Cleavable crosslinking reagents

Abstract

The present invention relates to the provision of trifunctional crosslinker compounds comprising (i) a moiety reactive with an amino acid side chain for selective labelling of proteins, (ii) a cleavable linker moiety and (iii) an affinity moiety for the detection, isolation and purification of captured proteins. The invention further relates to uses of the cleavable crosslinking reagents in methods for detecting, labelling, identifying and characterizing proteins and protein interactions in or on the surface of intact cells, cell lysates and / or protein mixtures.

Description

[0001] Applicant: ETH Zurich

[0002] Title: CLEAVABLE CROSSLINKING REAGENTS

[0003] Our ref.: DIS29349PCT

[0004] Date: December 1 , 2025

[0005] CLEAVABLE CROSSLINKING REAGENTS

[0006] Field of the Invention

[0007] The present invention relates to trifunctional crosslinker compounds comprising (i) a moiety reactive with an amino acid side chain for selective labelling of proteins, (ii) a cleavable linker moiety and (iii) an affinity moiety for the detection, isolation and purification of captured proteins, as well as their use in methods for detecting, labelling, identifying and characterizing proteins and protein interactions in or on the surface of intact cells, cell lysates and / or protein mixtures.

[0008] Background of the Invention

[0009] Spatial proteotyping, the specific identification of proteins that are present in a location of interest within a cell or on the surface of a cell, requires specific tagging of the pool of proteins present in this location at a particular point in time. Muller et al. disclose the recently developed LUX-MS technology which enables the opto-proteomic identification of such pools of proteins residing at the cell surface, using mass spectrometric strategies (Muller et al., Nature Communications, (2021)12:7036). The resulting data enables the identification of cell surface proteins for diagnostic and therapeutic purposes. In this method, cell surface residing proteins are being oxidized upon light induction by singlet oxygen generators (SOGs). SOGs modify several amino acids, including histidine, cysteine, tryptophan and methionine; however histidine is typically the most prominently oxidized amino acid (Muller et al., Nature Communications, (2021)12:7036, Supplementary Information). Oxidized proteins, now containing reactive carbonyl, hydroperoxide and / or endoperoxide functional groups (2-oxo-histidine, 2-hydroxy-tryptophan, etc.) are subsequently tagged with biocytin hydrazide. Tagged proteins can then be affinity-enriched using the biotin handle via streptavidin beads. Upon extensive washing, proteins bound to beads are being digested with trypsin, peptides upstream and downstream of the tagged site are released and used for identification of the bound protein(s). However, the specific peptide that has been tagged (or labelled) remains covalently bound to the bead and thus cannot be easily released. A release of the biotin-tagged peptide is only possible under harsh conditions, resulting in an undesirably high amount of impurities, which renders the peptide containing the biotin tag very hard to identify in a mass spectrometric analysis. Therefore, the tagged peptide itself cannot be identified and can only be determined indirectly, by mass-spectrometric detection of upstream or downstream peptides. This is undesired, since even after washing of the affinity matrix to eliminate unspecifically bound proteins hundreds of peptides / proteins can still be identified, hampering successful data analysis and determination of bona fide cell surface proteins.

[0010] Summary of the Invention

[0011] The present invention provides compounds that overcome the drawbacks of available biocompatible crosslinking reagents in that they enable the highly selective and efficient labelling of proteins of interest, enrichment of said proteins and subsequent identification and quantification by mass spectrometry.

[0012] To this end, the present invention relates to a cleavable crosslinking reagent comprising three moieties, wherein each moiety comprises a different functionality. A first moiety which is reactive with an amino acid side chain for the selective labelling of proteins. A second moiety comprises a cleavable linker. A third moiety comprises an affinity group for purification, preferably affinity purification of the proteins or peptides captured by the first functionality. These reagents are particularly advantageous due to the unique combination of these three different functionalities in one molecule find use in various biomedical applications such as the labeling, detection, identification and characterization of proteins, in particular surface proteins, in or on intact cells, cell lysates or protein mixtures.

[0013] In a first aspect, the invention provides a compound having a structure of Formula I:

[0014] X-S1-Z-S2-A wherein

[0015] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine;

[0016] Si and S2 are independently of each other a spacer group;

[0017] Z is a cleavable linker moiety; and

[0018] A is an affinity moiety.

[0019] In a further aspect, the invention provides the use of the compound of the invention for the isolation of one or more protein(s). In another aspect, the invention provides the use of the compound of the invention for the selective enrichment of one or more protein(s). In another aspect, the invention provides the use of the compound of the invention for the isolation and selective enrichment of one or more protein(s). In yet another aspect, the invention provides the use of the compound of the invention for the isolation, selective enrichment and identification of one or more protein(s). In yet another aspect, the invention provides the use of the compound of the invention for the isolation, selective enrichment, identification and quantification of one or more protein(s). In a further aspect, the invention provides a method for the isolation and identification of one or more protein(s), comprising the steps of:

[0020] (i) subjecting a sample comprising the one or more protein(s) to a chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional groups and / or one or more endoperoxide functional group(s) in an amino acid side chain comprised in the one or more protein(s), to obtain one or more oxidized protein(s);

[0021] (ii) reacting the one or more oxidized protein(s) with a compound of Formula I:

[0022] X-S1-Z-S2-A wherein

[0023] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine;

[0024] Si and S2 are independently of each other a spacer group;

[0025] Z is a cleavable linker moiety; and

[0026] A is an affinity moiety, wherein X reacts with the one or more carbonyl functional group(s), one or more hydroperoxide functional groups and / or one or more endoperoxide functional group(s) to form one or more labelled protein(s);

[0027] (iii) isolating and identifying the one or more labelled protein(s).

[0028] Brief description of the Figures

[0029] Figure 1 : (A) Schematic illustration for enrichment and oxidative release of a spike-in protein. (B) SDS-PAGE analysis of spike-in protein enrichment and release. Samples were separated on a 12% NuPAGE gel and stained with Silver Stain. Prominent bands around 77 kDa correspond to the spike-in protein transferrin. The gel demonstrates efficient binding and highly specific oxidative release from the affinity matrix.

[0030] Figure 2: Representative graphs of MS1 and MS2 scans. Highflyer peptides demonstrate favourable properties for mass spectrometry, including strong signal intensity in MS1 scans. Fragmentation of MS-cleavable bonds produces distinct reporter ions (indicated with a star) and characteristic remainder peptide patterns in MS2 scans, enhancing peptide identification and quantification. Figure 3: (A) Schematic illustration showing the process of labeling, enrichment, and selective release of "highflyer" peptides from a spike-in protein. (B) Detected highflyer peptides are mapped onto both the sequence and 3D structure of the spike-in protein (visualized in PyMOL, using PDB: 3QYT), showing that tagged amino acids localize to surface-accessible regions. (C) Graph demonstrating that the labile search pipeline identifies reporter ions from highflyer peptides specifically in oxidatively-released eluates (left), distinguishing them from tryptic peptides up- and downstream of labeling sites (right). This approach enhances peptide-spectrum matches (PSMs) compared to open search methods.

