODA. In some embodiments, a biological sample is enriched for a target peptide using field inversion gel electrophoresis (FIGE). In some embodiments, a biological sample is enriched for a target peptide using pulsed field gel electrophoresis (PFGE). In some embodiments, the matrix used during enrichment (e.g., a porous media, electrophoretic polymer gel) comprises immobilized affinity agents (also known as ‘immobilized capture probes’) that bind to target peptide present in the biological sample. In some embodiments, a matrix used during enrichment comprises 1, 2, 3, 4, 5, or more unique immobilized capture probes, each of which binds to a unique target molecule and/or bind to the same target molecule with different binding affinities.
In some embodiments, an immobilized capture probe is a peptide capture probe (e.g., an aptamer or an antibody) that binds to a target peptide. In some embodiments, a peptide capture probe binds to a target peptide with a binding affinity of 10−9 to 10−8 M, 10−8 to 10−7 M, 10−7 to 10−6 M, 10−6 to 10−5 M, 10−5 to 104 M, 104 to 10−3 M, or 10−3 to 10−2 M. In some embodiments, the binding affinity is in the picomolar to nanomolar range (e.g., between about 10−12 and about 10−9 M). In some embodiments, the binding affinity is in the nanomolar to micromolar range (e.g., between about 10−9 and about 10−6 M). In some embodiments, the binding affinity is in the micromolar to millimolar range (e.g., between about 10−6 and about 10−3 M). In some embodiments, the binding affinity is in the picomolar to micromolar range (e.g., between about 10−12 and about 10−6 M). In some embodiments, the binding affinity is in the nanomolar to millimolar range (e.g., between about 10−9 and about 10−3 M). In some embodiments, a single peptide capture probe may be used to enrich a plurality of related target peptides that share at least 50%, 60%, 70%, 80%, 90% 95%, or 99% sequence identity. In some embodiments, a single peptide capture probe may be used to enrich a plurality of related target peptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more related target proteins) that share at least 50%, 60%, 70%, 80%, 90% 95%, or 99% sequence homology.
In some embodiments, a target peptide to be functionalized may be released from the enrichment matrix after removal of non-target molecules. In some embodiments, a target peptide may be released from the enrichment matrix by increasing the temperature of the enrichment matrix. Adjusting the temperature of the matrix further influences migration rate as increased temperatures provide a higher capture probe stringency, requiring greater binding affinities between the target peptide and the capture probe. In some embodiments, when enriching related target molecules, the matrix temperature may be gradually increased in a step-wise manner in order to release and isolate target molecules in steps of ever-increasing homology. In some embodiments, temperature is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more in each step or over a period of time (e.g., 1-10 min, 1-5 min, or 4-8 min). In some embodiments, temperature is increased by 5%-10%, 5-15%, 5%-20%, 5%-25%, 5%-30%, 5%-40%, 5%-50%, 10%-25%, 20%-30%, 30%-40%, 35%-50%, or 40%-70% in each step or over a period of time (e.g., 1-10 min, 1-5 min, or 4-8 min). In some embodiments, temperature is increased by about 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C. in each step or over a period of time (e.g., 1-10 min, 1-5 min, or 4-8 min). In some embodiments, temperature is increased by 1-10° C., 1-5° C., 2-5° C., 2-10° C., 3-8° C., 4-9° C., or 5-10° C. in each step or over a period of time (e.g., 1-10 min, 1-5 min, or 4-8 min).
In certain embodiments, one or more of the methods described herein is automated. In certain embodiments, two, three, or four of the methods described herein are automated. Any combination of automated methods may be performed using the same, or different, apparatuses. In certain embodiments, more than one automated method is performed in a single apparatus, wherein the apparatus comprises a plurality of cartridges or compartments, and wherein one or more method is performed in a single cartridge.
Exemplary automation workflows are shown in
In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, one skilled in the art will understand that the invention may be practiced without these details.
Unless the context requires otherwise, throughout the present specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense (i.e., as “including, but not limited to”).
Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.
The term “aliphatic” refers to alkyl, alkenyl, alkynyl, and carbocyclic groups. Likewise, the term “heteroaliphatic” refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.
The term “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“C1-20 alkyl”) In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-10alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”). Examples of C1-6 alkyl groups include methyl (C1), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C6) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents (e.g., halogen, such as F). In certain embodiments, the alkyl group is an unsubstituted C1-10alkyl (such as unsubstituted C1-6 alkyl, e.g., —CH3 (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu or s-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is a substituted C1-10alkyl (such as substituted C1-6 alkyl, e.g., —CH2F, —CHF2, —CF3 or benzyl (Bn)). An alkyl group may be branched or unbranched.
