Acid-cleavable clickable linkers for efficient enrichment of alkyne-tagged biomolecules

Abstract

Presently disclosed is a linker useful for conjugating a protein or peptide that is labeled by an alkyne group to a biotin moiety, and methods for making and using the linker. One use of the linker is to identify or purify alkyne-tagged peptides. In such methods, the linker is joined to the alkyne group by a click reaction, and then the biotin-conjugated peptides are immobilized on an avidin- or streptavidin-coated substrate. Unbound peptides or proteins are washed away and then the immobilized peptides or proteins are cleaved from the substrate, preferably by a mild acid treatment. The thus isolated peptides can be analyzed by a variety of methods, e.g., to prepare a profile of labeled sites in a living cell.

Description

ACID-CLEA VABLE CLICKABLE LINKERS FOR EFFICIENT ENRICHMENT OF ALKYNE-TAGGED BIOMOLECULESSTATEMENT OF GOVERNMENT INTEREST

[0001] This invention was made with government support under Grant [DMR2005199] awarded by the National Science Foundation. The government has certain rights in the invention.FIELD

[0002] The subject matter disclosed herein relates to new molecules, compositions of them, methods for preparing them, and methods for their use.BACKGROUND

[0003] Alkyne-tagging has been widely used in chemical proteomics and structural proteomics of complex biological systems, since the tagged proteins / peptides can be enriched using biorthogonal click chemistry to greatly reduce the background, thus facilitating the subsequent analysis by liquid chromatography mass spectrometry (LCMS). [ 4-58] porexample, electrophilic probes with an alkyne tag, such as the probes I and II (Fig. 1 A), have been used for profiling protein-small molecule interactions across the proteome, aiming to discover new functionality and “ligandable” sites. To identify the binding sites of ligands / drugs, photoreactive probes III (Fig. 1 A) with an alkyne tag were used to allow enrichment of the captured fragments of the binding sites out of the vast background. [6-8] Further, crosslinkers with an alkyne tag IV (Fig. 1A) greatly facilitated profiling protein-protein interactions in live cells by crosslinking mass spectrometry (XLMS).[9, 10] Although currently proteome- wide studies using chemical probes are mostly performed in cell lysates and body fluids, increasing interest has been attracted to perform in vivo experiments using live cells and tissues, since evidence has indicated that cryptic druggable and functional sites on proteins may be only present in the native intracellular environment and vanish outside living cells. [^ H]

[0004] Upon tryptic digestion, peptides labeled with the clickable probes I-IV (Fig. IB) are enriched by attaching a handle for anchoring onto a solid support via a click reaction to the N3-group of a “click linker” (Fig. IB). The vast quantities of unlabeled peptides that interfere with LCMS analysis are then washed out. For releasing the enriched labeled peptides from the solid support, the click linker needs to be cleavable under mild conditions. A variety of click linkers have been reported, perhaps the best being DADPS azide biotin (Fig. 1C) in which the biotin handle strongly anchors the clicked peptides onto streptavidin- coated supports, while the dialkoxydiphenylsilane (DADPS) group can be subsequently cleaved under mild acidic conditions. [12- 16] However, this linker possesses a long hydrophobic chain at the azido end, which may promote nonspecific binding in the presence of a large amount of non-labeled hydrophobic peptides, thus hindering the click reaction and increasing sample loss and contamination.

[0005] The Examples in this disclosure exemplify the design and synthesis of a new click linker (APA biotin, Fig. 3), and demonstrate its high efficiency in a workflow for identifying alkyne-labeled sites in the proteome of living yeast cells by LCMS with unprecedented coverage.SUMMARY

[0006] Accordingly, one aspect of the present disclosure is a compound having the structural formula X:whereinR1 is hydrogen, hydroxyl, C1-C3 alkoxy, (CH2)n-C00H, NHCOR (R = C1-C3 alkyl, C1-C3 alkoxy, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH, NR3R4, or O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH;R2 is hydrogen, C1-C2 alkyl, C1-C3 alkoxy, (CH2)n-C00H, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH;R3 and R4 can be the same or different and are selected from hydrogen or C1-C3 alkyl, Cl- C3 alkoxy, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH; each R5 can be the same or different; and can be hydrogen, unsubstituted C1-C2 alkyl or Cl- C2 alkyl substituted by a 5- or 6-atom aryl or heteroaryl group; m = 0 to 3, and any of R1 can be the same or different; n = 0 to 3; o = 0 to 12 p = 1 or 2; q = 0 to 50.

[0007] Another aspect of the present disclosure is a method for making the molecule of formula X, comprising (see Fig. 3): i) coupling the pyridyl bromide 1 with the alkyl bromide acetate 2, employing nickel- catalyzed cross-electrophile coupling

[0021] promoted by the ligand 3

[0022] to provide the pyridyl acetate 4; ii) converting the alcohol 4 to the azide 5a using (PhO)2PON3 in the presence of 1,8- Diazabicyclo[5.4.0]undec-7-ene (DBU), and then hydrolyzing to the alcohol 5b; iii) separately preparing the symmetric acetal 7 from the protected 3- hydroxypropanamine 6 by transacetalation; iv) alkoxyalkylating the picolyl alcohol 5b with in-situ generated a-chloroalkyl ether from 7 in the presence of a Zn(II) catalyst to give the asymmetric acetal 8; v) deprotecting the phthalimide with hydrazine to obtain APA-NH2; vi) mixing APA-NH2 with the biotin-(PEG) ! 2-A-hydroxy succinimide ester 9 in 1 : 1 ratio in a solvent to obtain the compound of formula X.

[0008] A third aspect of this disclosure is a method for purifying alkyne-tagged proteins or peptides comprising: i) contacting proteins with an alkyne-tagging reagent to obtain alkyne-tagged proteins; ii) optionally digesting the alkyne-tagged proteins with a protease to obtain alkyne- tagged peptides; iii) reacting the alkyne-tagged proteins or peptides by a click reaction with a compound of the formula X to obtain biotinylated peptides; iv) contacting the biotinylated proteins or peptides with a biotin affinity reagent to form a complex; v) separating the complex from proteins or peptides that are not alkyne-tagged; vi) releasing the proteins or peptides from the complex to obtain purified alkyne- tagged peptides.

[0009] A fourth aspect of this disclosure is a method for diagnosing a disease associated with protein conformational change (such as by denaturation), aggregation or binding to an entity (such as another protein, a ligand, or a surface), comprising: i) contacting cells or cultured tissue of a subject or a lysate of cells of a subject with an alkyne-tagging reagent to obtain alkyne-tagged proteins; ii) digesting the alkyne-tagged proteins with a protease to obtain alkyne-tagged peptides; iii) reacting the alkyne-tagged peptides by a click reaction with a compound of the formula X to obtain biotinylated peptides; iv) contacting the biotinylated peptides with a biotin affinity reagent to form a complex; v) separating the complex from peptides that are not alkyne-tagged; vi) releasing the peptides from the complex to obtain purified alkyne-tagged peptides; vii) analyzing the alkyne-tagged peptides by one or more of high-performance liquid chromatography, capillary electrophoresis, gel electrophoresis or 2-D gelelectrophoresis and mass spectrometry to determine the amino acid sequence of the alkyne-tagged peptides; and viii) mapping the peptides onto 2- or 3-dimensional structures of the protein, the misfolding of the protein being associated with the disease, to characterize the conformation of the protein by a profile of alkyne-tagged peptides; and ix) comparing the profile of alkyne-tagged peptides in the cells of the subject to a reference profile of alkyne-tagged peptides in the normal conformation of the protein (i.e., the protein in cells of a healthy subjects); wherein a different profile in cells of the subject indicates a diagnosis of or risk for the disease.

[0010] A fifth aspect of the present disclosure is a reagent composition comprising: i) a compound of the formula X:whereinR1 is hydrogen, hydroxyl, C1-C3 alkoxy, (CH2)n-COOH, NHCOR (R = C1-C3 alkyl, C1-C3 alkoxy, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH, NR3R4, or O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH;R2 is hydrogen, C1-C2 alkyl, C1-C3 alkoxy, (CH2)n-COOH, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH;R3 and R4 can be the same or different and are selected from hydrogen or C1-C3 alkyl, Cl- C3 alkoxy, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH; each R5 can be the same or different; and can be hydrogen, unsubstituted C1-C2 alkyl or Cl- C2 alkyl substituted by a 5- or 6-atom aryl or heteroaryl group; m = 0 to 3, and any of R1 can be the same or different;n = 0 to 3; o = 0 to 12 p = 1 or 2; q = 0 to 50; ii) a copper salt or complex; and optionally iii) a click ligand.

[0011] A sixth aspect of the present disclosure is use of a compound of the formula X for conjugating an alkyne-tagged protein or peptide to biotin.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The invention will now be described in more detail with reference to working examples of the invention, given only by way of example, and with reference to the accompanying drawings, in which:

[0013] Fig. 1A shows common alkyne-tagging reagents; I and IV typically react with lysine, arginine, serine, and threonine side chains, and with the N-terminal amine group of proteins and peptides. II will react with cysteine side chains of proteins and peptide via the iodine atom. Ill is photo-reactive and can be used to label sites that bind the ligand moiety.

[0014] Fig. IB shows a general workflow of analysis of alkyne-tagged peptides.Chemical probes and crosslinkers, e.g., the K, R, S, T- (I, IV), cysteine- (II), and photo- reactive (III) ligand binding site probes shown in Fig. 1A, are contacted with proteins, which can be in complex biological systems. The labelled proteins are digested, e.g., with trypsin, and the resulting peptides are attached to a linker attached to an affinity moiety by a click reaction. (Alternatively, the peptides can be attached to a conjugation linker for covalently attaching the conjugated peptides to an immobilizing substrate by a click reaction.) The labeled peptides are enriched by immobilization via the affinity moiety (or other immobilizing substrate) and then analyzed by LCMS, for identification of the labeled sites across the proteome.

[0015] Fig. 1C shows the structural formula of the linker dialkoxydiphenylsilane (DADPS) azide biotin.

[0016] Fig. 2 shows a general workflow for application of the alkyne-tagging analysis to labeling proteins in live cells. In this workflow, the alkyne tagging reagent is added to live cells in culture or to cultured tissues. The cells are then collected and lysed and the lysate or the protein fraction of the lysate is digested with a protease or other digestion reagent or condition. The labeled peptides are then attached to the APA biotin linker by a click reaction and then immobilized on a streptavidin-coated bead. The beads are washed to remove unbound peptides and then the linker is cleaved by mild acid treatment and the freed peptides are recovered and analyzed by LCMS to determine the amino acid sequence of the recovered peptides. The peptides are then mapped by their sequence onto the 3D structure of the protein to ascertain structural changes in the protein by differences in the profile of the peptides that are recovered.

[0017] Fig. 3 shows the synthetic scheme of the APA biotin linker Formula Y. As explained below, this scheme is generalizable to synthesis of the biotinylated linker of formula X and the amine-labelling linker of formula Z.

[0018] Fig. 4A illustrates the workflow for application of the alkyne-tagging analysis to assessing changes in the yeast proteome during heat shock. The workflow of Fig. 2 is shown as applied to each of a sample of yeast cells grown under control conditions (30 °C - left side) and yeast cells grown under different heat treatments (right side).

[0019] Fig. 4B shows a graph of the abundance of each protein of all yeast proteins identified by bottom up proteomics vs. rank in yeast cells. Various yeast heat shock protein classes and other chaperone molecules (AAA+, CCT, HSP40, etc.) are located on the curve, with chaperone protein families highlighted by different patterns per the legend and nonchaperone proteins (“other”) indicated by ® .

[0020] Fig. 4C shows the distribution of the enriched labeled proteins recovered from streptavidin beads vs all detected proteins in major cellular compartments, shown as the percentage of proteins in each subcellular compartment in yeast in comparison to the percentage of the proteins that are labeled in each compartment by the alkyne-tagging method as performed in Example 2. “Labelled proteins” are those that are alkyne-tagged,fragmented, and purified by click reaction and streptavidin binding. “Proteome” is the total protein in each compartment quantified by mass spectrometry-based bottom up proteomics.

[0021] Fig. 4D shows profiles of labeled sites that significantly changed after 1, 2, 3, 4 and 5 minutes of heat shock at three temperatures compared to control samples (n=3) maintained at 30 °C as heatmaps. Proteins were categorized into chaperone proteins, client proteins of chaperones and other proteins. The treatment temperatures are labeled on top of the heatmaps. The color scale represents the log2 ratio of abundance of each labeled site between heat-shocked samples and control samples. Lighter bands indicate the abundance of labeled sites in heat-treated samples were significantly lower than those in control samples. Similarly, a darker band indicates higher abundance. Depth of the greyscale relates to the degree of change in abundance.