[0031] Figure 4: (A) Schematic illustration of the labeling and enrichment of cell surface proteins using a cleavable crosslinking reagent. (B) Volcano plot displaying relative abundance changes of quantified proteins from enriched tryptic digests of anti-IgM- vs. isotype-control-treated B lymphoma cells. Black triangles represent proteins localized to the cell surface. Gray crosses represent proteins localized to intracellular compartments. The primary binding targets, IgM and its heavy chain, are represented by black squares and labeled as IGHM and HV434, respectively. Proximal cell surface proteins that were identified to carry the specific mass motif on oxidatively released peptides are shown as gray circles. (C) Bar plot showing surface-annotated peptides uniquely identified with the defined mass motif in the IgM-targeted SOG-modified sample, compared to the isotype control-treated sample. Annotations of the proteins are consistent with those used in panel Fig. 4B (circles = “Mass Motif’, squares = “Target”).

[0032] Figure 5: Histogram depicting the selective oxidative release of a HIT-TCO-AF488 construct from surface proteins localized within CD45 nanoscale domains on Ramos B lymphoma cells. Cells targeted with an isotype control are represented in the histograms (1) and (4), while those targeted with CD45-SOG are shown in the histograms (2) and (3).

[0033] Figure 6: Direct comparison of proximity labeling with IgM as target protein on Ramos B-cells between non-cleavable biocytin hydrazide (A) and cleavable biocytin hydrazide (B). Both linkers were measured in quadruplicates and protein abundances were compared to an IgG isotype control. For the cleavable biocytin-hydrazide linker, proteins identified with the signature mass shift are additionally highlighted (triangles: proximal proteins with signature mass shift, squares: target protein(s) identified with signature mass shift). (C) Proportional Venn-diagram showing the overlap of significantly enriched and surfaceome annotated proteins using either cleavable or non-cleavable biocytin-hydrazide for the proximity labeling and enrichment workflow.

[0034] Figure 7: Schematic illustration showing the workflows for the peptide- and protein-centric detection of mass-shifts and diagnostic ions for different variants of the exemplary cleavable crosslinking reagents of the invention (cmTags). (A) Workflow for peptide-centric discovery. (B) Workflow for protein-centric discovery. Figure 8: Structures of cmTag variants and open search results for tryptic and oxidative release fractions of four different cmTag variants and a digest control. As moiety X, the different variants comprise (1) a hydrazide functional group (2) an aniline moiety (3) a hydrazide functional group and PEG-3 spacers, i.e. (-[O-(CH2)2]3-) linker (4) an alkoxyamine moiety. The bars show the number of PSMs identified with a specific mass shift in the different samples. Results were filtered for mass shifts that were specific to the oxidative release fractions of one cmTag variant family.

[0035] Figure 9A-9C: Schematic illustration showing the reporter ions of the cmTag variants comprising a hydrazide functional group. Peptide level evidence is shown as annotated spectra. The reporter ion (Fig. 9A, right) exhibited a mass shift of 161 .14 m / z. Further putative structures exhibited mass shifts of 174.112 m / z (Fig. 9A, left), 190.107 m / z (Fig. 9B, left) and 172.096 m / z (Fig. 9C, left). In all cases, the modifications were detected in the open search and confirmed by a hybrid search.

[0036] Figure 10A-10D: Schematic illustration showing the reporter ions of the cmTag variants comprising an alkoxyamine moiety. Peptide level evidence is shown as annotated spectra. For this cmTag a primary reporter ion (219.182 m / z) and secondary reporter ion (204.171 m / z) were identified (Fig. 10A, right). Further putative structures exhibited mass shifts of 232.154 m / z (Fig. 10A, left; open search and hybrid search), 247.162 m / z (Fig. 10B; open search), 248.148 m / z (Fig. 10C, left; hybrid search) and 230.138 m / z (Fig. 10D, left; hybrid search).

[0037] Figure 11A-11C: Schematic illustration showing the reporter ions of the cmTag variants comprising an aniline moiety. Peptide level evidence is shown as annotated spectra. The reporter ion (Fig. 11 A, right) exhibited mass shift of 237.171 m / z. Further putative structures exhibited mass shifts of 250.143 m / z (Fig. 11A, left; hybrid search), 266.138 m / z (Fig. 11 B; open search and hybrid search) and 248.127 m / z (Fig. 11 C, left; open search and hybrid search).

[0038] Detailed description of the Invention

[0039] Unless defined otherwise, the following definitions are used throughout the description.

[0040] The term “a” as used herein, refers to at least one, unless otherwise mentioned. The term “include” as used herein, refers to includes without limitation. The term “plurality” refers to a number of two or more.

[0041] The term “(interactive) binding” or “interaction” refers to any type of interactive association between a corresponding pair of molecules (e.g., reactive moiety / target protein) that exhibit mutual affinity or binding capacity. An interactive association may occur, e.g., between a corresponding pair of chemically reactive groups (donor / acceptor, acid / base, etc.) that exhibit mutual reactivity. Exemplary binding events include, without limitation, hydrophobic interactions, hydrophilic interactions, hydrogen bonds, van der Waals forces, ionic interactions, nonionic interactions, electrostatic interactions, covalent bonding, and the like. It is understood that depending of the nature of the binding event the interaction may be of different levels, i.e. transient or permanent, weak or strong binding.

[0042] The term "alkyl" as used herein refers to a straight or branched hydrocarbon containing 1-24, preferably 1 to 12 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, and t-butyl, pentyl, hexyl. The term "alkoxy" refers to an -O- alkyl group.

[0043] The term "alkylene" as used herein refers to a divalent radical derived from a hydrocarbon, for example -CHR-(CHR)n- with R being H or a substituent of choice. Typically, an alkylene group will have from 1 to 24 carbon atoms (i.e., n=24), preferably 10 to 24 carbon atoms. The term "heteroalkylene" as used herein refers to an alkylene having one or more heteroatoms, such as O, N or S, preferably O or N, inserted into the alkylradicals.

[0044] The term "aryl" as used herein refers to a 6-carbon monocyclic, 10-carbon bicyclic, 14-carbon tricyclic aromatic ring system wherein each ring may be unsubstituted or have 1 to 4 substituents. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. Phenylene, as used in the context of the present invention, preferably denotes a 1 ,2-, 1 ,3- or 1 ,4- phenylene group, which is optionally substituted.

[0045] The term "cycloalkyl" refers to a saturated and partially unsaturated cyclic hydrocarbon group having 3 to 12 carbons. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0046] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having one or more heteroatoms (such as O, N, or S). Examples of heteroaryl groups include pyridyl, furyl, imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl, indolyl, and thiazolyl. Pyridyl includes 2-pyridyl, 3-pyridyl and 4-pyridyl, preferably 2- pyridyl. The term "heteroaralkyl" refers to an alkyl group substituted with a hetero aryl group.

[0047] The term "heterocycloalkyl" refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having one or more heteroatoms (such as O, N or S). Examples of heterocycloalkyl groups include, but are not limited to, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, and tetrahydro furanyl, glucosyl.