The term “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 20 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, an alkenyl group has 1 to 20 carbon atoms (“C1-20 alkenyl”). In some embodiments, an alkenyl group has 1 to 12 carbon atoms (“C1-12 alkenyl”). In some embodiments, an alkenyl group has 1 to 11 carbon atoms (“C1-11 alkenyl”). In some embodiments, an alkenyl group has 1 to 10 carbon atoms (“C1-10 alkenyl”). In some embodiments, an alkenyl group has 1 to 9 carbon atoms (“C1-9 alkenyl”). In some embodiments, an alkenyl group has 1 to 8 carbon atoms (“C1-8 alkenyl”). In some embodiments, an alkenyl group has 1 to 7 carbon atoms (“C1-7 alkenyl”). In some embodiments, an alkenyl group has 1 to 6 carbon atoms (“C1-6 alkenyl”). In some embodiments, an alkenyl group has 1 to 5 carbon atoms (“C1-5 alkenyl”). In some embodiments, an alkenyl group has 1 to 4 carbon atoms (“C1-4 alkenyl”). In some embodiments, an alkenyl group has 1 to 3 carbon atoms (“C1-3 alkenyl”). In some embodiments, an alkenyl group has 1 to 2 carbon atoms (“C1-2 alkenyl”). In some embodiments, an alkenyl group has 1 carbon atom (“C1 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C1-4 alkenyl groups include methylidenyl (C1), ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C1-6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C1-20 alkenyl. In certain embodiments, the alkenyl group is a substituted C1-20 alkenyl. In an alkenyl group, a C═C double bond for which the stereochemistry is not specified (e.g., —CH═CHCH3 or
may be in the (E)- or (Z)-configuration.
The term “heteroalkenyl” refers to an alkenyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 20 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-20 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 12 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-12 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 11 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-11 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-10 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 9 carbon atoms at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-9 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-8 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-7 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-6 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-5 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-4 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC1-3 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 2 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC1-2 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-6 alkenyl”). Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a “substituted heteroalkenyl”) with one or more substituents. In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC1-20 alkenyl. In certain embodiments, the heteroalkenyl group is a substituted heteroC1-20 alkenyl.
The term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 20 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C1-20 alkynyl”). In some embodiments, an alkynyl group has 1 to 10 carbon atoms (“C1-10 alkynyl”). In some embodiments, an alkynyl group has 1 to 9 carbon atoms (“C1-9 alkynyl”). In some embodiments, an alkynyl group has 1 to 8 carbon atoms (“C1-8 alkynyl”). In some embodiments, an alkynyl group has 1 to 7 carbon atoms (“C1-7 alkynyl”). In some embodiments, an alkynyl group has 1 to 6 carbon atoms (“C1-6 alkynyl”). In some embodiments, an alkynyl group has 1 to 5 carbon atoms (“C1-5 alkynyl”). In some embodiments, an alkynyl group has 1 to 4 carbon atoms (“C1-4 alkynyl”). In some embodiments, an alkynyl group has 1 to 3 carbon atoms (“C1-3 alkynyl”). In some embodiments, an alkynyl group has 1 to 2 carbon atoms (“C1-2 alkynyl”). In some embodiments, an alkynyl group has 1 carbon atom (“C1 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C1-4 alkynyl groups include, without limitation, methylidynyl (C1), ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C1-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C1-20 alkynyl. In certain embodiments, the alkynyl group is a substituted C1-20 alkynyl.
The term “heteroalkynyl” refers to an alkynyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 1 to 20 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-20 alkynyl”). In certain embodiments, a heteroalkynyl group refers to a group having from 1 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-10alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-9 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-8 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-7 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-6 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-5 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-4 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC1-3 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 2 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC1-2 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-6 alkynyl”). Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a “substituted heteroalkynyl”) with one or more substituents. In certain embodiments, the heteroalkynyl group is an unsubstituted heteroC1-20 alkynyl. In certain embodiments, the heteroalkynyl group is a substituted heteroC1-20 alkynyl.
“Aralkyl” is a subset of “alkyl” and refers to an alkyl group substituted by an aryl group, wherein the point of attachment is on the alkyl moiety
As used herein, the term “alkoxy” refers to an alkyl group having an oxygen atom that connects the alkyl group to the point of attachment: i.e., alkyl-O—. As for the alkyl portions, alkoxy groups can have any suitable number of carbon atoms, such as C1-6 or C1-4. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. Alkoxy groups are unsubstituted, but can be described, in some embodiments as substituted. “Substituted alkoxy” groups can be substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, nitro, cyano, and alkoxy.
The term “cycloalkyl” refers to cyclic alkyl radical having from 3 to 10 ring carbon atoms (“C3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10 cycloalkyl”). Examples of C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-6 cycloalkyl groups include the aforementioned C5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is unsubstituted C3-10 cycloalkyl. In certain embodiments, the cycloalkyl group is substituted C3-10 cycloalkyl.