[0022] The data show the time course change in the profile of alkyne-tagged peptides identified in yeast cells exposed to heat shock of 37 °C, 42 °C and 47 °C after culture at 30 °C. One row in a heat map represents on labeled site. “Clustered Labeled Sites” are grouped together algorithmically in the heat map to group sites changing in approximately the same way (direction and intensity) together; “time” is the time at the indicated heat shock temperature. Separate analyses are shown for chaperone proteins, client proteins, and “nonclient” proteins not known to have a chaperone.

[0023] Fig. 4E presents a rearrangement of the data in Fig. 4D, showing all of the proteins together in one panel (left side), and wherein labeled sites within one protein are grouped together. The bar at the left grey scales the classification of the proteins along the panel as chaperones, clients, or non-client proteins. Note that in Fig. 4E and 5 data are rearranged to group labeled sites by individual protein.

[0024] The right panel shows a “zoom in” on the data for a single protein, chaperone protein Hsp72 (P10592, gene SSA2), at domain level (middle column) and the individual peptide fragment level (numbered by the alkyne-tagged amino acid) in the right column. Alkyne-tagged amino acids are greyscale coded (K = lysine, R = arginine, S = serine, T = threonine) as shown in the legend.

[0025] The three domains of Hsp72, namely nucleotide-binding domain (NBD, from sequence location 1 to 383), substrate-binding domain (SBD, from sequence location 384 to 546), and C-terminal domain (acts as a “Lid” over the SBD, from sequence location 547 to 642), are indicated at the right of the side bar labeled as Domain. The labeled amino acids (K, R, S, and T) are indicated by the side bar labeled as AA.

[0026] Fig. 5 shows the 3D structure of Hsp72 as calculated by AlphaFold in free form showing lysine residues with significant changes of reactivity with 10 vs. the control samples (30°C) (13), and lysine residues without statistically significant changes of reactivity with 10 vs the control samples (30°C) (E3 ), after subj ecting to heat shock at 37 °C for 5 min.

[0027] Fig. 6 shows structures of a number of ligands that might be used in a click reaction for complexing the metal ion catalyst. Arg is arginine; Tat is Tat peptide, corresponding to the poly cationic region (aa49-57) of the Tat protein.DETAILED DESCRIPTION

[0028] The present disclosure provides a compound having the structural formula X:whereinR1 is hydrogen, hydroxyl, C1-C3 alkoxy, (CH2)n-COOH, NHCOR (R = C1-C3 alkyl, C1-C3 alkoxy, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH, NR3R4, or O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH;R2 is hydrogen, C1-C2 alkyl, C1-C3 alkoxy, (CH2)n-COOH, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH;R3 and R4 can be the same or different and are selected from hydrogen or C1-C3 alkyl, Cl- C3 alkoxy, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH; each R5 can be the same or different; and can be hydrogen, unsubstituted C1-C2 alkyl or Cl- C2 alkyl substituted by a 5- or 6-atom aryl or heteroaryl group; m = 0 to 3, and any of R1 can be the same or different; n = 0 to 3; o = 0 to 12 p = 1 or 2; q = 0 to 50; though preferably q can be from 1 to 24 or more preferably from 10 to 15.

[0029] In some embodiments of the compound X, R1 preferably is hydroxyl and m = 1, 2 or 3.

[0030] In some preferred embodiments, additionally or alternatively, R2 is Cl alkoxy.

[0031] In some preferred embodiments, additionally or alternatively, the compound can have the formula X’ :

[0032]

[0033] In any of the embodiments of compound X, R5 can be methyl or 2-pyridylmethyl.

[0034] In one preferred embodiment of the compound, X has the structural formula Y:

[0035] Also disclosed herein is a compound having the structural formula Z:wherein:R1 is hydrogen, hydroxyl, C1-C3 alkoxy, (CH2)n-COOH, NHCOR (R = C1-C3 alkyl, C1-C3 alkoxy, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH, NR3R4, or O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH;R2 is hydrogen, hydroxyl, C1-C2 alkyl, C1-C3 alkoxy, (CH2)n-COOH, CH2CH2-O- (CH2)o-CH3, or CH2CH2-O-(CH2)o-OH;R3 and R4 can be the same or different and are selected from hydrogen or C1-C3 alkyl, Cl- C3 alkoxy, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH; each R5 can be the same or different; and can be hydrogen, unsubstituted C1-C2 alkyl or Cl- C2 alkyl substituted by a 5- or 6-atom aryl or heteroaryl group; m = 0 to 3, and any of R1 can be the same or different; n = 0 to 3; o = 0 to 12 p = 1 or 2;R6 is an amine group that can react with an epoxide or succinimide group, such as NHR7, (CH2)SNHR7 or (CH2)SN(R7)2, wherein R7 can be hydrogen or C1-C2 alkyl and s = 0 to 2.

[0036] Typically, R6 is NH2.

[0037] The linker compound Z can be a compound of the formula Z’ :Z' wherein R6 is an amine group that can react with an epoxide or succinimide group, such as NHR7, (CH2)SNHR7 or (CH2)SN(R7)2, wherein R7 can be hydrogen or C1-C2 alkyl and s = 0 to 2. Typically, R6 is NH2.

[0038] The compound Z or Z’ can be one wherein R1 is hydroxyl and m = 1, 2 or 3.

[0039] Additionally or alternatively, the compound Z or Z’ can be one wherein R2 is Cl alkoxy.

[0040] Further, the compound Z or Z’ can be any one of these embodiments wherein R5 is methyl or 2-pyridylmethyl.

[0041] Also disclosed herein is a method for making the compound X, illustrated for the synthesis in Example 1 of APA biotin. With reference to Fig. 3, the disclosed method comprises coupling the pyridyl bromide 1 with the alkyl bromide acetate 2, employing nickel-catalyzed cross-electrophile coupling

[0021] promoted by the ligand 3

[0022] to provide the pyridyl acetate 4; converting the alcohol 4 to the azide 5a in 52% yield using (PhO)2PON3 in the presence of l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), and then hydrolyzing to the alcohol 5b; separately preparing the symmetric acetal 7 from the protected 3 -hydroxy propanamine 6 by transacetalation; alkoxyalkylating the picolyl alcohol 5b with in-situ generated a-chloroalkyl ether from 7 in the presence of a Zn(II) catalyst to give the asymmetric acetal 8; deprotecting the phthalimide with hydrazine to obtain APA- NH2; mixing APA-NH2 with the biotin-(PEG)12-A-hydroxy succinimide ester 9 in 1 : 1 ratio in a solvent to obtain the compound of formula Y.

[0042] In this method, the pyridyl bromide 1 and alkyl bromide acetate 2 having the desired substituents Rl, R2 and R5, and appropriate numbers of groups m, n, o, and p, for making compounds within the scope of structural formula X can be purchased commercially. Also, the biotin-(PEG)12-A-hydroxy succinimide ester 9 can be purchased with various numbers of PEG residues q, including from 0 to 50 as described above.

[0043] Also disclosed herein is a method for making a compound Z , comprising: i) coupling of the pyridyl bromide 1 with the alkyl bromide acetate 2, employing nickel- catalyzed cross-electrophile coupling promoted by the ligand 3 to provide the pyridyl acetate 4; ii) converting the alcohol 4 to the azide 5a in 52% yield using (PhO)2PON3 in the presence of l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), and then hydrolyzing to the alcohol 5b; iii) separately preparing the symmetric acetal 7 from the protected 3-hydroxypropanamine 6 by transacetalation; iv) alkoxyalkylating the picolyl alcohol 5b with in-situ generated a-chloroalkyl ether from 7 in the presence of a Zn(II) catalyst to give the asymmetric acetal 8; v) deprotecting the phthalimide with hydrazine to obtain APA-NH2.

[0044] In this method for making a compound Z, the pyridyl bromide 1 and alkyl bromide acetate 2 having the desired substituents Rl, R2 and R5, and appropriate numbers of groups m, n, o, and p, for making compounds within the scope of structural formula Z can be purchased commercially.

[0045] Also disclosed herein is a method for purifying alkyne-tagged proteins or peptides comprising: contacting proteins with an alkyne-tagging reagent under conditions sufficient to obtain alkyne-tagged proteins; optionally digesting the alkyne-tagged proteins with a protease to obtain alkyne- tagged peptides;reacting the alkyne-tagged proteins or peptides by a click reaction with a compound of the formula X to obtain biotinylated peptides; contacting the biotinylated proteins or peptides with a biotin affinity reagent to form a complex; separating the complex from proteins or peptides that are not alkyne-tagged; releasing the proteins or peptides from the complex to obtain purified alkyne- tagged proteins or peptides.

[0046] An “alkyne tagging reagent” is a bifunctional or multifunctional reagent that includes at least one group that reacts specifically with one or more amino acid side chain groups in a protein or with the amino terminal amine, and an alkyne group spatially separated from the amino acid reactive group(s). Examples of alkyne tagging reagents are I, II, III and IV in Fig. 1A and compound 10 in Fig. 4A.

[0047] An alternative method for purifying alkyne-tagged proteins or peptides uses the linker compound Z to conjugate the alkyne-tagged proteins or peptides to an amine group, which is in turn reacted with a functional group attached to a substrate. Such a method comprises: i) contacting proteins with an alkyne-tagging reagent under conditions sufficient to obtain alkyne-tagged proteins; ii) optionally digesting the alkyne-tagged proteins with a protease to obtain alkyne-tagged peptides; iii) reacting the alkyne-tagged proteins or peptides by a click reaction with a compound of any one of claims

[0035] to

[0040] obtain amine-labelled proteins or peptides; iv) contacting and reacting the amine-labelled proteins or peptides with a functionalized substrate, wherein r = 1 to 20, to obtain immobilized proteins or peptides covalently attached to the substrate; v) separating the immobilized proteins or peptides from proteins or peptides that are not alkyne-tagged; vi) releasing the immobilized proteins or peptides from the substrate to obtain purified alkyne-tagged peptides.

[0048] In an alternative embodiment of this method, the compound of the formula Z can be reacted directly with the functionalized substrate, to form an immobilized linker Z. Then the immobilized linker is contacted and reacted by a click reaction with proteins or peptides that have been labelled with an alkyne tag. Such a method comprises: i) contacting proteins with an alkyne-tagging reagent under conditions sufficient to obtain alkyne-tagged proteins; ii) optionally digesting the alkyne-tagged proteins with a protease to obtain alkyne-tagged peptides; iii) separately reacting a linker compound of any one of claims

[0035] to

[0040] ; iv) with a functionalized substrate, wherein r = 1 to 20, to obtain the linker compound covalently attached to the substrate; v) contacting and reacting the alkyne-tagged proteins or peptides with the linker compound covalently attached to the substrate to obtain immobilized proteins or peptides; vi) separating the immobilized proteins or peptides from proteins or peptides that are not alkyne-tagged; vii) releasing the immobilized proteins or peptides from the substrate to obtain purified alkyne-tagged peptides.

[0049] The functional group is preferably set off from the substrate by a hydrophilic linker, e.g., PEG linkers of various length. The functional group can be any amine reactive group, but is preferably a succinimide or epoxide group. For example, the functionalized substrate can be:or, wherein r = 1 to 20, or r = 1 to 12 or 4 to 12.

[0050] Functionalized substrates as described above are commercially available, for example Thermo Fisher Scientific provides Pierce™ NHS-Activated Magnetic Beads (Catalog number: 88826).

[0051] In either of these purification methods, when the optional step of digesting the alkyne-tagged protein to obtain alkyne-tagged peptides is performed, the protease that is used can be any known protease, preferably one that cleaves the protein within a specific amino acid sequence. For example, any of trypsin, chymotrypsin, pepsin, Glu-C, LysN, Lys-C or Asp-N, or any mixture of these, can be used.

[0052] There are many known variations of “click” reactions for joining the linker X or the linker Z to the alkyne tag.[53’581Conditions for conducting the click reaction can be any of them commonly used in the art.

[0053] For using the present method for identifying or purifying alkyne tagged proteins or peptides, especially in or from living cells or cultured tissues, use of a copper catalyst is preferred. Additionally or alternatively the click reaction is performed at 4 °C to 50 °C, preferably at room temperature.

[0054] As well, the click reaction is preferably performed at pH 5-9 in a buffer, e.g., a Phosphate Buffered Saline (PBS) buffer.

[0055] Thiols should be kept from the click reaction, as these quench the catalyst.

[0056] A “biotin affinity reagent” is a reagent that specifically binds to biotin. A biotin affinity reagent will preferably show a specificity of at least 1000-fold for binding biotin to binding a non-biotin compound or to a surface that does not include a biotin molecule. Commonly used biotin affinity reagents are avidin and streptavidin.