[0048] Cycloalkyl, heterocycloalkyl, aryl, heteroaryl may be unsubstituted or have 1 to 4 substituents.

[0049] Examples of substituents include, but are not limited to, at least one halo, hydroxyl, amino, cyano, nitro, mercapto, carboxy, or a hydrocarbyl group selected from an alkyl, alkenyl, alkylamino, dialkylamino, or alkoxy group having one to six carbon atoms.

[0050] Exemplary hydrocarbyl-substituted cycloalkyl groups include 2-methylcyclopropyl, 2- ethylcyclopropyl, 2-methylcyclobutyl, 3-methylcyclobutyl, 2-methylcyclopentyl, 2,3- dimethylcyclopentyl, 3-iso-propylcyclopentyl, 2,6-dimethylcyclohexyl, 4-(t-butyl)cyclohexyl, 2- vinylcyclohexyl, 3-allylcyclopentyl, 3,4-diallylcyclopentyl, l-(4-pyridinyl)piperidinyl, l-(4- pyridinylmethyl)piperidinyl, 4-(4-pyridinyl)piperidinyl, 4-(4-pyridinyl)piperazin-1-yl, and bicyclohexyl groups.

[0051] Exemplary hydrocarbyl-substituted cycloalkenyl groups include 3-methyl-3-cyclopenten-1-yl, 3.4- dimethyl-3-cyclopenten-1 -yl, 2-iso-propyl-2-cyclopenten-1 -yl, 2,3-diethyl-2-cyclopenten-1 -yl, 4-vinyl- 1-cyclohexen-1-yl, 3,4-diethyl-3-cyclopenten-1-yl, and 3,4-diallyl-3-cyclopenten-1-yl groups.

[0052] Exemplary hydrocarbyl-substituted aryl groups include tolyl, mesityl, xylyl, cumenyl, cymenyl, 3,5- di(t-butyl)phenyl, 2-methylnaphthyl, 2-vinylphenyl, 2-vinylbenzyl, 2-vinylnaphthyl, 4- cyclohexylphenyl, biphenyl, 4-(4-piperidinyl)pyridinyl, and p-terphenyl groups.

[0053] Exemplary hydrocarbyl-substituted heteroaryl groups include 2-methylpyridin-1 -yl, 2-ethylpyridin-1 - yl, 3-vinylimidazol-1-yl, 2-methylimidazol-1-yl, 2-methylquinoxalin-1-yl, 1 -allylbenzotriazolyl, 2,2'- bipyridyl, 4,4'-bipyridyl, 4-methylpyrazinyl, 4-(pyridinylmethyl)-pyridinyl, 4-benzylpyrazinyl, nicotinamidyl, 2-methylfuranyl, 5-methylfurfurylamino, 2-methylthiopheneyl, 4-methyloxazolyl, 2,5 - diphenyl-4-methyloxazolyl, and 4-methylthiazolyl groups.

[0054] The term "halogen" denotes a chloro, fluoro, bromo or iodo substituent, preferably a chloro or fluoro substituent.

[0055] The term "optionally substituted" as used herein typically refers to substitution by Hal, -OR, -CN, - NO2, -COOR, C(1-8)alkyl, C(1-8)alkylene, C(1-8)alkoxy, wherein R is from 1 to 8 carbon atoms.

[0056] In preferred embodiments, the compound of the invention is water soluble and biocompatible.

[0057] The term “water soluble” typically refers to a solubility of a material in water of greater than 1 wt% based on the total weight of the material and water at 24° C. It is understood that water solubility is imparted by the hydrophilic nature of the compound of the invention, more specifically by the hydrophilic nature of the moieties A, X, Z, Si and S2.

[0058] The term "biocompatible" refers to chemical inertness with respect to most components of cells (e.g., human cells), tissues or body fluids and minimal toxic effects of the compounds according to the invention towards cells or tissues. As used herein, the terms functional group and moiety are used interchangeably.

[0059] The phrases “capable of reacting with”, “capable of specifically reacting with”, “(specifically) reacting with”, “(specifically) reacts with” or “(specifically) reactive towards” are used interchangeably herein and indicate that a first compound or functional group will undergo a specific chemical reaction with a second compound or functional group when brought in contact under suitable conditions. For example, a moiety X as disclosed herein is capable of reacting with carbonyl, hydroperoxide and / or endoperoxide functional groups, i.e., it will undergo a specific chemical reaction with carbonyl, hydroperoxide and / or endoperoxide functional groups when brought in contact under suitable conditions.

[0060] Functional groups or moieties can be chemically coupled to each other by using activators, or activating reagents. Examples of activating reagents used for activating a functional group include but are not limited to 1 -hydroxybenzotriazole (HOBt), 3-hydroxy-3,4-dihydro-1 ,2,3-benzotriazine-4- one (HOOBt), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), 2-(1 H-7- azabenztriazol-1-yl)-1 ,1 ,3,3-tetramethyluronium hexafluoro phosphate (HATU), 2-(1 H-benzotriazol- 1-yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate (HBTU), 3,4-dihydro-1 ,2,3-benzotriazin-4- one-3-oxy tetramethyluronium hexafluorophosphate (HDTU), benzotriazol-1- yloxytris(dimethylamino)phosphonium hexafluoro phosphate (BOP), benzotriazol-1-yloxytris- (pyrrolidino)-phosphonium hexafluoro phosphate (PyBop), (3,4-dihydro-1 ,2,3-benzotriazin-4-one-3- oxy)diethyl phosphate (DEPBt), 3,4-dihydro-1 ,2,3-benzotriazin-4-one-3-oxy tris-(pyrrolidino)- phosphonium hexafluorophosphate (PDOP), 2-(benzotriazol-1-yloxy)-1 ,3-dimethyl-2-pyrrolidin-1 -yl- 1 ,3,2-diazaphosph-olidinium hexafluorophosphonate (BOMP), 5-(1 H-7-azabenzotriazol-1-yloxy)- 3, 4-dihydro-1 -methyl 2H-pyrrolium hexachloroantimonate (AOMP), (1 H-7-azabenzotriazol-1- yloxy)tris(dimethylamino) phosphonium hexafluoroposphate (AOP), 5-(1 H-Benzotriazol-1-yl)-3,4- dihydro-1 -methyl 2H-pyrrolium hexachloroantimonate N-oxide (BDMP), 2-bromo-3-ethyl-4-methyl thiazolium tetrafluoroborate (BEMT), 2-bromo-1 -ethyl pyridinium tetrafluoroborate (BEP), 2-bromo-1 -ethyl pyridinium hexachloroantimonate (BEPH), N-(1 H-benzotriazol-1-ylmethylene)-N- methylmethanaminium hexachloroantimonate N-oxide (BOMI), N,N'-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-CI), 1-(1 H-benzotriazol-1-yloxy)phenylmethylene pyrrolidinium hexachloro antimonate (BPMP), 1 ,1 ,3,3-bis(tetramethylene) fluorouronium hexafluorophosphate (BTFFH), chloro(4-morphoino)methylene morpholinium hexafluorophosphate (CMMM), 2-chloro-1 ,3-dimethyl- 1 H-benzimidazolium hexafluorophosphate (CMBI), 2-fluoro-1 -ethyl pyridinium tetrafluoroborate (FEP), 2-fluoro-1 -ethyl pyridinium hexachloroantimonate (FEPH), 1 -(1 -pyrrolidinyl-1 H-1 ,2,3- triazolo[4,5-b]pyridin-1-ylmethylene)pyrrolidinium hexafluorophosphate N-oxide (HAPyU), O-(1 H- benzotriazol-1-yl)-N,N,N',N;-bis(pentamethylene)uronium hexafluorophosphate (HBPipU), O-(1 H- benzotriazol-1-yl)-N,N,N0,N0-bis(tetramethylene)urinium hexafluorophosphate (HBPyU), (1 H-7- azabenzotriazol-1 -yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOp), chlorotripyrrolidinophosphonium hexafluorophosphate (PyClOP), 1 ,1 ,3,3-bis(tetramethylene) chlorouronium hexafluorophosphate (PyCIU), tetramethylfluoromamidinium hexafluorophosphate (TFFH), triphosgene, triazine-based reagents [cyanuric chloride, cyanuric fluoride, 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4- methylmorpholinium chloride (DMT-MM), 2-chloro-4,6-dimethoxy-1 ,3,5-triazine (CDMT)], bis(2- chlorophenyl) phosphorochloridate, diphenyl phosphorochloridate, diphenyl phosphoroazide (DPPA) and any combination thereof.