The term “heteroalkyl,” as used herein, refers to an alkyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by a heteroatom or optionally substituted heteroatom, e.g., nitrogen (e.g.,
oxygen (e.g.,
or sulfur (e.g.,
Heteroalkyl groups may be optionally substituted with one, two, three, or, in the case of alkyl groups of two carbons or more, four, five, or six substituents independently selected from any of the substituents described herein. Heteroalkyl group substituents include: (1) carbonyl; (2) halo; (3) C6-C10 aryl; and (4) C3-C10 carbocyclyl. A heteroalkylene is a divalent heteroalkyl group.
The term “alkoxy,” as used herein, refers to —ORa, where Ra is, e.g., alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclyl, heterocyclyl, or heteroaryl. Examples of alkoxy groups include methoxy, ethoxy, isopropoxy, tert-butoxy, phenoxy, and benzyloxy.
The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C14 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents (e.g., —F, —OH or —O(C1-6 alkyl). In certain embodiments, the aryl group is an unsubstituted C6-14 aryl. In certain embodiments, the aryl group is a substituted C6-14 aryl.
The term “aryloxy” refers to an —O-aryl substituent.
The term “heteroaryl” refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, e.g., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). In certain embodiments, the heteroaryl is substituted or unsubstituted, 5- or 6-membered, monocyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur. In certain embodiments, the heteroaryl is substituted or unsubstituted, 9- or 10-membered, bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur.
In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.
The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl is substituted or unsubstituted, 3- to 7-membered, monocyclic heterocyclyl, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently oxygen, nitrogen, or sulfur, as valency permits.
In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
The term “carbonyl” refers a group wherein the carbon directly attached to the parent molecule is sp2 hybridized, and is substituted with an oxygen, nitrogen or sulfur atom, e.g., a group selected from ketones (e.g., —C(═O)Raa), carboxylic acids (e.g., —CO2H), aldehydes (—CHO), esters (e.g., —CO2Raa, —C(═O)SRaa, —C(═S)SRaa), amides (e.g., —C(═O)N(Rbb)2, —C(═O)NRbbSO2Raa, —C(═S)N(Rbb)2), and imines (e.g., —C(═NRbb)Raa, —C(═NRbb)ORaa), —C(—NRbb)N(Rbb)2), wherein Raa and Rbb are as defined herein.
The term “amino,” as used herein, represents —N(RN)2, wherein each RN is, independently, H, OH, NO2, N(RN0)2, SO2ORN0, SO2RN0, SORN0, an N-protecting group, alkyl, alkoxy, aryl, cycloalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), wherein each of these recited RN groups can be optionally substituted; or two RN combine to form an alkylene or heteroalkylene, and wherein each RN0 is, independently, H, alkyl, or aryl. The amino groups of the disclosure can be an unsubstituted amino (i.e., —NH2) or a substituted amino (i.e., —N(RN)2).
The term “substituted” as used herein means at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. “Substituted” also means one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, in some embodiments “substituted” means one or more hydrogen atoms are replaced with NRgRh, NRgC(═O)Rh, NRgC(═O)NRgRh, NRgC(═O)ORh, NRgSO2Rh, OC(═O)NRgRh, ORg, SRg, SORg, SO2Rg, OSO2Rg, SO2ORg, ═NSO2Rg, and SO2NRgRh. “Substituted also means one or more hydrogen atoms are replaced with C(═O)Rg, C(═O)ORg, C(═O)NRgRh, CH2SO2Rg, CH2SO2NRgRh.
In the foregoing, Rg and Rh are the same or different and independently hydrogen, alkyl, alkoxy, alkylaminyl, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. “Substituted” further means one or more hydrogen atoms are replaced by a bond to an aminyl, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylaminyl, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl group. In addition, each of the foregoing substituents may also be optionally substituted with one or more of the above substituents.
The terms “salt thereof” or “salts thereof” as used herein refer to salts which are well known in the art. For example, Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Additional information on suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference. Salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counter ions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
A “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds. The terms refer to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein or peptide will be at least three amino acids in length. In some embodiments, a peptide is between about 3 and about 100 amino acids in length (e.g., between about 5 and about 25, between about 10 and about 80, between about 15 and about 70, or between about 20 and about 40, amino acids in length). In some embodiments, a peptide is between about 6 and about 40 amino acids in length (e.g., between about 6 and about 30, between about 10 and about 30, between about 15 and about 40, or between about 20 and about 30, amino acids in length). In some embodiments, a plurality of peptides can refer to a plurality of peptide molecules, where each peptide molecule of the plurality comprises an amino acid sequence that is different from any other peptide molecule of the plurality. In some embodiments, a plurality of peptides can include at least 1 peptide and up to 1,000 peptides (e.g., at least 1 peptide and up to 10, 50, 100, 250, or 500 peptides). In some embodiments, a plurality of peptides comprises 1-5, 5-10, 1-15, 15-20, 10-100, 50-250, 100-500, 500-1,000, or more, different peptides. A protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification. A protein may also be a single molecule or may be a multi-molecular complex. A protein or peptide may be a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, synthetic, or any combination of these.