[0057] Preferably, the biotin affinity reagent is covalently bound to an immobilizing substrate, such as a bead or a pipette tip or a microwell tray. However, in some embodiments the biotin affinity reagent can also be non-covalently bound, for example by complexation, to an immobilizing substrate.

[0058] The step of separating the alkyne-tagged proteins or peptides can be one wherein the biotin affinity reagent comprises an immobilizing substrate and the immobilizing substrate is separated from peptides that are not alkyne-tagged. For example, the immobilizing substrate can be in the form of a microtiter plate, and peptides that are not alkyne-tagged can be washed out of the wells of the microtiter plate.

[0059] The step of separating the alkyne-tagged proteins or peptides can be one wherein the immobilizing substrate is an avidin- or streptavidin-coated microbead and the complex is separated from the peptides that are not alkyne-tagged by centrifugation.

[0060] The step of separating the alkyne-tagged proteins or peptides can be one wherein the immobilizing substrate is an avidin- or streptavidin-coated chromatography matrix, and the complex is separated from the peptides that are not alkyne-tagged by an affinity chromatography protocol. Both of matrices for biotin affinity chromatography and protocols for using them are considered well known in the art. For instance, a number of avidin- and streptavidin-coated matrices and also protocols for using them for biotin affinity chromatography are described in Thermo Scientific Avidin-Biotin Technical Handbook

[0052] , In some embodiments, the chromatography matrix can be packed into a pipette tip or centrifuge tube, such as a FastPure™ Mini Spin Column from Thomas Scientific or the matrix can be a streptavidin-coated filter membrane or microtiter capture plate, such as SAM® Biotin Capture Membrane or a SAM® Biotin Capture Plate available from Promega

[0053] .

[0001] The immobilizing substrate can be a wall of a microfluidic chamber or a pipette tip or other tube that is coated with avidin or streptavidin, or that is functionalized with the amine-reactive functional group, and the step of separating the alkyne-tagged proteins or peptides can be performed by washing the coated or functionalized wall with a washingsolution until all of the proteins that are not covalently or affinity bound to the wall are removed.

[0062] In any of the methods for identifying or purifying alkyne-tagged proteins or peptides herein, the step of releasing the alkyne tagged protein or peptide from the complex or from the immobilized substrate can be performed by a mild acid cleavage. Such mild acid cleavage can be performed using 1% to 10% formic acid or 0.1% to 10% Trifluoroacetic acid (TFA), for example 0.1% to 1% TFA.

[0063] Use of higher acid concentrations allows the use of shorter cleavage reaction times. For example, cleavage using 10% TFA can be done within minutes.

[0064] Chromatography steps useful to analyze the cleaved peptides often use 0.1% formic acid buffer as a mobile phase. In these instances use of 1% - 10% formic acid in the cleavage reaction is advantageous, even if slower, for avoiding a subsequent desalting step before analyzing the peptides by chromatography. For example, using 1% formic acid, about 92% cleavage is obtained at 150 minutes; using 10% formic acid, complete cleavage is obtained in 1 hour.

[0065] In any of the methods for alkyne-tagging proteins or peptides disclosed herein, the step of contacting the protein with the alkyne-tagging reagent can be performed by adding the alkyne-tagging reagent to living cells in culture or to a cultured tissue.

[0066] The alkyne-tagged proteins or peptides that are prepared by the methods disclosed herein can be analyzed by one or more of high-performance liquid chromatography, capillary electrophoresis, gel electrophoresis or 2-D gel electrophoresis, mass spectrometry and amino acid sequence determination. Applications of these methods to protein and peptide analysis are well-known in the art.

[0067] These analytic methods can be applied individually or serially, and can be employed quantitatively or qualitatively.

[0068] For example, the peptides can be separated and their amino acid sequence determined by two rounds of mass spectrometry. Or, labeled whole proteins can be separated from unlabeled proteins in the same way as separating the labeled peptides from theunlabeled peptides. The detectable proteins or peptides can be quantitated by mass spectrometry to observe changes in expression levels of a number of proteins at one time. Or, the peptides can be separated by HPLC and then their amino acid sequence determined by mass spectrometry.

[0069] In instances when the amino acid sequences of the alkyne-labeled peptides are determined, the labeled sites can be mapped onto 2- or 3 -dimensional structures of a protein to characterize the conformational changes or aggregation of the protein, as exemplified in Fig.s 4E and 5.

[0070] Aggregations of misfolded cytosolic or nuclear proteins have been associated with a broad range of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS)

[0050] ,

[0071] Thus, analysis of the alkyne-tagged peptide profile of certain proteins in a subject provides a method for diagnosing a disease associated with misfolding, conformational change or aggregation of a protein, comprising: contacting cells of a subject or a lysate of cells of a subject with an alkyne-tagging reagent to obtain alkyne-tagged proteins; digesting the alkyne-tagged proteins with a protease to obtain alkyne-tagged peptides; reacting the alkyne-tagged peptides by a click reaction with a compound of the formula X or formula Z to obtain biotinylated or amine-labelled peptides; separating the biotinylated or amine-labelled peptides from peptides that are not alkyne-tagged, as described above; releasing the peptides from the biotin or amine label to obtain purified alkyne- tagged peptides; separating the alkyne-tagged peptides by one or more of high-performance liquid chromatography, capillary electrophoresis, gel electrophoresis, 2-D gel electrophoresis, mass spectrometry and determining the amino acid sequence of the alkyne-tagged peptides;mapping the peptides onto 2- or 3-dimensional structures of the protein, the misfolding of the protein being associated with the disease, to characterize the conformation of the protein by a profile of alkyne-tagged peptides; and comparing the profile of alkyne-tagged peptides in the cells of the subject to a reference profile of alkyne-tagged peptides in the normal conformation of the protein (i.e. the conformation of the protein in cells of a healthy subjects); wherein a different profile in cells of the subject indicates the presence of or risk of the disease.

[0072] In such a method it is anticipated that the reference profile of alkyne-tagged peptides that represents the normal conformation of the protein will be determined from a statistical analysis of the profile of the protein obtained from a sufficient number of healthy individuals.

[0073] Such a method as above can be used to diagnose Alzheimer’s disease by analyzing the profile of tau protein or [J-amyloid protein; or to diagnose Parkinson’s disease by analyzing the profile of a-synuclein; or to diagnose Amyotropic Lateral Sclerosis by analyzing the profile of superoxide dismutase 1 (SOD1) and / or Tar DNA Binding Protein-43 or Dipeptide Repeat Proteins

[0051] ; to diagnose Huntington’s disease by analyzing the profile of Huntingtin; or to diagnose a transmissible spongiform encephalopathy disease, such as Creutzfeld-Jakob disease or Bovine Spongiform Encephalopathy or scrapie, by analyzing the profile of a prion protein.

[0074] The linker compound of the formula X or of the formula Z is conveniently used in a click reaction as a reagent composition comprising: i) a copper salt or complex; ii) a linker that is a compound of the formula X:or a compound of the formula Z:whereinR1 is hydrogen, hydroxyl, C1-C3 alkoxy, (CH2)n-C00H, NHCOR (R = C1-C3 alkyl, C1-C3 alkoxy, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH, NR3R4, or O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH;R2 is hydrogen,-Cl-C2 alkyl, C1-C3 alkoxy, (CH2)n-C00H, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH;R3 and R4 can be the same or different and are selected from hydrogen or C1-C3 alkyl, Cl- C3 alkoxy, CH2CH2-O-(CH2)o-CH3, or CH2CH2-O-(CH2)o-OH; each R5 can be the same or different; and can be hydrogen, unsubstituted C1-C2 alkyl or Cl- C2 alkyl substituted by a 5- or 6-atom aryl or heteroaryl group; m = 0 to 3, and any of R1 can be the same or different; n = 0 to 3; o = 0 to 12p = 1 or 2; q = 0 to 50;R6 is an amine group that can react with an epoxide or succinimide group, such as NHR7, (CH2)SNHR7 or (CH2)SN(R7)2, wherein R7 can be hydrogen or C1-C2 alkyl and s = 0 to 2; and optionally iii) a click reaction ligand.

[0075] Typically, R6 is NH2;

[0076] A “click reaction ligand” is a complexing agent that complexes a metal catalyst for a click reaction. The complex is advantageously resistant to metal binding ligands that might be present in the click reaction.

[0077] Examples of click reaction ligands that might be used in a reagent composition are shown in Fig. 6

[0042] ,

[0078] The reagent composition can be one wherein in the compound of formula X or formula Z, R1 is hydroxyl and m = 1, 2 or 3.

[0079] Additionally or alternatively, in a reagent composition the compound of the formula X or formula Z can be one wherein R2 is Cl alkoxy.

[0080] Still further, a reagent composition can be one in which the linker is the compound of the formula Y :

[0081] Or, a reagent composition can be one in which the linker compound Z is a compound of the formula Z’ :wherein R6 is an amine group that can react with an epoxide or succinimide group, such as NHR7, (CH2)SNHR7 or (CH2)SN(R7)2, wherein R7 can be hydrogen or C1-C2 alkyl and s = 0 to 2. Typically, R6 is NH2.

[0082] In reagent composition embodiments, the click reaction ligand can be present in a concentration from 0.01 to 1 mM, e.g. 0.1 to 0.5 mM; the copper salt or complex can be present at from 0.01 to 1 mM, e.g. from 0.1 to 0.5 mM; and the linker compound of formula X or formula Z can be present at from 0.1 to 5 mM, e.g. from about 0.25 to 1 mM.

[0083] In the reagent composition used in the Example 2 below, the click reaction ligand is present in a concentration of about 0.5 mM, the copper salt or complex is present at about 0.25 mM and the compound of formula Y is present at about 0.15 mM.

[0084] Additionally or alternatively, in a reagent composition the click reaction ligand can bethe copper salt can be copper sulfate, and the linker can be the compound of the Y :, or the linker can be the compound of the formula Z’ :wherein:R1 is hydrogen, hydroxyl, C1-C3 alkoxy, (CH2)n-C00H, NHCH=O, NR3R4, or O-(CH2)o- CH3;R2 is hydrogen, hydroxyl, C1-C2 alkyl, C1-C3 alkoxy or (CH2)n-C00H;R3 and R4 can be the same or different and are selected from hydrogen or C1-C3 alkyl; each R5 can be the same or different; and can be hydrogen, unsubstituted C1-C2 alkyl or Cl- C2 alkyl substituted by a 5- or 6-atom aryl or heteroaryl group; m = 0 to 3, and any of R1 can be the same or different; n = 0 to 3; o = 0 to 3; p = 1 or 2; andR6 is an amine group that can react with an epoxide or succinimide group, such as NHR7, (CH2)SNHR7 or (CH2)SN(R7)2, wherein R7 can be hydrogen or C1-C2 alkyl and s = 0 to 2. Typically, R6 is NH2.

[0085] Such reagent compositions as above can be used for conjugating an alkyne-tagged protein or peptide to biotin in a click reaction.

[0086] A mild reducing agent is added to the click reaction following addition of the reagent composition to a concentration of from 0.1 to 100 mM, typically from about 0.5 to 5 mM.

[0087] In Example 2, sodium ascorbate is added to a concentration of 2.5 mM.

[0088] Reagents for performing a click reaction composition can be provided in the form of a kit that comprises at least one container of a reagent composition comprising i) a compound of formula X or of formula Z; ii) a copper salt or complex; and optionally iii) a click ligand.

[0089] Such a kit can comprise separate containers, a first container that contains i) a compound of formula X;ii) a copper salt or complex; and optionally iii) a click ligand; and a second container that contains i) a compound of formula Z; ii) a copper salt or complex; and optionally iii) a click ligand.

[0090] Additionally or alternatively a kit can include a separate container that contains a mild reducing agent, such as sodium or potassium ascorbate.

[0091] Alternatively, a kit can include separate containers, each containing one of a compound of the formula X, a copper salt or complex, a click ligand and optionally a container containing a reducing agent. Such a kit might further include another container containing a compound of the formula Z.

[0092] Alternatively, a kit can include separate containers, each containing one of a compound of the formula Z, a copper salt or complex, a click ligand and optionally a container containing a reducing agent.EXAMPLESMaterials and reagents

[0093] Reagents and solvents were purchased from Sigma-Aldrich, Fisher Scientific, Vector Lab, and TCI. They were used without further purification unless otherwise noted. Reactions were run under N2 using standard Schlenk techniques. Methylene chloride, THF and toluene were purified by an JC Meyer solvent purification system. Flash chromatography (FC) was performed on 60 A silica gel (SiliCycle Inc.). Thin layer chromatography (TLC) was performed on silica gel Al foils and visualized under 254 nm UV light. 1H- and 13C- NMR spectra were recorded on a JEOL EC-400 and EC-600 spectrometer using residual CHC13 as internal reference. High resolution mass spectrometry (HRMS) was performed on a Bruker timsTOF Pro system equipped with a NanoElute liquid chartography system. For proteomic sample preparation and analysis, LC-MS grade water, acetonitrile (ACN), formicacid (FA), and sequencing grade trypsin were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). All other chemicals were purchased from Millipore Sigma (St. Louis, MO) and used without further purification unless noted otherwise.Example 1 - Synthesis of the APA biotin Linker.