[0061] The invention provides a compound having a structure of Formula I:

[0062] X-S1-Z-S2-A wherein

[0063] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine;

[0064] Si and S2 are independently of each other a spacer group;

[0065] Z is a cleavable linker moiety; and

[0066] A is an affinity moiety.

[0067] In specific embodiments, the compound of the invention has a structure of Formula I:

[0068] X-S1-Z-S2-A wherein

[0069] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA);

[0070] Si and S2 are independently of each other a spacer group;

[0071] Z is a cleavable linker moiety comprising a functional group having a formula selected from the group consisting of: wherein Ri is selected from H or CH3; and

[0072] A is an affinity moiety comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s).

[0073] The compound according to the invention is a cleavable crosslinking reagent.

[0074] Moiety reactive with an amino acid side chain

[0075] As used herein, the moiety X is a moiety (or functional group) that is capable of reacting with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine. In a more preferred embodiment, the amino acid is histidine, tryptophan or tyrosine. In an even more preferred embodiment, the amino acid is histidine or tryptophan. In a further highly preferred embodiment, the amino acid is histidine. The moiety X is capable of reacting with carbonyl functional group(s), hydroperoxide functional group(s) and / or with endoperoxide functional group(s). A carbonyl functional group is a functional group with the formula C=O, composed of a carbon atom double-bonded to an oxygen atom. A moiety is capable of reacting with a carbonyl functional group when said moiety is able to undergo a specific chemical reaction with a carbonyl functional group when said moiety comes into contact with the carbonyl functional group. An endoperoxide functional group is a functional group with the formula -0-0- in which two oxygen atoms are connected by a single bond to each other and are also bonded to neighboring carbon atoms within a cyclic structure. A moiety is capable of reacting with an endoperoxide functional group when said moiety is able to undergo a specific chemical reaction with an endoperoxide functional group when said moiety comes into contact with the endoperoxide functional group. A hydroperoxide functional group is a functional group with the formula -O-OH. A moiety is capable of reacting with a hydroperoxide functional group when said moiety is able to undergo a specific chemical reaction with a hydroperoxide functional group when said moiety comes into contact with the hydroperoxide functional group. The moiety X is capable of reacting with a carbonyl functional group, a hydroperoxide functional group and / or an endoperoxide functional group under suitable experimental conditions. Various combinations of a moiety X and carbonyl, hydroperoxide and / or endoperoxide functional group(s) are technically feasible, and a skilled person is able to select appropriate combinations. In some embodiments, the amino acid side chain has been subjected to a chemical reaction to obtain an oxidized amino acid side chain, relative to the native amino acid side chain. In some embodiments, the amino acid side chain comprises a carbonyl functional group, a hydroperoxide functional group and / or an endoperoxide functional group. In some embodiments, X is capable of reacting with a carbonyl functional group comprised in an amino acid side chain of 2-oxo-histidine or 2-hydroxy-tryptophan. In some embodiments, X is capable of reacting with a carbonyl functional group comprised in an amino acid side chain of 2- oxo-histidine. In some embodiments, X is capable of reacting with a carbonyl functional group comprised in an amino acid side chain of 2-hydroxy-tryptophan. In some embodiments, the amino acid is (a) histidine and the amino acid side chain is selected from the group consisting of

[0076] (b) tryptophan and the amino acid side chain is selected from the group consisting of (c) tyrosine and the amino acid side chain is selected from the group consisting of

[0077] (d) methionine and the amino acid side chain is

[0078] ; and / or

[0079] (f) cysteine and the amino acid side chain is selected from the group consisting of

[0080] (Wavy lines mark the bonds connecting the amino acid side chain to the protein or peptide backbone.)

[0081] In some embodiments, the amino acid is (a) histidine and the amino acid side chain is selected from the group consisting of and / or (b) tryptophan and the amino acid side chain is selected from the group consisting of In some embodiments, the amino acid is histidine and the amino acid side chain is selected from the group consisting of

[0082] In some embodiments, the amino acid side chain comprises a furan functional group. A furan functional group can, for example, be introduced into an amino acid side chain by reacting an amino acid with furan-2-carboxylic acid N-hydroxysuccinimide ester. N-hydroxysuccinimide esters react with free amine groups, e.g., free amine groups comprised within lysine side chains.

[0083] In some embodiments, X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine or 3- ethylaminoaniline (3-EA) or derivatives thereof. A hydrazide functional group is a functional group having a structure -CO-NH-NH2.

[0084] In some embodiments, X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine or 3- ethylaminoaniline (3-EA).

[0085] In some embodiments, X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3- ethylaminoaniline (3-EA) or derivatives thereof.

[0086] In some embodiments, X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3- ethylaminoaniline (3-EA).

[0087] In preferred embodiments, X is selected from the group consisting of a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine or 3-ethylaminoaniline (3-EA).

[0088] In preferred embodiments, X is selected from the group consisting of a hydrazide functional group, alkoxyamine, aniline or 3-ethylaminoaniline (3-EA).

[0089] In further preferred embodiments, X is selected from the group consisting of a hydrazide functional group, alkoxyamine or aniline. Accordingly, in some embodiments, X is selected from the group consisting of

[0090] (Wavy lines mark the bonds connecting the moiety reactive with an amino acid side chain X to the spacer group Si.)