With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to plural as is appropriate to the context and/or application. The various singular/plural permutations can be expressly set forth herein for sake of clarity.
It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (for example, bodies of the appended claims) are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims can contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (for example, “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”
In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
Those skilled in the art will appreciate that certain compounds described herein can exist in one or more different isomeric (e.g., stereoisomers, geometric isomers, tautomers) and/or isotopic (e.g., in which one or more atoms has been substituted with a different isotope of the atom, such as hydrogen substituted for deuterium) forms. Unless otherwise indicated or clear from context, a depicted structure can be understood to represent any such isomeric or isotopic form, individually or in combination.
While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
Example 1. QP126, a synthetic peptide fragment from a hypothetical Lys-C only digest of full length CRP, was used as a substrate. Cysteine residues were capped with iodoacetamide. The functionalization of QP126 was then performed as shown in
Example 2. A crude digest (Lys-C/Trypsin) of truncated CRP was reacted with 4-nitrovinyl sulfonamide coupling reagent and incubated for 4 hours at 37° C., followed by LC/HRMS analysis. Four fragments from a predicted Lys-C/Trypsin digest were observed in their labeled forms, demonstrating the lysine reactive probe is capable of labeling fragments from a complex digestion mixture. See
Example 3. This procedure converts C-terminal Lysines to ε-azido lysines via a diazotransfer using imidazole-1-sulfonyl azide. Lysine labeling methods often also result in labeling the N-terminal of proteins and peptides. However, in this method the diazo transfer can be directed to either the N-terminal or the ε-amine of lysine by modulating the pH of the reaction buffer. Specifically, the pKa of the N-terminal amino group ranges from −7-8.5 (depending on the amino acid at the N-terminal), while the pKa of the ε-amine of lysine is ˜11. As such, reactions performed at neutral pH direct the diazo transfer to the N-terminus, while reactions occurring at pH 10-11 will direct the diazo transfer to the ε-amine of lysine (
Example 4. A representative cysteine protection procedure involves:
1. 90 μL of CRP (1 mg/mL), 360 μL of 25 mM HEPES pH 7.8 w/10% acetonitrile, 0.23 μL of 0.5 M TCEP were combined and incubated at room temperature for 20 min; and
2. 9 μL of 0.5 M iodoacetamide was added, and the reaction was incubated in the dark for 30 min at room temperature.
Example 5. Sample denaturation for enzymatic digestion: In order to denature proteins for protease digestion the disulfide bonds need to be broken. This involves the reduction and alkylation of cysteine residues. Using Insulin as a model system, the following method was developed to reduce cysteines with TCEP (2 mM, 37° C., 30 min) and alkylate them with iodoacetamide (62 mM, r.t, 30 min in the dark). This protocol was validated on our Insulin sample using HPLC and Mass Spectrometry and complete reduction of disulfides was observed. Alkylation is nearly complete, with a single free-cysteine observed in no more than 30% of Insulin peptides. These conditions are compatible with automation and the buffers necessary for enzymatic digestion of samples from serum/plasma.
Example 6. Insulin as a model system for sample preparation: Insulin was used as a model system. Insulin is a complex small protein that allows testing of sample preparation procedures in a controlled fashion with QC after every operation and without the need for enzymatic digestion. Using the sample preparation protocols described herein (e.g., Examples 3 and 5), Insulin libraries were prepared that result in the isolation of a single peptide, i.e., a single peptide having a terminal lysine (chain B;
Example 7. Insulin sample preparation workflows with and without a pre-formed DNA-SV complex are shown in
Example 8. Preparation of a peptide-Q24ds-SV complex (
As shown in
As shown in
In certain workflows, starting with Q24-BisBt-DBCO, conjugation to SV and purification on ion exchange HPLC provided pure 1:1 DNA:SV complex, which was then hybridized to Q24-comp-Cy3B. This entire complex was then conjugated by click chemistry with azido-peptides of interest.
Example 9. Exemplary C-terminal immobilization of peptides via diazotransfer.
This application is a Continuation application of application Ser. No. 17/153,490, filed Jan. 20, 2021, issuing as U.S. Pat. No. 11,358,981, issue date Jun. 14, 2022, which claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent applications U.S. Ser. No. 62/964,075, filed Jan. 21, 2020, and U.S. Ser. No. 63/041,244, filed Jun. 19, 2020, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63041244 | Jun 2020 | US | |
| 62964075 | Jan 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17153490 | Jan 2021 | US |
| Child | 17837360 | US |