[0094] The click linker APA biotin features an Acetal linkage and a Picolyl Azide head, thus termed APA. We use a Cu(I) chelating picolyl azido group to greatly accelerate the click reaction,

[0001] ashas been demonstrated in many systems.^, 17-19] jnaddition, under LCMS conditions with 0.1% formic acid in the mobile phase, the resultant pyridinium group in the enriched peptides will often enhance the MS sensitivity for their detection.

[0095] The small acetal linkage in APA combined with a sufficiently long oligo(ethylene glycol) chain is expected to greatly reduce non-specific binding to proteins and peptides as well as to the solid support coated with streptavidin for capturing the labeled peptides. In general, the linker of formula X is more hydrophilic than the linkers of the prior art, and this quality is thought to reduce the non-specific binding of the linker to proteins or peptides or to a protein-coated solid support.

[0096] Upon removal of the non-labeled peptides from the solid support by washing, the acetal linker enables release of the labeled peptides under mild acidic conditions. p0] Although picolyl azide biotin derivatives without a cleavable linkage are commercially available, their usage requires a cleavable linkage to be incorporated in the alkyne tag,P, 18, 1 ] since direct release of the biotinylated peptides from an avidin support requires harsh conditions causing contamination and sample loss.

[0097] The overall synthesis scheme of the APA biotin linker is illustrated in Fig. 3.

[0098] The synthesis of Biotin APA begins by coupling of the pyridyl bromide 1 with the alkyl bromide 2, employing a reported nickel -catalyzed cross-electrophile couplingpl] promoted by the ligand 3p2] to provide the pyridyl acetate 4 in a low yield. The low yield is in part due to product loss during drying under high vacuum and difficulties to separate the 2- hydroxymethylpyridine bioproduct by chromatography. The alcohol 4 is converted to the azide 5a in 52% yield using (PhO)2PON3 in the presence of DBU, which was hydrolyzed tothe alcohol 5b. On the other hand, the symmetric acetal 7 is readily prepared from the protected 3 -hydroxy propanamine 6 by transacetalation.pO] With the picolyl alcohol 5b and the symmetrical acetal 7 in hand, the asymmetric acetal 8 is efficiently prepared in 54% yield by alkoxyalkylation with in-situ generated a-chloroalkyl ether from 7, catalyzed by Zn(II).p3] Deprotection of the phthalimide with hydrazine gave APA-NH2 in 54% yield. Mixing APA-NH2 with the bi otin-(PEG) 12-A' -hydroxy succinimide ester 9 in 1 : 1 ratio in CDC13 furnishes the final product APA biotin.

[0099] Within 2 h of reaction, ^H- and ^C-NMR showed the complete disappearance of the characteristic triplet at d 3.83 ppm and the signal at 65.71 ppm from the NHS-ester 9. High resolution LCMS showed the crude product was sufficiently pure for use without further purification.

[0100] Compound 4. The synthetic procedure was based on a reported one

[0021] with modification. Under N2 atmosphere, to a 100 mL Schlenk tube equipped with a magnetic stirbar was added in the following order, Zn powder (2.61 g, 40 mmol), LiCl (572 mg, 13.5 mmol), 3-bromo-5-hydroxymethylpyridine (cmpd. 1, 2.519 g, 13.3 mmol) in dimethylacetamide (DMAc, 25 mL), the ligand 3 (156 mg, 0.661 mmol, prepared according to the literature

[0022] ), NiBr2 DME (206 mg, 0.667 mmol). The mixture was heated with an oil bath at 70°C. Under rigorous stirring, 3-bromopropyl acetate (2, 3.705 g, 20.5 mmol) in DMA (15 mL) was dropwise added to the reaction mixture via a syringe pump for 1.5 h. The mixture was stirred overnight (14 h) at 70°C (bath temperature). After cooling to room temperature, the mixture was filtered through a pad of Celite in a glass filter, and washed with EtOAc (3 x 60 mL). The filtrate was washed with 5% NH40H (2 x 25 mL) and then brine (3 x 20 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated in a rotavap, and the mixture was separated by FC (3% - 4% MeOH / CH2C12) to give 4 (520 mg, 19%). TLC: 5% MeOH / CH2C12, Rf 0.50. 1H-NMR (400 MHz, CDC13) 8 8.38 (br. s, 1H), 7.50 (dd, J = 8.0, 1.6 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 4.72 (s, 2H), 4.08 (t, J = 6.4 Hz, 2H), 2.70 (t, J = 7.6 Hz, 2H), 2.05 (s, 3 H), 1.97-1.91 (m, 2H), 1.63 (br. s, 1H).13C NMR (100 MHz, CDC13) 5 171.21, 157.14, 148.56, 136.79, 135.24, 120.44, 64.16, 63.49, 30.00, 29.13, 21.04.

[0101] Compound 5a. At 0°C under N2, to a stirred soln, of 4 (120 mg, 0.574 mmol) in anhydrous THF (2 mL) were simultaneously dropwise added in 3 min (PhO)2PON3 (288 uL, 1.34 mmol) and DBU (200 uL, 1.34 mmol) in separate 500 uL syringes. The resultant suspension was stirred for 30 min. After the cooling bath was removed, the mixture was stirred for 2 h. Usual workup and FC (20% - 30% AcOEt / hexane) gave 5a (70 mg, 52%). TLC: 5% MeOH / CH2C12, Rf 0.35. 1H-NMR (400 MHz, CDC13) 8 8.64 (br. s, 1H), 7.52 (dd, J = 8.0, 2.4 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 4.42 (s, 2H), 4.06 (t, J = 6.4 Hz, 2H), 2.68 (t, J - 7.6 Hz, 2H), 2.02 (s, 3H), 1.97-1.90 (m, 2H). 13C NMR (100 MHz, CDC13) 8 171.14, 153.54, 149.83, 136.94, 135.98, 121.91, 63.45, 55.50, 29.89, 29.16.

[0102] Compound 5b. To a stirred soln, of 5a (60 mg, 0.30 mmol) in MeOH (2.7 mL) was added a MeOH soln, of NaOMe (0.5 M, 0.12 mL, 0.06 mmol). After stirring for 5 h at r. t., the mixture was neutralized with 10% H2SO4, and dried to give 5b (56 mg, 99%). TLC: 5% MeOH / CH2C12, Rf 0.35. 1H-NMR (400 MHz, CDC13) 8 8.40 (d, J = 2.0 Hz, 1H), 7.54 (dd, J = 7.6, 2.0 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 4.42 (s, 2H), 3.64 (t, J = 6.4 Hz, 2H), 2.72 (br. s, 1H), 2.70 (t, J = 7.6 Hz, 2H), 1.88-1.79 (m, 2H). 13C NMR (100 MHz, CDC13) 6 153.13, 149.69, 137.27, 136.88, 122.03, 61.46, 55.40, 33.81, 28.95.

[0103] Compound 7. The procedure was based a reported one

[0020] with modification. A mixture of N-(3-hydroxypropyl)-phthalimide (6, 10.26 g, 50 mmol, 2 eq ), 2,2- dimethoxyethane (4.05 g, 45 mmol, 1.8 eq.), and Amberlyst 15 (H-form, 4.7 meg / g, 300 mg, -0.056 eq.) in dry toluene (400 mL) was stirred in a 1 L round bottle flask equipped with a Dean-Stark distillatory. The mixture was refluxed at 120°C (oil bath temperature) for 30 min. The temperature was increased to allow azeotropic distillation of 400 mL toluene in total during 4.5 h; upon every 100 mL toluene was distilled, the same amount of anhydrous toluene containing 2, 2-dimethoxy ethane (0.41 g, 4.4 mmol) was added to the mixture. The mixture was cooled to r.t., treated with Et3N (1 mL), filtered a pad of Celite, and washed with EtOAc. FC (20% EtOAc / hexane) gave 7 (7.1 g, 63%). TLC: 4:6 EtOAc / Hexane, Rf0.12. 1H-NMR (400 MHz, CDC13) 5 7.83-7.80 (m, 4H), 7.70-7.66 (m, 4H), 4.60 (q, J = 5.4 Hz, 1H ), 3.82-3.71 (m, 4H), 3.64-3.59 (m, 2H), 3.46-3.40 (m, 2H), 1.92 (quintet, J = 6.4 Hz, 4H), 1.18 (d, J = 5.6 Hz, 3 H). 13C NMR (100 MHz, CDC13) 5 168.47, 133.92, 132.26, 123.22, 100.09, 62.93, 35.66, 28.86, 19.53.

[0104] Compound 8. Under N2, to a dried 10 mL Schlenk tube was added a soln, of 7 (145 mg, 0.315 mmol) in anhydrous CH2C12 (0.3 mL). Under stirring, a soln, of ZnC12 (50 mM in anhydrous THF, 7 pL. 0.35 pmol) was added, and the soln, was cooled with an ice bath. Under stirring, AcCl (23 uL, 0.32 mmol) was added dropwise. The ice bath was removed and the mixture was stirred at r.t. for 1.5 h. To this stirred soln., a soln, of 5b (56 mg, 0.29 mmol) and DIPEA (61 uL, 0.35 mmol) in anhydrous CH2C12 (0.3 mL) was rapidly added. The mixture was stirred overnight at r.t. FC (13%-40% EtOAc / hexane) gave 8 (83 mg, 68%). TLC: 4:6 EtOAc / Hexane, Rf 0.45. 1H-NMR (400 MHz, CDC13) 5 8.41 (d, J = 2.0 Hz, 1H), 7.85-7.80 (m, 2H), 7.72-7.68 (m, 2H), 7.53 (dd, J = 8.0, 2.0 Hz, 1H), 7.24 (d, J ~ 8.0 Hz, 1H), 4.63 (q, J = 5.4 Hz, 1H), 4.41 (s, 2H), 3.78 (t, J = 6.8 Hz, 2H), 3.63-3.50 (m, 2H), 3.47-3.38 (m, 2H), 2.68 (t, J = 7.6 Hz, 2H), 1.98-1.91 (m, 2H), 1.89-1.80 (m, 2H), 1.24 (d, J = 5.2 Hz, 3H). 13C NMR (100 MHz, CDC13) 8 168.50, 153.20, 149.89, 136.99, 136.71, 133.99, 132.22, 123.24, 121.82, 100.06, 64.05, 63.19, 55.56, 35.65, 31.20, 29.39, 28.86, 19.69.

[0105] APA-NH2. Under N2, to 8 (80 mg, 0.19 mmol) in a Schlenk tube with a stirbar was added a soln, of hydrazine (IM in THF, 1.9 mL, 19 mmol). The stirred soln, was heated with a 40° oil bath for 20 min and 50°C for 30 min. After cooling down to r.t., 10 N KOH (0.5 mL) was added, and the soln, was stirred overnight (15 h). The mixture was dried in a rotavap, and the residue was dissolved in water (0.2 mL) and extracted with ether (10 mL). The ether layer was washed with water (0.2 mL) and dried over anhydrous Na2SO4. High vacuum drying gave APA-NH2 (30 mg, 54%). 1H-NMR (400 MHz, CDC13) 6 8.43 (d, J = 2.0 Hz, 1H), 7.54 (dd, J = 8.0, 2.4 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 4.65 (q, J = 5.4 Hz, 1H), 4.45 (s, 2H), 3.66-3.55 (m, 2H), 3.50-3.40 (m, 2H), 2.78 (t, J =6.8 Hz, 2H), 2.71 (t, J = 7.8 Hz, 2H), 1.92-1.85 (m, 2H), 1.70 (quintet, J = 6.8 H, 2H), 1.39 (br. 2H), 1.30 (d, J = 5.2 Hz,3H). 13C NMR (100 MHz, CDC13) 8 153.25, 149.92, 136.97, 136.65, 121.82, 99.99, 64.18, 63.53, 55.56, 39.69, 33.76, 31.16, 29.39, 19.85.