[0091] In some embodiments, X is 1-methyl-4-aryl-urazole (MAUra) or a derivative thereof. In some embodiments, X is an alkoxyamine or a derivative thereof. In some embodiments, X is an aniline or a derivative thereof. In some embodiments, X is a 3-ethylaminoaniline or a derivative thereof. In some embodiments, X is 1-methyl-4-aryl-urazole (MAUra). In some embodiments, X is alkoxyamine. In some embodiments, X is 3-ethylaminoaniline (3-EA). In some embodiments, X is aniline.

[0092] In preferred embodiments, X is a hydrazide functional group, or a derivative thereof. In further preferred embodiments, X is a hydrazide functional group.

[0093] In some embodiments, the compound of the invention has a structure selected from the group consisting of

[0094] wherein

[0095] Si and S2 are independently of each other a spacer group;

[0096] Z is the cleavable linker moiety; and

[0097] A is the affinity moiety.

[0098] In specific embodiments, the compound of the invention has a structure selected from the group consisting of wherein Si and S2 are independently of each other a spacer group; Z is the cleavable linker moiety comprising a functional group having a formula selected from the group consisting of: wherein Ri is selected from H or CH3; and

[0099] A is the affinity moiety comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s).

[0100] In preferred embodiments, the compound of the invention has a structure according to formula wherein

[0101] Si and S2 are independently of each other a spacer group;

[0102] Z is the cleavable linker moiety; and

[0103] A is the affinity moiety.

[0104] In preferred embodiments, the compound of the invention has a structure according to formula wherein

[0105] Si and S2 are independently of each other a spacer group;

[0106] Z is the cleavable linker moiety comprising a functional group having a formula selected from the group consisting of: wherein Ri is selected from H or CH3; and

[0107] A is the affinity moiety comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s).

[0108] Compounds comprising a hydrazide functional group, are particularly useful for efficient labeling of proteins or peptides. Hydrazides in the compounds of the invention are capable of selectively reacting with carbonyl functional groups, hydroperoxide functional groups and / or endoperoxide functional groups comprised in amino acid side chains. A specific advantage of hydrazides or their derivatives in the context of the present invention is that hydrazides react with oxidized amino acid side chains (e.g., hydroperoxide or endoperoxide functional groups) to form an adduct containing a hydrazide bond, that is amenable to efficient fragmentation during mass-spectrometric analysis, resulting in unique ions that can be used as reporter ions (also called signature ions) to specifically identify labelled peptide fragments with very high sensitivity and specificity. Specific reporter ions also form through the reaction between hydrazides or further embodiments of X described herein (e.g., aniline, alkoxyamine, 1-methyl-4-aryl-urazole (MAUra), or 3-ethylaminoaniline (3-EA)) and amino acid side chains comprising carbonyl, hydroperoxide and / or endoperoxide functional groups. Both moiety X and cleavable linker moiety Z contribute to the formation of the specific reporter ions of the invention. Exemplary reporter ions are provided in Figures 8, 9 and 10.

[0109] Cleavable linker moiety

[0110] As used herein, cleavable linker moiety (Z) is a moiety (or functional group) that covalently connects two or more further moieties or functional groups (e.g., X and A) and that is cleavable by exposure to cleavage conditions and / or a cleavage reagent. Cleavage refers to the breaking or disruption of covalent chemical bonds within a compound to form fragments of said compound.

[0111] In some embodiments, Z is cleavable by exposure to one or more oxidizing agent(s). In preferred embodiments, Z is cleavable by exposure to one or more periodate(s) (e.g., periodate salt(s) and / or cyclic periodate(s)). In some embodiments, Z is cleavable by exposure to one or more cyclic periodate(s). In some embodiments, Z is cleavable by exposure to periodate ions. A periodate ion is a compound having the formula lO . In more preferred embodiments, Z is cleavable by exposure to sodium periodate.

[0112] Cleavage can be performed at a range of suitable concentrations that may be titrated to identify the most suitable concentration. In some embodiments, Z is cleavable by exposure to one or more periodate(s) (e.g., sodium periodate), wherein the one or more periodate(s) is / are used at a concentration of between about 0.01 nM and about 100 nM, preferably between about 0.1 nM and about 50 nM, more preferably between about 1 nM and about 10 nM. In a specific embodiment, the one or more periodate(s) is / are used at a concentration of about 2 nM.

[0113] In some embodiments, Z comprises a geminal diol or a secondary amino alcohol. In some embodiments, Z comprises a secondary amino alcohol. In some embodiments, Z comprises a secondary amino alcohol derived from serine. In some embodiments, Z comprises a secondary amino alcohol derived from isoserine. In some embodiments, Z comprises a secondary amino alcohol derived from threonine.

[0114] In some embodiments, Z comprises a functional group having a formula selected from the group consisting of: wherein R1 is selected from H or CH3.

[0115] (wavy lines mark the bonds connecting the cleavable linker moiety Z to the spacer groups Si and S2).

[0116] In preferred embodiments, Z comprises a functional group having a formula selected from the group consisting of: wherein Ri is selected from H or CH3.

[0117] In other preferred embodiments, Z comprises a functional group having the formula In other preferred embodiments, Z comprises a functional group having the formula wherein R1 is selected from H or CH3.

[0118] In preferred embodiments, Z comprises a functional group having a formula selected from the group consisting of: wherein Ri is selected from H or CH3.

[0119] (Wavy lines mark the bonds connecting the cleavable linker moiety Z to the spacer groups Si and S2.) In further preferred embodiments, Z comprises a functional group having a formula selected from the group consisting of: wherein R1 is selected from H or CH3.

[0120] In more preferred embodiments, Z comprises a functional group having the formula

[0121] In further more preferred embodiments, Z comprises a functional group having the formula wherein R1 is selected from H or CH3. In preferred embodiments, Z comprises a functional group having a formula selected from the group consisting of: wherein R1 is selected from H or CH3.

[0122] In further preferred embodiments, Z comprises a functional group having a formula selected from the group consisting of: wherein Ri is selected from H or CH3.

[0123] In more preferred embodiments, Z comprises a functional group having the formula

[0124] This linker structure is also referred to as isoseramox.

[0125] In further more preferred embodiments, Z comprises a functional group having the formula wherein Ri is selected from H or CH3.

[0126] This linker structure is also referred to as seramox, when Ri is H orthreamox when Ri is CH3.

[0127] In specific embodiments, the compound of the invention has a structure selected from the group consisting of wherein

[0128] Si and S2 are independently of each other a spacer group;

[0129] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0130] A is the affinity moiety, and wherein Ri is selected from H or CH3.

[0131] In further specific embodiments, the compound of the invention has a structure selected from the group consisting of

[0132] wherein

[0133] Si and S2 are independently of each other a spacer group;

[0134] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0135] A is the affinity moiety, and wherein R1 is selected from H or CH3.

[0136] In further specific embodiments, the compound of the invention has a structure according to the formula wherein

[0137] Si and S2 are independently of each other a spacer group;

[0138] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0139] A is the affinity moiety.

[0140] In further specific embodiments, the compound of the invention has a structure according to the formula

[0141] wherein

[0142] Si and S2 are independently of each other a spacer group;

[0143] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0144] A is the affinity moiety, and wherein R1 is selected from H or CH3.