[0106] APA biotin. To a stirred soln, of APA-NH2 (12 mg, 0.041 mmol) in CDC13 (0.5 mL) in a 1 mL flask was added the biotin-EG12-NHS (9, 40 mg, 0.043 mmol) in CDC13 (0.4 mL). The soln, was stirred at r.t. for 2 h. NMR spectra were recorded, and the solvent was removed to give APA biotin (41 mg), which was sufficiently pure for use without further purification. HRMS: [M + Na]+ C51H90N8O17SNa m / z calculated 1141.60; measured 1141.60; [M + H]+ C51H90N8O17SH m / z calculated 1119.62; measured 1119.62; [M + 2H]2+ C51H92N8O17S m / z calculated 560.313; measured 560.315. 1H-NMR (600 MHz, CDC13) 8 8.38 (s, 1H), 7.52 (d, J = 8.0, 1H), 7.24 (d, J = 8.0 Hz, 1H), 6.78 (t, J =5.4 Hz, 1H), 6.67 (br. t, J = 5.2 Hz, 1 H), 6.43 (br. s, 1H), 6.25 (br., 1H), 5.84 (br., 1H), 4.61(q, J = 5.4 Hz, 1H), 4.48-4.45 (m, 1H), 4.42 (s, 2H), 4.28-4.25 (m, 1H), 3.67 (t, J = 5.8 Hz, 2H), 3.67-3.49 (m, 48H), 3.42-3.34 (m, 4H), 3.27 (q, J = 7.4 Hz, 2H), 3.13-3.07 (m, 1H), 2.84 (dd, J = 12.8, 4.4 Hz, 1H), 2.82 (s, 4H, the NHS-OH biproduct), 2.70 (d, J ~ 12.8 Hz, 1H), 2.67 (t, J = 6.8 Hz, 2H), 2.40, (t, J = 6.0 Hz, 2H), 2.17 (t, J = 7.4 Hz, 2H), 1.83 (quintet, J = 6.4 Hz, 2H), 1.70 (quintet, J = 6.8 H, 2H), 1.69-1.57 (m, 2H), 1.42-1.34 (m, 2H), 1.25 (d, J = 5.2 Hz, 3H). 13C NMR (150 MHz, CDC13) 8 173.49, 171.62, 164.32, 153.12, 149.63, 136.97, 137.29, 136.81, 122.05, 100.06, 70.58 (br.), 70.49, 70.44, 70.37, 70.27, 70.12, 69.94, 67.34, 64.25, 63.66, 61.91, 60.36, 55.65, 55.31, 40.56, 39.21, 37.28, 37.02, 35.93, 31.10, 29.66, 29.35, 28.27, 28.10, 25.62, 25.49, 19.87.Example 2 - Detection of Altered Protein Conformation and / or Aggregation and / or Partner Binding During Heat Shock

[0107] The performance of the linker APA biotin was evaluated by performing chemical labeling mass spectrometry (CLMS) for profding the proteome structural change in yeast S. cerevisiae cells after subjecting them to heat shock from 30 °C rapidly to 37 °C, 42 °C, and 47 °C for short durations of 1, 2, 3, 4, and 5 min (Fig. 4A). After each duration, the samples were labeled with the A-hydroxysuccinimide ester probe 10 that mainly reacts with lysine but also with threonine, serine, and arginine. [4] Unlike the previous chemicalproteomic studies aiming to discover covalent drugs and “ligandable” sites by developing probes with a high selectivity, [1-5] our goal is to use CLMS for profiling the proteome-wide structural change of live cells in response to external stimuli. Therefore, probes such as 10 with a wide spectrum reactivity (in this instance, reactive with amino terminal groups, and K, R, S and T side chains) is desirable.[00108J The overall experimental design of this Example is illustrated in Fig. 4A.Yeast culture

[0109] The yeast Saccharomyces cerevisiae wide type strain BY4741 (a gift from Dr. Vishal Gohil at the Texas A&M University) was cultured in YPD (1% yeast extract, 2% peptone, and 2% dextrose) medium at 30 °C for overnight. The culture was washed and resuspended in phosphate buffered saline (PBS) with an OD of 1 at 600 nm. For heat shock at 37 °C, 42 °C, and 47 °C, prewarmed PBS at the specific temperature was used. The tube with yeast cells was then immediately placed in water bath of specific temperatures and incubated for 1-5 min. At the end of heat shock, the yeast cells were spun down, and PBS of 30 °C was added with 500 uM of labeling agent 10. The cell culture was incubated at 30 °C for 15 min, and 1 mM of hydroxyl amine was added to the culture to quench the reaction.Protein extraction and digestion

[0110] The proteomic sample preparation was performed by following the Sample Preparation by Easy Extraction and Digestion (SPEED) procedure

[0041] , Briefly, the yeast cells were centrifuged and supernatant was removed as much as possible. Ten microliters of trifluoroacetic acid was added to the cell pellet and vortexed until all samples were dissolved. One hundred microliters of 2 M Tris base was added followed by a mixture of TCEP (10 mM) and CAA (40 mM). The samples were heated at 95 °C for 5 min followed by adding 500 pF of HPLC grade water. Protein concentration was then determined using the Bradford reagent, and 20 pg were used for tryptic digestion. Trypsin (0.4 pg) was added into each sample and incubated at 37 °C for overnight. The digested peptides were then desalted using Cl 8 ziptips and vacuum dried using a CentriVap (Labconco Corporation, Kansas City, MO).A small portion (10%) of each sample was used to profile protein abundance by DIA MS (see below), and the rest was used to enrich labeled peptides.Enrichment of labeled peptides

[0111] A click reaction was performed to attach handles to labeled peptides by following the published procedure

[0042] . Briefly, peptide samples were dissolved in water to a concentration of 0.1 mM. A solution of reagents (0.15 mM APA biotin, 0.25 mM CuSO4, 0.5 mM tris igand (Fig. 6) was added. A sodium ascorbate stock solution was prepared freshly and added at a final concentration of 2.5 mM to initiate the click reaction. After incubation on a shaker for 15 minutes at room temperature, the reaction was quenched using 0.3 mM pentasodium DTPA. The reaction mixture was added to streptavidin sepharose high performance beads (GE Healthcare, USA) that were prewashed with 20 mM sodium phosphate buffer (pH7.4) ten times vigorously. After incubation on a shaker at 1200 rpm for 2 h at room temperature, the beads were washed with 100 pL 20 mM sodium phosphate buffer ten times. After removing supernatant, 10% formic acid was added to the beads and the beads were incubated at room temperature for 1 h; cleavage of APA biotin in 10% formic acid completed in 1 h at room temperature. The supernatant containing cleaved labeled peptides was collected, and the beads were washed with 20 mM sodium phosphate buffer. The wash solution was combined with the supernatant. The labeled peptides were then desalted using Cl 8 stage tips and vacuum dried as above. Each dried sample was resuspended in 2% ACN with 0.1% FA for LC-MS analysis. nanoLC-MS / 'MS

[0112] The liquid chromatography -mass spectrometry procedure was published elsewhere.

[0043] Specifically, a NanoElute LC system coupled to a timsTOF Pro (Bruker Daltonics, Germany) via a CaptiveSpray source was used. Samples (100 ng) were loaded onto an in-house packed column (75 pm x 15 cm, 1.9 pm ReproSil-Pur C18 particle (Dr. Maisch GmbH, Germany), column temperature 40 °C) with buffer A (0.1% FA in water) and buffer B (0.1% FA in ACN) as mobile phases. The 21 -min gradient was 17.8 min from 2% B to 30% B, 18.3 min to 95% B, and maintained for another 2.4 min.

[0113] Mass spectrometry (MS) data acquisition for protein abundance data was achieved using a data-independent acquisition approach (diaPASEF) for deep coverage of the yeast proteome without enrichment

[0046] , The collected protein abundance data were used for the normalization of the labeled proteome data to remove the effects of protein abundance changes on labeled peptide abundances. Specifically, a diaPASEF scheme with 16 m / z and ion mobility windows was used. The electrospray voltage was 1.6 kV, and the ion transfer tube temperature was 180 °C. Full MS scans were acquired over the range m / z of 100-1700. The collision energy was ramped linearly as a function of the mobility from 27 eV at 1 / K0 = 0.85 Vscm-2 to 45 eV at 1 / K0 = 1 .3 Vscm-2.

[0114] MS data acquisition for labeled peptides was achieved using a data-dependent acquisition approach (ddaPASEF).

[0044] The parallel accumulation- serial fragmentation (PASEF) mode with 4 PASEF scans per cycle was used. The electrospray voltage was 1.4 kV, and the ion transfer tube temperature was 180 °C. Full MS scans were acquired over the mass-to-charge (m / z) range of 100-1700. The target intensity value was 2.0 xlO4with a threshold of 2500. A fixed cycle time was set to 0.53 s, and a dynamic exclusion duration was 0.4 min with ± 0.015 amu tolerance. Only peaks with charge state > 2 were selected for fragmentation.Peptide and protein identification and quantification

[0115] The software DIANN version 1.8

[0045] was used with default settings for peptide and protein identification and quantification from diaPASEF data DirectDIA approach was employed, and the data were searched against a UniProt-SwissProt protein database without isoforms (Saccharomyces cerevisiae, downloaded on 09 / 12 / 2022, 6175 entries). Cysteine carbamidomethylation was listed as a fixed modification, and methionine oxidation and acetylation as variable modifications. The false discovery rate was controlled at <1% at both peptide and protein levels. Only proteolytic peptides were used for quantification.

[0116] The software MSFragger

[0046] (version 4.0) was used for labeled peptide identification and quantification. The same protein database was used. Cysteine carbamidomethylation was used as a fixed modification, and methionine oxidation andacetylation as variable modifications. Additionally, mass shifts of 80.0262 and 272.1273 on lysine, threonine, serine, tyrosine, and arginine residues and protein N-terminal were used as variable modifications to identify labeled peptides. Peptide length was set to be 7-50, and 2 missed cleavages were allowed. Precursor charges were 1-5, and m / z range was 100-1800. Precursor tolerance was 20 ppm, and ion match tolerance was 0.05 m / z. The precursor and product ion masses were set to be monoisotopic. The false discovery rate (FDR) was controlled at <1% at peptide spectrum match, peptide, and protein levels.Statistical analysis

[0117] All statistical analyses were performed using R software version 4.1.0. Log2- transformation and median normalization were used to normalize the protein abundance quantification data. For labelled peptide quantification data, an in-house python script was used to extract only labelled sites, and the abundance of each labelled site was calculated by summing all labelled peptides containing the specific labelled site. The data were then log2- transformed and normalized to total ion intensity. The labelled site quantification data were further normalized with protein abundance data. Statistical analysis was then performed using empirical Bayes moderated tests as implemented in the R / Bioconductor limma package [47, 48], The p-values were further adjusted using the Benjamini-Hochberg method, and only labelled sites with an adjusted p-value below the threshold (alpha = 0.05) was considered statistically significant.

[0118] The list of 63 yeast chaperones

[0049] was used as reference set to identify the presence of chaperone proteins. The presence of chaperone-client relationships was also investigated using the list of yeast chaperone physical interactors retrieved from the BioGRID database (v3.5, https: / / thebiogrid.org / ). The clients of chaperones were restricted to those interactions detected either by “affinity capture-MS” or by “affinity capturewestern” experimental methods. The presence of disordered regions on proteins were investigated using the UniProt database.

[0119] Using APA biotin (compound Y, Fig. 3) in the workflow shown in Fig. 4A, we identified 1239 proteins, including all classes of chaperones, that contained >1 sites labeled by 10. The labeling was throughout all major cellular compartments as shown in Fig. 4C.

[0120] Significantly, over 9400 labeled sites were identified, mostly on lysine (69%) but also on serine, threonine, tyrosine, and arginine in the proteome of the living yeast cells. In comparison, the most comprehensive lysine labeling (14,000 sites in totalP, 3])usec[ excessive lysates of two human cell lines (3.5 times of the S. cerevisiae proteome) for labeling by -100 lysine-reactive probes in many experiments.

[0121] In comparison, the most comprehensive lysine labeling quantified 14,000 sites in total [2, 3]) used excessive lysates of two human cell lines, containing 2.5 times more number of proteins than that of the S. cerevisiae proteome, and -100 different kinds of lysine-reactive probes in many experiments. Furthermore, only 12% unlabeled peptides were found among all peptides collected from the streptavidin beads aftereptides with modification on these amino acids, acid cleavage. In addition, among all labeled peptides collected from the streptavidin beads after acid cleavage, only 0.5% of them that did not react with APA biotin were enriched.

[0122] Overall, APA biotin demonstrated a high efficiency for enrichment of the labeled peptides leading to the quantification of an unprecedented number of labeled peptides in yeast cells, which greatly facilitated the profiling of the protein structural change associated with the change of reactivity of the probe 10 at these sites.

[0123] The vast improvement by this work over the reported CLMS method using a N- hydroxysuccinimide (NHS) ester probed 24] could beattributed to the highly efficient click linker APA biotin that greatly increased coverage of the labeling sites and reduced background noise for LCMS analysis. One or multiple labeled sites in >900 proteins exhibited significant changes in reactivity to the probe at each temperature. Moreover, 68- 73% of the proteins with significant changes of reactivity were heat shock proteins (Hsps) or chaperone proteins (38-39, total 63 in this yeast strain), and their “client” proteins (592-618). The “client” proteins from the database are those bind to immobilized chaperones asdetermined by affinity capturing MS and western blot.

[0025] Fig 40 shows the heatmap of the clustered labeled site intensity fold change (heat shock vs control) for the chaperones, their client proteins, and the other proteins upon heat shock at the three temperatures for 1, 2, 3, 4, and 5 min. The temporal profile shows that increasing the heat shock temperature and duration generally resulted in more sites with a significant decrease in labeling intensity, particularly for the non-client proteins, due to their reduced accessibility / reactivity. This result suggests that the chaperones' protective role for their client vs. non-client proteins were in effect rapidly (within a minute). [26-30]

[0124] Significantly, for the over 900 proteins with altered reactivity to 10 upon heat shock, details on all the labeled sites of individual proteins can be obtained and mapped to their known or predicted 3D structures. For example, Fig. 5 shows all labeled sites of chaperone protein Hsp72 (Pl 0592, gene SSA2) during the 5 min heat shock at 37°C, which are mapped to an AlphaFold predicted 3D structure of the free chaperone protein (without binding to ADP / ATP and substrate).[00125J This Example demonstrates that the chemical labeling mass spectrometry (CLMS) method using APA biotin allows for a deep coverage of labeled sites for profiling the proteome structural changes in living cells. Use of APA biotin in this method provides efficient enrichment of the labeled peptides thus greatly enhancing the sensitivity and data quality for identifying and quantifying the binding region of low abundant protein targets in living cells.

[0126] Further, this Example illustrates that CLMS performed in live cells using APA linkers for enrichment is a powerful tool for profiling proteome-wide structural changes in live cells in response to environmental stimuli; here heat shock, but this can include drug treatments1241, ischemia, and others. It is complementary to the well-known methods, including thermal proteome profiling,133 341limited proteolysis MS (LiP-MS),135391and hydroxyl radical footprinting,1401with the advantages of being capable to perform the measurement in live cells and probe the reactivity of certain residues (K, T, S, R) on individual proteins across the proteome.ENUMERATED EMBODIMENTS

[0127] Particular enumerated embodiments (Ees) encompassed by the present disclosure are set out below.

[0128] Ee 1. A compound having the structural formula X:whereinR1 is hydrogen, hydroxyl, C1-C3 alkoxy, (CH2)n-COOH, NHCH=O, NR3R4, or O-(CH2)o- CH3;R2 is hydrogen, hydroxyl, C1-C2 alkyl, C1-C3 alkoxy or (CH2)n-COOH;R3 and R4 can be the same or different and are selected from hydrogen or C1-C3 alkyl; each R5 can be the same or different; and can be hydrogen, unsubstituted C1-C2 alkyl or Cl-C2 alkyl substituted by a 5- or 6-atom aryl or heteroaryl group; m = 0 to 3, and any of R1 can be the same or different; n = 0 to 3; o = 0 to 3 p = 1 or 2; q = 0 to 50.

[0129] Ee 2. The compound of Ee Error! Reference source not found., wherein R1 is hydroxyl and m = 1, 2 or 3.

[0130] Ee 3. The compound of Ee Error! Reference source not found, or Error!Reference source not found., wherein R2 is Cl alkoxy.

[0131] Ee 4. The compound of any one of Ees 1 to 3 that has the structural formula X’ :

[0132] Ee 5. The compound of any one of Ees 1 to

[0132] , wherein R5 is methyl or 2- pyridylmethyl.

[0133] Ee 6. The compound of Ee 1 that has the structural formula Y :

[0134] Ee 7. A compound that has the structural formula Z:wherein:R1 is hydrogen, hydroxyl, C1-C3 alkoxy, (CH2)n-COOH, NHCH=O, NR3R4, or O-(CH2)o- CH3;R2 is hydrogen, hydroxyl, C1-C2 alkyl, C1-C3 alkoxy or (CH2)n-COOH;R3 and R4 can be the same or different and are selected from hydrogen or C1-C3 alkyl; each R5 can be the same or different; and can be hydrogen, unsubstituted C1-C2 alkyl or Cl- C2 alkyl substituted by a 5- or 6-atom aryl or heteroaryl group;m = 0 to 3, and any of R1 can be the same or different; n = 0 to 3; o = 0 to 3; p = 1 or 2; andR6 is an amine group that can react with an epoxide or succinimide group, such as NHR7, (CH2)SNHR7 or (CH2)SN(R7)2, wherein R7 can be hydrogen or C1-C2 alkyl and s = 0 to 2.

[0135] Ee 8. The compound of Ee 7, wherein R1 is hydroxyl and m = 1, 2 or 3.

[0136] Ee 9. The compound of Ee 7 or 8, wherein R2 is Cl alkoxy.

[0137] Ee 10. The compound of any one of Ees 7 to 9, wherein R5 is methyl or 2- pyridylmethyl.

[0138] Ee 11. The compound of Ee 7 that has the formula Z’ :wherein R6 is an amine group that can react with an epoxide or succinimide group, such as NHR7, (CH2)SNHR7 or (CH2)SN(R7)2, wherein R7 can be hydrogen or C1-C2 alkyl and s = 0 to 2.

[0139] Ee 12. A method for making the molecule of Ee 1, comprising: i) coupling of the pyridyl bromide 1 with the alkyl bromide acetate 2, employing nickel- catalyzed cross-electrophile coupling

[0021] promoted by the ligand 3

[0022] to provide the pyridyl acetate 4; ii) converting the alcohol 4 to the azide 5a in 52% yield using (PhO)2PON3 in the presence of l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), and then hydrolyzing to the alcohol 5b;iii) separately preparing the symmetric acetal 7 from the protected 3- hydroxypropanamine 6 by transacetalation; iv) alkoxyalkylating the picolyl alcohol 5b with in-situ generated a-chloroalkyl ether from 7 in the presence of a Zn(II) catalyst to give the asymmetric acetal 8; v) deprotecting the phthalimide with hydrazine to obtain APA-NH2; vi) mixing APA-NH2 with the biotin-(PEG)12-A-hydroxy succinimide ester 9 in 1 : 1 ratio in a solvent to obtain the compound of formula X.Ee 13. A method for making the compound of Ee 7, comprising: i) coupling of the pyridyl bromide 1 with the alkyl bromide acetate 2, employing nickel- catalyzed cross-electrophile coupling promoted by the ligand 3 to provide the pyridyl acetate 4; ii) converting the alcohol 4 to the azide 5a in 52% yield using (PhO)2PON3 in the presence of l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), and then hydrolyzing to the alcohol 5b; iii) separately preparing the symmetric acetal 7 from the protected 3-hydroxypropanamine 6 by transacetalation; iv) alkoxyalkylating the picolyl alcohol 5b with in-situ generated a-chloroalkyl ether from 7 in the presence of a Zn(II) catalyst to give the asymmetric acetal 8; v) deprotecting the phthalimide with hydrazine to obtain the compound of formula Z.Ee 14. A method for purifying alkyne-tagged proteins or peptides comprising: i) contacting proteins with an alkyne-tagging reagent to obtain alkyne-tagged proteins; ii) optionally digesting the alkyne-tagged proteins with a protease to obtain alkyne- tagged peptides; iii) reacting the alkyne-tagged proteins or peptides by a click reaction with a compound of any one of Ees 1 to 6 to obtain biotinylated peptides; iv) contacting the biotinylated proteins or peptides with a biotin affinity reagent to form a complex;v) separating the complex from proteins or peptides that are not alkyne-tagged; vi) releasing the proteins or peptides from the complex to obtain purified alkyne- tagged peptides.

[0140] Ee 15. The method of Ee 14, wherein the biotin affinity reagent comprises an immobilizing substrate and the immobilizing substrate is separated from peptides that are not alkyne-tagged.

[0141] Ee 16. The method of Ee 15, wherein the immobilizing substrate is an avidin- or streptavidin-coated microbead and the complex is separated from the peptides that are not alkyne-tagged by centrifugation.

[0142] Ee 17. The method of Ee 15, wherein the immobilizing substrate is an avidin- or streptavidin-coated chromatography matrix, and the complex is separated from the peptides that are not alkyne-tagged by an affinity chromatography protocol.

[0143] Ee 18. The method of Ee 15, wherein the chromatography matrix is packed into a pipette tip or centrifuge tube or is a filter membrane, and the complex is separated from the peptides that are not alkyne-tagged by an affinity chromatography protocol.

[0144] Ee 19. A method for purifying alkyne-tagged proteins or peptides comprising: i) contacting proteins with an alkyne-tagging reagent under conditions sufficient to obtain alkyne-tagged proteins; ii) optionally digesting the alkyne-tagged proteins with a protease to obtain alkyne- tagged peptides; iii) reacting the alkyne-tagged proteins or peptides by a click reaction with a compound of any one of Ees 8 to 12 obtain amine-labelled proteins or peptides; iv) contacting and reacting the amine-labelled proteins or peptides with a functionalized substrateor, wherein r = 1 to 20, to obtain immobilized proteins or peptides covalently attached to the substrate; v) separating the immobilized proteins or peptides from proteins or peptides that are not alkyne-tagged; and vi) releasing the immobilized proteins or peptides from the substrate to obtain purified alkyne-tagged peptides.

[0145] Ee 20. The method of any one of Ees 12 to 19, wherein the releasing step vi) is performed by a mild acid cleavage.

[0146] Ee 21. The method of any one of Ees 12 to 20, wherein the alkyne-tagged proteins are digested to obtain alkyne-tagged peptides.

[0147] Ee 22. The method of any one of Ees 12 to 21, wherein the contacting step i) is performed by adding the alkyne-tagging reagent to living cells in culture or to a cultured tissue.

[0148] Ee 23. The method of any one of Ees 12 to 22, that further comprises analysing the alkyne-tagged peptides by one or more of high-performance liquid chromatography, capillary electrophoresis, gel electrophoresis or 2-D gel electrophoresis, mass spectrometry and amino acid sequencing.

[0149] Ee 24. The method of Ee 23 that further comprises mapping peptides onto 2- or 3- dimensional structures of a protein to characterize the conformation of the protein.

[0150] Ee 25. A method for diagnosing a disease associated with misfolding, conformational change or aggregation of a protein, comprising: i) contacting cells of a subject or a lysate of cells of a subject with an alkyne- tagging reagent to obtain alkyne-tagged proteins; ii) digesting the alkyne-tagged proteins with a protease to obtain alkyne-tagged peptides;iii) reacting the alkyne-tagged peptides by a click reaction with a compound of any one of Ees 1 to 11 to obtain biotinylated or amine-labelled peptides; iv) separating the biotinylated or amine-labelled peptides from peptides that are not alkyne-tagged; v) releasing the peptides from the biotin or amine label to obtain purified alkyne- tagged peptides; vi) analyzing the alkyne-tagged peptides by one or more of high-performance liquid chromatography, capillary electrophoresis, gel electrophoresis or 2-D gel electrophoresis and mass spectrometry; vii) determining the amino acid sequence of the alkyne-tagged peptides; viii) mapping the peptides onto 2- or 3 -dimensional structures of the protein, the of the misfolding, conformational change, or aggregation of the protein being associated with the disease, to characterize the conformation of the protein by a profile of alkyne-tagged peptides; and ix) comparing the profile of alkyne-tagged peptides in the cells of the subject to a reference profile of alkyne-tagged peptides in the normal conformation of the protein; wherein a different profile in cells of the subject indicates the presence or risk of the disease.

[0151] Ee 26. The method of Ee 25, wherein: the disease is Alzheimer’s disease and the protein is tau or 0-amyloid protein; or the disease is Parkinson’s disease and the protein is a-synuclein; or the disease is Amyotropic Lateral Sclerosis and the protein is superoxide dismutase 1 (SOD1) or Tar DNA Binding Protein-43 or Dipeptide Repeat Proteins; the disease is Huntington’s disease and the protein is Huntingtin; the disease is transmissible spongiform encephalopathy disease and the protein is a prion.

[0152] Ee 27. A reagent composition comprising: i) a copper salt or complex; and ii) a linker compound of any one of Ees 1 to 11.

[0153] Ee 28. The reagent composition of Ee 27, that further comprises a click ligand.

[0154] Ee 29. The reagent composition of Ee 28, wherein the click ligand is present at from 0.1 to 0.5 mM.

[0155] Ee. 30. The reagent composition of Ee 29, wherein the click ligand is present in a concentration of about 0.15 mM, the copper salt or complex is present at about 0.25 mM and the compound of formula X is present at about 0.5 mM.