[0145] In preferred embodiments, the compound of the invention has a structure selected from the group consisting of wherein

[0146] Si and S2 are independently of each other a spacer group;

[0147] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0148] A is the affinity moiety, and wherein R1 is selected from H or CH3.

[0149] In further preferred embodiments, the compound of the invention has a structure selected from the group consisting of

[0150] wherein

[0151] Si and S2 are independently of each other a spacer group;

[0152] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0153] A is the affinity moiety, and wherein R1 is selected from H or CH3.

[0154] In preferred embodiments, the compound of the invention has a structure according to the formula wherein

[0155] Si and S2 are independently of each other a spacer group;

[0156] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0157] A is the affinity moiety, and wherein R1 is selected from H or CH3.

[0158] In further preferred embodiments, the compound of the invention has a structure according to the formula wherein

[0159] Si and S2 are independently of each other a spacer group;

[0160] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0161] A is the affinity moiety.

[0162] In further preferred embodiments, the compound of the invention has a structure selected from the group consisting of wherein

[0163] Si and S2 are independently of each other a spacer group;

[0164] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0165] A is the affinity moiety, and wherein R1 is selected from H or CH3.

[0166] In further preferred embodiments, the compound of the invention has a structure selected from the group consisting of wherein

[0167] Si and S2 are independently of each other a spacer group;

[0168] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0169] A is the affinity moiety, and wherein R1 is selected from H or CH3.

[0170] In further preferred embodiments, the compound of the invention has a structure according to the formula wherein

[0171] Si and S2 are independently of each other a spacer group;

[0172] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0173] A is the affinity moiety, and wherein R1 is selected from H or CH3.

[0174] In further preferred embodiments, the compound of the invention has a structure according to the formula wherein Si and S2 are independently of each other a spacer group;

[0175] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0176] A is the affinity moiety.

[0177] In any of the specific and preferred embodiments disclosed herein, X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X may be selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA); and A may be an affinity moiety comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s).

[0178] Seramox and isoseramox-type linkers are particularly useful as cleavable linkers. These linkers offer very high cleavage rates under oxidative conditions (e.g., when treated with one or more periodate(s)) under mild conditions. Quantitative and highly selective cleavage within minutes can be achieved with these linkers.

[0179] Isoseramox-type linkers allow for traceless cleavage that releases a free amine as a cleavage product. Peptide fragments carrying such a free amine (e.g., a primary amine) are so-called highflyer peptides that possess superior properties for mass spectrometric analysis. The free amine is efficiently ionized during mass spectrometry analysis and the resulting ions exhibit superior signal-to-noise ratios. Seramox-type linkers result in aldehyde functional groups on the resulting fragments, which allow for subsequent functionalization with suitable reagents.

[0180] Moreover, seramox and isoseramox-type linkers possess broad substrate compatibility. They are compatible with in vitro cell culture or cell lysates and are able to penetrate cell membranes.

[0181] Moreover, seramox and isoseramox-type linkers form part of the reporter ions in the methods of the invention.

[0182] Affinity moiety

[0183] Affinity moiety A of the compound of the invention is an affinity moiety for isolation and purification, preferably for affinity purification of proteins or peptides.

[0184] The term “affinity moiety” or "affinity group" as used herein refers to an identifiable tag, group, or moiety that is capable of being specifically bound by another functional group (optionally attached or linked to a solid support, such as a bead, a filter, a plate, a membrane, a chromatographic resin, etc.) for detection, identification and purification purposes. It is understood that many different types of affinity groups are known in the art and may be used, either individually or a combination of one or more different affinity groups for the present invention. Exemplary affinity moieties include, but are not limited to, small chemical compounds (such as biotin / avidin and derivatives thereof, glutathione / GST) and short amino acid sequences, typically 2 to 20 amino acids in length, and preferably 4 to 12 amino acids in length (such as antibody fragments or the (His)etag, (Leu)3tag, the FLAG tag or the c-Myc tag), nucleic acid sequences (e.g., DNA, RNA, or PNA), or fluorescent tags. All these affinity tags are well established in the art and commercially available. In some embodiments, the affinity group is selected from the group consisting of biotin and derivatives thereof, carbohydrates, and glycans.

[0185] In some embodiments, the affinity moiety A comprises a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s). In one embodiment, the second functional group is covalently bound to a solid support, such as a bead, a filter, a plate, a membrane or a chromatographic resin. In a preferred embodiment, the second functional group is covalently bound to a bead or to a chromatographic resin.

[0186] Particularly suitable affinity group are based on click-chemistry, i.e., they are able to form one or more covalent bond(s) to an affinity resin through a cycloaddition reaction. In some embodiments, A comprises a first functional group capable of reacting with a second functional group to form one or more covalent bond(s) through a cycloaddition reaction. In one embodiment, the second functional group is covalently bound to a solid support, such as a bead, a filter, a plate, a membrane or a chromatographic resin. In a preferred embodiment, the second functional group is covalently bound to a bead or a chromatographic resin.

[0187] The formation of a covalent bond during affinity purification results in a very strong attachment to an affinity resin (e.g., a solid support) and thus enables very stringent washing procedures thereby reducing impurities in downstream processing steps.

[0188] In some embodiments, the cycloaddition reaction is a [4+2] cycloaddition reaction (also termed Diels-Alder reaction). The cycloaddition reaction may be a copper(l)-catalyzed azide-alkyne cycloaddition (CuAAC), or a strain-promoted cycloaddition reaction, such as a strain-promoted azide-alkyne cycloaddition (SPAAC), a strain-promoted alkyne-nitrone cycloaddition (SPANC) or a strain-promoted alkene-tetrazine cycloaddition. In preferred embodiments, the cycloaddition reaction is a strain-promoted cycloaddition reaction. Strain-promoted cycloaddition reactions do not require an addition of reactive catalysts, such as copper(l), thereby avoiding potential undesired side reactions within a sample. Strain-promoted alkene-tetrazine cycloadditions are entropically particularly favorable and moreover irreversible due to the release of nitrogen gas as one reaction product. In some embodiments, A comprises a functional group selected from biotin, biocytin, tetrazine, methyltetrazine, trans-cyclooctene (TCO), azide (-N3), a terminal alkyne (-CCH) or derivatives thereof. In some embodiments, A comprises a functional group selected from biotin, biocytin, tetrazine, methyltetrazine, trans-cyclooctene (TCO), azide (-N3) or a terminal alkyne (-CCH).

[0189] In preferred embodiments, A comprises a functional group selected from tetrazine, methyltetrazine, trans-cyclooctene (TCO), azide (-N3), a terminal alkyne (-CCH) or derivatives thereof. In further preferred embodiments, A comprises a functional group selected from tetrazine, methyltetrazine, trans-cyclooctene (TCO), azide (-N3) or a terminal alkyne (-CCH). These functional groups provide long-term stability in aqueous buffers.