[0156] Ee 31. The reagent composition of Ee 29 or Ee 30, wherein the click ligand isthe copper salt is copper sulfate; and the linker compound has the formula Y:

[0157] Ee 32. The reagent composition of Ee 29 or Ee 30, wherein the click ligand isthe copper salt is copper sulfate; and the linker compound has the formula Z’:wherein:R1 is hydrogen, hydroxyl, C1-C3 alkoxy, (CH2)n-C00H, NHCH=O, NR3R4, or O-(CH2)o- CH3;R2 is hydrogen, hydroxyl, C1-C2 alkyl, C1-C3 alkoxy or (CH2)n-C00H;R3 and R4 can be the same or different and are selected from hydrogen or C1-C3 alkyl; each R5 can be the same or different; and can be hydrogen, methyl or 2-pyridylmethyl; m = 0 to 3, and any of R1 can be the same or different; n = 0 to 3; o = 0 to 3; p = 1 or 2; andR6 is an amine group that can react with an epoxide or succinimide group, such as NHR7, (CH2)SNHR7 or (CH2)SN(R7)2, wherein R7 can be hydrogen or C1-C2 alkyl and s = 0 to 2.

[0158] Ee 33. A kit comprising at least one container of the reagent composition of any one of Ees 27 to 32, and a separate container of a reducing agent.

[0159] Ee 34. Use of a compound of any one of Ees Error! Reference source not found, to 6 for conjugating an alkyne-tagged protein or peptide to biotin.

[0160] Ee 35. Use of a compound of any one of Ees 7 to 11 for covalently attaching an alkyne-tagged protein or peptide to an immobilizing substrate.

[0161] Ee 36. The use of Ee 35, wherein the attachment is viaherein r = 1 to 20.

[0162] REFERENCES[1] K. M. Backus, B. E. Correia, K. M. Lum, S. Forli, B. D. Horning, G. E. Gonzalez-Paez,S. Chatterjee, B. R. Lanning, J. R. Teijaro, A. J. Olson, D. W. Wolan, B. F. Cravatt, ‘Proteome-wide covalent ligand discovery in native biological systems’, Nature 2016, 534, 570-574.[2] M. E. Abbasov, M. E. Kavanagh, T. A. Ichu, M. R. Lazear, Y. Tao, V. M. Crowley, C.W. Am Ende, S. M. Hacker, J. Ho, M. M. Dix, R. Suciu, M. M. Hayward, L. L. Kiessling, B. F. Cravatt, ‘A proteome-wide atlas of lysine-reactive chemistry’, Nat Chem 2021, 13, 1081-1092.[3] S. M. Hacker, K. M. Backus, M. R. Lazear, S. Forli, B. E. Correia, B. F. Cravatt, ‘Global profiling of lysine reactivity and ligandability in the human proteome’, NatChem 2017, 9, 1181-1190.[4] C. C. Ward, J. I. Kleinman, D. K. Nomura, ‘NHS-Esters As Versatile Reactivity -Based Probes for Mapping Proteome-Wide Ligandable Hotspots’, ACS Chem Biol 2017, 12, 1478-1483.[5] K. C. Tang, J. Cao, L. M. Boatner, L. Li, J. Farhi, K. N. Houk, J. Spangle, K. M. Backus,M. Raj, ‘Tunable Amine-Reactive Electrophiles for Selective Profiling of Lysine’, Angew Chem Int Ed Engl 2022, 61, e202112107.[6] Z. Li, P. Hao, L. Li, C. Y. Tan, X. Cheng, G. Y. Chen, S. K. Sze, H. M. Shen, S. Q. Yao, ‘Design and synthesis of minimalist terminal alkyne-containing diazirine photocrosslinkers and their incorporation into kinase inhibitors for cell- and tissue-based proteome profiling’, Angew Chem Ini Ed Engl 2013, 52, 8551-8556.[7] T. Kambe, B. E. Correia, M. J. Niphakis, B. F. Cravatt, ‘Mapping the protein interaction landscape for fully functionalized small-molecule probes in human cells’, J Am Chem Soc 2014, 136, 10777-10782.[8] J. M. Wozniak, W. Li, P. Governa, L. Y. Chen, A. Jadhav, A. Dongre, S. Forli, C. G. Parker,‘Enhanced mapping of small-molecule binding sites in cells’, Nat Chem Biol 2024.[9] A. Wheat, C. Yu, X. Wang, A. M. Burke, I. E. Chemmama, R. M. Kaake, P. Baker, S.D. Rychnovsky, J. Yang, L. Huang, ‘Protein interaction landscapes revealed by advanced in vivo cross-linking-mass spectrometry’, Proc Natl Acad Sci U S A 2021, 118.

[0010] H. Gao, L. Zhao, B. Zhong, B. Zhang, Z. Gong, B. Zhao, Y. Liu, Q. Zhao, L. Zhang,Y. Zhang, ‘In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane- Permeable, and Alkynyl-Enrichable Crosslinker’, Anal Chem 2022, 94, 7551-7558.

[0011] B. F. Cravatt, ‘Activity -based protein profiling - finding general solutions to specific problems’, I sr J Chem 2023, 63, e202300029.

[0012] J. Szychowski, A. Mahdavi, J. J. L. Hodas, J. D. Bagert, J. T. Ngo, P. Landgraf, D. C.Dieterich, E. M. Schuman, D. A. Tirrell, ‘Cleavable Biotin Probes for Labeling of Biomolecules via Azide-Alkyne Cycloaddition’, J Am Chem Soc 2010, 132, 18351- 18360.

[0013] N. R. Burton, D. A. Polasky, F. Shikwana, S. Ofori, T. Yan, D. J. Geiszler, F. D. VeigaLeprevost, A. I. Nesvizhskii, K. M. Backus, ‘Solid-Phase Compatible Silane-Based Cleavable Linker Enables Custom Isobaric Quantitative Chemoproteomics’, J Am Chem Soc 2023, 145, 21303-21318.

[0014] Z. Li, K. Liu, P. Xu, J. Yang, ‘Benchmarking Cleavable Biotin Tags for Peptide-CentricChemoproteomics’, JProteome Res 2022, 21, 1349-1358.

[0015] N. Gertsik, C. W. Am Ende, K. F. Geoghegan, C. Nguyen, P. Mukherjee, S. Mente, U. Seneviratne, D. S. Johnson, Y. M. Li, ‘Mapping the Binding Site of BMS-708163 on gamma-Secretase with Cleavable Photoprobes’, Cell Chem Biol 2017, 24, 3-8.

[0016] S. Huangfu, X. Yu, Z. Sun, B. Jiang, H. Chen, ‘Chemical reagents for the enrichment of modified peptides in MS-based identification’, Chem Commim (Camb) 2024, 60, 1509-1516.

[0017] G. C. Kuang, P. M. Guha, W. S. Brotherton, J. T. Simmons, L. A. Stankee, B. T.Nguyen, R. J. Clark, L. Zhu, ‘Experimental Investigation on the Mechanism of Chelation-Assisted, Copper(II) Acetate- Accel erated Azide-Alkyne Cycloaddition’, J Am Chem Soc 2011, 133, 13984-14001.

[0018] D. K. Miyamoto, H. A. Flaxman, H. Y. Wu, J. Gao, C. M. Woo, ‘Discovery of a CelecoxibBinding Site on Prostaglandin E Synthase (PTGES) with a Cleavable Chelation-Assisted Biotin Probe’, ACS Chem Biol 2019 , 14, 2527-2532.

[0019] B. Yang, S. Tang, C. Ma, S. T. Li, G. C. Shao, B. Dang, W. F. DeGrado, M. Q. Dong,P. G. Wang, S. Ding, L. Wang, ‘Spontaneous and specific chemical cross-linking in live cells to capture and identify protein interactions’, Nat Commun 2017, 8, 2240.

[0020] B. Liu, S. Thayumanavan, ‘Substituent Effects on the pH Sensitivity of Acetals and Ketals and Their Correlation with Encapsulation Stability in Polymeric Nanogels’, J Am Chem Soc 2017, 139, 2306-2317.

[0021] E. C. Hansen, C. Li, S. Yang, D. Pedro, D. J. Weix, ‘Coupling of Challenging HeteroarylHalides with Alkyl Halides via Nickel -Catalyzed Cross- Electrophile Coupling’, J OrgChem 2017, 82, 7085-7092.

[0022] E. C. Hansen, D. J. Pedro, A. C. Wotal, N. J. Gower, J. D. Nelson, S. Caron, D. J.Weix, ‘New ligands for nickel catalysis from diverse pharmaceutical heterocycle libraries’, Nat Chem 2016, 8, 1126-1130.

[0023] M. A. Berliner, K. Belecki, ‘ Simple, rapid procedure for the synthesis of chloromethyl methyl ether and other chloro alkyl ethers’, J. Org. Chem.2005, 70, 9618-9621.

[0024] C. F. Ruan, J. H. Zhou, Z. X. Li, K. J. Li, Z. Fang, X. L. Zhang, M. L. Ye, ‘Proteome-WideDeconvolution of Drug Targets and Binding Sites by Lysine Reactivity Profiling’, Analytical Chemistry 2022, 94, 3352-3359.

[0025] Y. C. Gong, Y. Kakihara, N. Krogan, J. Greenblatt, A. Emili, Z. Zhang, W. A. Houry, ‘An atlas of chaperone-protein interactions in Saccharomyces cerevisiae: implications to protein folding pathways in the cell’, Molecular Systems Biology 2009, 5, 14.

[0026] A. Horovitz, T. H. Reingewertz, J. Cuellar, J. M. Valpuesta, ‘Chaperonin Mechanisms:Multiple and (Mis)Understood?’, Anna Rev Biophys 2W22, 51, 115-133.

[0027] A. L. Horwich, ‘Molecular chaperones in cellular protein folding: the birth of a field’, Cell 2014, 157, 285-288.

[0028] B. Bukau, J. Weissman, A. Horwich, ‘Molecular chaperones and protein quality control’, Ce / Z 2006, 125, 443-451.

[0029] D. Balchin, M. Hayer-Hartl, F. U. Hartl, ‘In vivo aspects of protein folding and quality control’, Science 2016, 353, aac4354.

[0030] C. L. Klaips, M. H. M. Gropp, M. S. Hipp, F. U. Hartl, ‘Sisi potentiates the stress response to protein aggregation and elevated temperature’, Nat Commun 2020, 11, 6271.

[0031] Nitika, C. M. Porter, A. W. Truman, M. C. Truttmann, ‘Post-translational modifications of Hsp70 family proteins: Expanding the chaperone code’, J Biol Chem 2020, 295, 10689-10708.

[0032] C. Sahin, A. Leppert, M. Landreh, ‘Advances in mass spectrometry to unravel the structure and function of protein condensates’, Nat Protoc 2023, 75, 3653-3661.

[0033] K. V. Huber, K. M. Olek, A. C. Muller, C. S. Tan, K. L. Bennett, J. Colinge, G. Superti-Furga, ‘Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling’, Nat Methods 2015, 72, 1055-1057.

[0034] A. Mateus, N. Kurzawa, J. Perrin, G. Bergamini, M. M. Savitski, ‘Drug TargetIdentification in Tissues by Thermal Proteome Profiling’, AnnuRev Pharmacol Toxicol 2022, 62, 465-482.

[0035] V. Cappelletti, T. Hauser, I. Piazza, M. Pepelnjak, L. Malinovska, T. Fuhrer, Y. Li, C. Dorig,P. Boersema, L. Gillet, J. Grossbach, A. Dugourd, J. Saez- Rodriguez, A. Beyer, N. Zamboni, A. Caflisch, N. de Souza, P. Picotti, ‘Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ’, Ce / / 2021, 184, 545-559 e522.

[0036] I. Piazza, K. Kochanowski, V. Cappelletti, T. Fuhrer, E. Noor, U. Sauer, P. Picotti, ‘A Map of Protein-Metabolite Interactions Reveals Principles of Chemical Communication’, Ce / / 2018, 172, 358-372 e323.

[0037] Y. Feng, G. De Franceschi, A. Kahraman, M. Soste, A. Melnik, P. J. Boersema, P. P. de Laureto, Y. Nikolaev, A. P. Oliveira, P. Picotti, ‘Global analysis of protein structural changes in complex proteomes’, Nat Biotechnol 2014, 32, 1036-1044.

[0038] M. Pepelnjak, N. de Souza, P. Picotti, ‘Detecting Protein-Small Molecule Interactions UsingLimited Proteolysis-Mass Spectrometry (LiP-MS)’,Trends Biochem Sci 2020, 45, 919-920.

[0039] I. Piazza, N. Beaton, R. Bruderer, T. Knobloch, C. Barbisan, L. Chandat, A. Sudau, I. Siepe,O. Rinner, N. de Souza, P. Picotti, L. Reiter, ‘A machine learning-based chemoproteomic approach to identify drug targets and binding sites in complex proteomes’, Nature Communications 2020, 11, #4200.

[0040] A. McKenzie-Coe, N. S. Montes, L. M. Jones, ‘Hydroxyl Radical Protein Footprinting:A Mass Spectrometry-Based Structural Method for Studying the Higher Order Structure of Proteins’, Chem Rev 2022, 122, 7532-7561.

[0041] J. Doellinger, A. Schneider, M. Hoeller, P. Lasch, ‘Sample Preparation by EasyExtraction and Digestion (SPEED) - A Universal, Rapid, and Detergent-free Protocol for Proteomics Based on Acid Extraction’, Mol. Cell. Proteomics 2020, 19, 209-222.

[0042] S. H. Li, L. Wang, F. Yu, Z. L. Zhu, D. Shobaki, H. Q. Chen, M. Wang, J. Wang, G. T.Qin, U. J. Erasquin, L. Ren, Y. J. Wang, C. Z. Cai, ‘Copper- catalyzed click reaction on / in live cells’, Chemical Science 2017, 5, 2107-2114.

[0043] G. Qin, C. Chao, L. J. Lattery, H. Lin, W. Fu, K. Richdale, C. Cai, ‘Tear proteomic analysis of young glasses, orthokeratology, and soft contact lens wearers’, Journal of Proteomics 2023, 270, 104738.

[0044] G. T. Qin, P. Z. Zhang, M. X. Sun, W. J. Fu, C. Z. Cai, ‘Comprehensive spectral libraries for various rabbit eye tissue proteomes’, Scientific Data 2022, 9, 6.

[0045] V. Demichev, C. B. Messner, S. I. Vernardis, K. S. Lilley, M. Raiser, ‘DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput’, Nat. Methods 2020, 77, 41-44.

[0046] F. C. Yu, S. E. Haynes, G. C. Teo, D. M. Avtonomov, D. A. Polasky, A. I. Nesvizhskii,‘Fast Quantitative Analysis of timsTOF PASEF Data with MSFragger and lonQuant’, Mol. Cell. Proteomics 2020, 19, 12.

[0047] M. E. Ritchie, B. Phipson, D. Wu, Y. Hu, C. W. Law, W. Shi, G. K. Smyth, Timma powers differential expression analyses for RNA-sequencing and microarray studies’, Nucleic Acids Research 2015, 43, e47-e47.

[0048] V. Schwammle, I. R. Leon, O. N. Jensen, ‘Assessment and Improvement of Statistical Tools for Comparative Proteomics Analysis of Sparse Data Sets with Few Experimental Replicates’, Journal of Proteome Research 2013, 72, 3874-3883.

[0049] Y. Gong, Y. Kakihara, N. Krogan, J. Greenblatt, A. Emili, Z. Zhang, W. A. Houry, ‘An atlas of chaperone-protein interactions in Saccharomyces cerevisiae: implications to protein folding pathways in the cell’, Mol Syst Biol 2009, 5, 275.

[0050] Feng Z, Chen X, Wu X, Zhang M. Formation of biological condensates via phase separation: Characteristics, analytical methods, and physiological implications. J Biol Chem. 2019 Oct 4;294(40): 14823-14835.

[0051] Parakh S, Atkin JD. Protein folding alterations in amyotrophic lateral sclerosis. Brain Res. 2016 Oct 1 1648(P1 B):633-649.

[0052] 1601675-Avidin-Biotin-Handbook.pdf https: / / tools.thermofisher.com / content / sfs / brochures / 1601675-Avidin-Biotin-Handbook.pdf.

[0053] "From One to 9,000 Samples: Using High-Denisity Streptavidin-Coated Membranes for Kinase Detection” Promega Cell Notes Issue 5 2003, pp. 1-11; https: / / www.promega.eom / ~ / media / files / resources / cell%20notes / cn005 / from%20one%20to %209000%20samples%20using%20high-density%20streptavidin- coated%20membranes%20for%20kinase%20detection.pdf?la=en Promega Cell Notes Issue 5 2003, pp. 1-11.

[0054] C. Fang, M. Fantin, X. Pan, K. de Fiebre, M. L. Coote, K. Matyjaszewski, P. Liu, ‘Mechanistically Guided Predictive Models for Ligand and Initiator Effects in Copper- Catalyzed Atom Transfer Radical Polymerization (Cu-ATRP)’, J Am Chem Soc 2019, 141, 7486-7497.

[0055] L. M. Schiapparelli, D. B. McClatchy, H. H. Liu, P. Sharma, J. R. Yates, H. T. Cline, ‘Direct Detection of Biotinylated Proteins by Mass Spectrometry’, Journal ofProteome Research 2014, 13, 3966-3978.

[0056] L. Nebhani, C. Barner-Kowollik, ‘Orthogonal Transformations on Solid Substrates: Efficient Avenues to Surface Modification’, Advanced Materials 2009, 21, 3442-3468.

[0057] A. Mahdavi, J. Szychowski, J. T. Ngo, M. J. Sweredoski, R. L. J. Graham, S. Hess, O. Schneewind, S. K. Mazmanian, D. A. Tirrell, ‘Identification of secreted bacterial proteins by noncanonical amino acid tagging’, Proceedings of the National Academy of Sciences of the United States of America 2014, 111, 433-438.

[0058] J. Szychowski, A. Mahdavi, J. J. L. Hodas, J. D. Bagert, J. T. Ngo, P. Landgraf, D. C. Dieterich, E. M. Schuman, D. A. Tirrell, ‘Cleavable Biotin Probes for Labeling of Biomolecules via Azide-Alkyne Cycloaddition’, J Am Chem Soc 2010, 132, 18351-18360.

Claims

CLAIMSWhat is claimed is:

1. A compound having the structural formula X:wherein:R1 is hydrogen, hydroxyl, C1-C3 alkoxy, (CH2)n-C00H, NHCH=O, NR3R4, or 0-(CH2)o- CH3;R2 is hydrogen, hydroxyl, C1-C2 alkyl, C1-C3 alkoxy or (CH2)n-C00H;R3 and R4 can be the same or different and are selected from hydrogen or C1-C3 alkyl; each R5 can be the same or different; and can be hydrogen, unsubstituted C1-C2 alkyl or Cl-C2 alkyl substituted by a 5- or 6-atom aryl or heteroaryl group; m = 0 to 3, and any of R1 can be the same or different; n = 0 to 3; o = 0 to 3; p = 1 or 2; q = 0 to 50.

2. The compound of claim 1 that has the structural formula X’:

3. The compound of claim 1 that has the structural formula Y:

4. A compound that has the structural formula Z:wherein:R1 is hydrogen, hydroxyl, C1-C3 alkoxy, (CH2)n-C00H, NHCH=O, NR3R4, or O-(CH2)o- CH3;R2 is hydrogen, hydroxyl, C1-C2 alkyl, C1-C3 alkoxy or (CH2)n-C00H;R3 and R4 can be the same or different and are selected from hydrogen or C1-C3 alkyl; each R5 can be the same or different; and can be hydrogen, unsubstituted C1-C2 alkyl or Cl- C2 alkyl substituted by a 5- or 6-atom aryl or heteroaryl group;m = 0 to 3, and any of R1 can be the same or different; n = 0 to 3; o = 0 to 3; p = 1 or 2; andR6 is an amine group that can react with an epoxide or succinimide group, such as NHR7, (CH2)SNHR7 or (CH2)SN(R7)2, wherein R7 can be hydrogen or C1-C2 alkyl and s = 0 to 2.

5. The compound of claim 4 that has the formula Z’ :R6 is an amine group that can react with an epoxide or succinimide group, such as NHR7, (CH2)SNHR7 or (CH2)SN(R7)2, wherein R7 can be hydrogen or C1-C2 alkyl and s = 0 to 2.

6. A method for making the molecule of claim 1, comprising: i) coupling of the pyridyl bromide 1 with the alkyl bromide acetate 2, employing nickel- catalyzed cross-electrophile coupling promoted by the ligand 3 to provide the pyridyl acetate 4; ii) converting the alcohol 4 to the azide 5a in 52% yield using (PhO)2PON3 in the presence of l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), and then hydrolyzing to the alcohol 5b; iii) separately preparing the symmetric acetal 7 from the protected 3-hydroxypropanamine6 by transacetalation; iv) alkoxyalkylating the picolyl alcohol 5b with in-situ generated a-chloroalkyl ether from7 in the presence of a Zn(II) catalyst to give the asymmetric acetal 8; v) deprotecting the phthalimide with hydrazine to obtain APA-NH2; vi) mixing APA-NH2 with the biotin-(PEG)12-A-hydroxysuccinimide ester 9 in 1 : 1 ratio in a solvent to obtain the compound of formula X.

7. A method for purifying alkyne-tagged proteins or peptides comprising: i) contacting proteins with an alkyne-tagging reagent under conditions sufficient to obtain alkyne-tagged proteins; ii) optionally digesting the alkyne-tagged proteins with a protease to obtain alkyne-tagged peptides; iii) reacting the alkyne-tagged proteins or peptides by a click reaction with a compound of of any one of claims 1 to 3 to obtain biotinylated peptides; iv) contacting the biotinylated proteins or peptides with a biotin affinity reagent to form a complex; v) separating the complex from proteins or peptides that are not alkyne-tagged; vi) releasing the proteins or peptides from the complex to obtain purified alkyne-tagged peptides.

8. A method for purifying alkyne-tagged proteins or peptides comprising: i) contacting proteins with an alkyne-tagging reagent under conditions sufficient to obtain alkyne-tagged proteins; ii) optionally digesting the alkyne-tagged proteins with a protease to obtain alkyne-tagged peptides; iii) reacting the alkyne-tagged proteins or peptides by a click reaction with a compound of claim 4 or 5 obtain amine-labelled proteins or peptides; iv) contacting and reacting the amine-labelled proteins or peptides with a functionalized substrateor, wherein r = 1 to 20, to obtain immobilized proteins or peptides covalently attached to the substrate; v) separating the immobilized proteins or peptides from proteins or peptides that are not alkyne-tagged; vi) releasing the immobilized proteins or peptides from the substrate to obtain purified alkyne-tagged peptides.

9. A method for diagnosing a disease associated with misfolding, conformational change or aggregation of a protein, comprising: i) contacting cells of a subject or a lysate of cells of a subject with an alkyne-tagging reagent to obtain alkyne-tagged proteins; ii) digesting the alkyne-tagged proteins with a protease to obtain alkyne-tagged peptides; iii) reacting the alkyne-tagged peptides by a click reaction with a compound of any one of claims 1 to 3 to obtain biotinylated or amine-labelled peptides; iv) separating the biotinylated or amine-labelled peptides from peptides that are not alkyne-tagged; v) releasing the peptides from the biotin or amine label to obtain purified alkyne-tagged peptides; vi) analyzing the alkyne-tagged peptides by one or more of high-performance liquid chromatography, capillary electrophoresis, gel electrophoresis or 2-D gel electrophoresis and mass spectrometry; vii) determining the amino acid sequence of the alkyne-tagged peptides; viii) mapping the peptides onto 2- or 3 -dimensional structures of the protein, the of the misfolding, conformational change, or aggregation of the protein being associated with the disease, to characterize the conformation of the protein by a profile of alkyne- tagged peptides; andix) comparing the profile of alkyne-tagged peptides in the cells of the subject to a reference profile of alkyne-tagged peptides in the normal conformation of the protein; wherein a different profile in cells of the subject indicates the presence or risk of the disease.

10. A reagent composition comprising i) a copper salt or complex; ii) a linker compound of any one of claims 1 to 5; and optionally iii) a click ligand.

11. The reagent composition of claim 10 wherein the copper salt or complex is present at a concentration from 0.1 to 1 mM and the linker compound of formula X or the formula Z is present at a concentration from 0.1 to 5 mM, and, if present, the click ligand is present at a concentration from 0.1 to 1 mM.

12. The reagent composition of claim 10 or 11, wherein the click ligand is present and isorthe copper salt is copper sulfate, and the linker compound has the formula Y:, or the linker compound has the formula Z’ :wherein each R5 can be the same or different and is hydrogen, methyl or 2-pyridylmethyl; and R6 is NH2.

13. A kit comprising at least one container of the reagent composition of any one of claims 10 to 12, and a separate container of a reducing agent.

14. Use of a compound of any one of claims Error! Reference source not found, to 3 for conjugating an alkyne-tagged protein or peptide to biotin.

15. Use of a compound of claim 4 or 5 for covalently attaching an alkyne-tagged protein or peptide to an immobilizing substrate.

16. The use of claim 15, wherein the attachment is viawherein r = 1 to 20.

Images