[0190] In some embodiments, the affinity moiety is selected from the group of tetrazine, methyltetrazine, trans-cyclooctene (TCO) or derivatives thereof. In some embodiments, the affinity moiety is tetrazine or a derivative thereof. In some embodiments, the affinity moiety is methyltetrazine or a derivative thereof. In some embodiments, the affinity moiety is TCO or derivatives thereof. In some embodiments, the affinity moiety is tetrazine. In some embodiments, the affinity moiety is methyltetrazine. In some embodiments, the affinity moiety is TCO. These affinity moieties are characterized by their fast reaction kinetics. Like many cycloadditions, the TCO-tetrazine reaction is highly selective, allowing it to proceed without disturbing natural biological processes.

[0191] In some embodiments, A is biotin or a derivative thereof. In some embodiments, A is biocytin or a derivative thereof. In some embodiments, A is biotin. In some embodiments, A is biocytin. An advantage of biotin and biocytin is their strong and specific interaction with avidin or streptavidin proteins. Biotin-tagged proteins can be easily isolated using streptavidin-coated beads or columns.

[0192] In specific embodiments, the compound of the invention has a structure selected from the group consisting of wherein

[0193] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine;

[0194] Si and S2 are independently of each other a spacer group; and Z is the cleavable linker moiety; and wherein

[0195] R2 is selected from H, CH3, or (CH2)2-COOH.

[0196] Preferably, in specific embodiments, X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA); and Z is a cleavable linker moiety comprising a functional group having a formula selected from the group consisting of: wherein R1 is selected from H or CH3.

[0197] Spacers

[0198] Attached to the cleavable linker Z are two spacer groups Si and S2 which covalently link the cleavable linker moiety to the respective functionalities A (affinity moiety) and X (moiety reactive with an amino acid side chain) according to Formula I. Generally, a spacer is a moiety or group that separates or connects functional moieties in a compound.

[0199] The length of the spacers Si and S2 determines the distance between the moiety X, the cleavable linker moiety Z and the affinity moiety A. Si and S2 may be chosen such that steric crowding is minimized and the reactivity of the three moieties A, Z and X are not compromised.

[0200] Thus, varying the lengths of spacers Si and S2 allows for adjusting the spatial accessibility of the individual moieties comprised in the compound according to the invention. Longer spacers can result in an increased accessibility of the individual moieties and thereby facilitate, e.g., an affinity purification through the affinity moiety A. Shorter spacers result in a reduced overall size of the compound, which may facilitate penetration in a molecularly crowded environment like a cell surface.

[0201] In addition, introducing functional groups that carry a charge under common buffer conditions into the spacers Si and S2 allows for adjusting solubility, conformation, and electrostatic interactions of the compound of the invention. Spacers comprising charged functional groups generally improve solubility in aqueous media and can either attract or repel nearby ions or molecules, impacting binding affinity, stability or biological activity. For example, positively charged spacers might improve solubility and facilitate interactions with negatively charged cell membranes.

[0202] In some embodiments, the spacer groups Si and S2 are independently of each other (i) a single bond or (ii) a straight-chain or branched, substituted or unsubstituted C(1 -24) hydrocarbon, wherein one or more, preferably non-adjacent, -CH2- groups independently from each other may be replaced by one or more bridging groups and / or an unsubstituted or substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl. A bridging group may replace a -CH2- group within the alkylene chain or a terminal -CH2- group.

[0203] In some embodiments, the bridging group is selected from -CH(OH)-, -O-, -CO-, -CH2(CO)-, -SO-, - CH2(SO)-, -SO2-, -CH2(SO2)-, -COO-, -OCO-, -S-CO-, -CO-S-, -SOO-, -OSO-, -SOS-, -O-CO-O-, - OCH2-, -CH2O-, -NR3-, -NR3-CO-, -CO-NR3-, -NR3-CO-O-, -O-CO-NR3-, -NR3-CO-NR3-, -CH=CH-, - C=C-, -CH=CH-COO-, -OCO-CH=CH-, -CH=N-, -C(CH3)=N-, -N=N-, wherein each R3represents H or C(1-6)alkyl. Preferred bridging groups include -CH(OH)-, -O-, -CO-, -CH2(CO)-, -COO-, -OCO-, - O-CO-O-, -OCH2-, -CH2O-, -NR3-, -NR3-CO-, -CO-NR3-, -NR3-CO-O-, -O-CO-NR3-, -NR3-CO-NR3-, -CH=CH-, -CH=N-, -C(CH3)=N-, wherein R3 represents H or C(1 -6)alky I , or combinations thereof. More preferred bridging groups include -CH(OH)-, -O-, -CO-, -CH2(CO)-, -COO-, -OCO-, -O-CO-O-, -OCH2-, -CH2O-, NR3-, -NR3-CO-, -CO-NR3-, wherein R3 represents H or C(1 -6)alkyl, or combinations thereof.

[0204] In specific embodiments, the spacer groups Si and S2 are independently of each other a substituted or unsubstituted chain having 6 to 30 carbon atoms, preferably comprising a polyethylene glycol group (having 2 to 24 ethylene glycol monomers in a linear configuration), a polyalcohol group, a polyamine group (e.g., spermine, spermidine and polymeric derivatives thereof), a polyester group (e.g., poly(ethyl acrylate) having from 3 to 15 ethyl acrylate monomers in a linear configuration), a polyamino acid group or a combination thereof. In some embodiments, the spacer groups Si and S2 are independently of each other a substituted or unsubstituted hydrocarbon chain having 6 to 30 carbon atoms and comprising 2, 3, 4 or 5 polyalcohol groups, polyamine groups (e.g., spermine, spermidine and polymeric derivatives thereof), a polyester group (e.g., poly(ethyl acrylate) having from 3 to 5 ethyl acrylate monomers in a linear configuration), a polyamino acid group or a combination thereof.

[0205] In some embodiments, the spacer groups Si and S2 independently of each other comprise a polyamino acid comprising 1 to 8 amino acids (i.e., an amino acid or a di-, tri-, tetra-, penta-, hexa-, hepta- or octapeptide). In preferred embodiments, Si and S2 are each independently selected from a linear chain comprising one or more repeating units of Formula II:

[0206] -[Yl-(CH2)n]p- and / or Formula III:

[0207] -[Y2-(CH2)m-Y3]q-, or combinations thereof, wherein

[0208] Y1, Y2, Y3 are independently of each other a group selected from -O-, -CO-, COO-, -OCO-, -O-CO- O-, -OCH2-, -CH2O-, NR4-, -NR4-CO-, -CO-NR4-, wherein R4 represents H or C(1 -6)-alkyl, and n, m, p, and q are independently of each other an integer from 1 to 10.

[0209] In preferred embodiments, Si and S2are each independently selected from a linear chain comprising one or more repeating units of Formula II

[0210] -[Yl-(CH2)n]p-, wherein

[0211] Y1 is -O-, n is 2, and p is an integer from 1 to 10, preferably from 1 to 5, most preferably from 1 to 3.

[0212] In preferred embodiments, Si and S2are each independently selected from a linear chain comprising one or more repeating units of Formula II

[0213] -[Yl-(CH2)n]p-, wherein Y1 is -O-; n is 2; and p is 3.

[0214] The structure of the spacer groups Si and S2can be adapted to enhance the solubility of the compound according to the invention in polar solvents such as water. For example, polyethylene glycol based spacers (-[O-(CH2)2]p-) are known to increase the solubility of organic compounds in polar solvents.

[0215] Further Embodiments In some embodiments, the compound comprises one or more isotopic label(s). In some embodiments, the one or more isotopic label(s) are each independently selected from18O,13C,15N or2H. In some embodiments, the one or more isotopic label(s) are comprised within the spacer group Si, the spacer group S2 and / or within the moiety X. In preferred embodiments, the one or more isotopic label(s) are comprised within the spacer group Si. In some embodiments, the one or more isotopic label(s) are comprised within the moiety X.

[0216] Compounds of the invention that comprise one or more isotopic label(s) can be used for massdifference labelling or for isobaric labelling. Mass-difference labeling approaches introduce a mass difference for the same protein or peptide by incorporating a light or heavy isotopic form of the labeling reagent (e.g., to mark different experimental conditions). Resulting light and heavy labeled peptides are combined prior to MS analysis, and quantitation is accomplished by comparing the extracted ion chromatogram peak areas of light and heavy forms of the same peptide.

[0217] For isobaric labelling, peptides or proteins are labeled with chemical groups that are structurally identical and have nominally identical mass (i.e., they are isobaric), but vary in terms of distribution of heavy isotopes in their structure. When using isobaric labels (or tags), each sample (e.g., from different experimental conditions) comprises an individual isobaric label, different samples are pooled together and introduced into the mass spectrometer for quantitative analysis. Since samples are isobarically labeled, the same peptide from multiple samples produces a single peak in MS mode (a mixture of first ions having the same mass-to-charge ration, m / z), but upon MS2fragmentation, each labeled sample gives rise to a unique reporter ion (a mixture of second ions having different m / z ratios). Relative quantitation is achieved by correlating the relative abundance of each reporter ion with its originating sample.

[0218] Isotopic labels can for example be used for the labelling of different experimental conditions.

[0219] In some embodiments, the compound of the invention has a structure according to the Formula I:

[0220] X-S1-Z-S2-A wherein

[0221] Si and S2 are independently of each other a spacer group; and wherein independently from each other

[0222] X is selected from the group consisting of

[0223]

[0224] Z is selected from the group consisting of wherein Ri is selected from H or CH3; and

[0225] A is selected from the group consisting of wherein R2 is selected from H, CH3, or (CH2)2-COOH.

[0226] (Wavy lines mark the bonds connecting the moieties X, Z and A to the spacer group(s) Si and / or S2.)

[0227] In specific embodiments, the compound of the invention has a structure of Formula IV:

[0228]

[0229] Formula IV wherein

[0230] Si and S2 are independently of each other a spacer group. In specific embodiments, the compound of the invention has a structure according to the formula wherein

[0231] Si and S2 are independently of each other a spacer group. In specific embodiments, the compound of the invention has a structure according to the formula wherein

[0232] Si and S2 are independently of each other a spacer group. In specific embodiments, the compound of the invention has a structure according to the formula wherein

[0233] Si and S2 are independently of each other a spacer group. In a particularly preferred embodiment, the compound of the invention has a structure according to the formula

[0234] In a further particularly preferred embodiment, the compound of the invention has a structure according to the formula

[0235] In a further particularly preferred embodiment, the compound of the invention has a structure according to the formula

[0236]

[0237] In a further particularly preferred embodiment, the compound of the invention has a structure according to the formula In a further particularly preferred embodiment, the compound of the invention has a structure according to the formula

[0238] In a further particularly preferred embodiment, the compound of the invention has a structure according to the formula

[0239]

[0240] In a further particularly preferred embodiment, the compound of the invention has a structure according to the formula In a further particularly preferred embodiment, the compound of the invention has a structure according to the formula

[0241] It is understood that for use in the preparation of the compounds of the invention, the spacer group is preferably provided with terminal functional groups which can be selectively protected or activated for attachment to any one of the functionalities A, Z, or X. Thus, in some embodiments, the spacer groups may be coupled to A, Z, or X through a bridging group, preferably through groups selected from -COO-, -CO-NR5-, -O-, -NR5-, -NR5-COO-, and -S-S- linkages, wherein R5 represents H or C(1-6)alkyl. Methods and uses of the invention

[0242] In a further aspect, the invention provides the use of the compound of the invention for the isolation of one or more protein(s). In another aspect, the invention provides the use of the compound of the invention for the selective enrichment of one or more protein(s). In another aspect, the invention provides the use of the compound of the invention for the isolation and selective enrichment of one or more protein(s). In yet another aspect, the invention provides the use of the compound of the invention for the isolation, selective enrichment and identification of one or more protein(s). In yet another aspect, the invention provides the use of the compound of the invention for the isolation, selective enrichment, identification and quantification of one or more protein(s).

[0243] In some embodiments, the uses comprise:

[0244] (i) subjecting a sample comprising the one or more protein(s) to a chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain comprised in the one or more protein(s), to obtain one or more oxidized protein(s);

[0245] (ii) reacting the one or more oxidized protein(s) with a compound of Formula I:

[0246] X-S1-Z-S2-A wherein

[0247] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine;

[0248] Si and S2 are independently of each other a spacer group;

[0249] Z is a cleavable linker moiety; and

[0250] A is an affinity moiety, wherein the moiety X reacts with the one or more carbonyl functional group(s), the one or more hydroperoxide functional group(s) and / or the one or more endoperoxide functional group(s) to form one or more labelled protein(s);

[0251] (iii) isolating and identifying the one or more labelled protein(s).

[0252] In some embodiments, the uses comprise:

[0253] (i) subjecting a sample comprising the one or more protein(s) to a chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain comprised in the one or more protein(s), to obtain one or more oxidized protein(s);

[0254] (ii) reacting the one or more oxidized protein(s) with a compound of Formula I: X-S1-Z-S2-A wherein

[0255] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA);

[0256] Si and S2 are independently of each other a spacer group;

[0257] Z is a cleavable linker moiety comprising a functional group having a formula selected from the group consisting of: wherein R1 is selected from H or CH3; and

[0258] A is an affinity moiety comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s), wherein the moiety X reacts with the one or more carbonyl functional group(s), the one or more hydroperoxide functional group(s) and / or the one or more endoperoxide functional group(s) to form one or more labelled protein(s);

[0259] (iii) isolating and identifying the one or more labelled protein(s).

[0260] In a further aspect, the invention provides a method for the isolation and identification of one or more protein(s), comprising the steps of:

[0261] (i) subjecting a sample comprising the one or more protein(s) to a chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain comprised in the one or more protein(s), to obtain one or more oxidized protein(s); (ii) reacting the one or more oxidized protein(s) with a compound of Formula I:

[0262] X-S1-Z-S2-A wherein

[0263] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine;

[0264] Si and S2 are independently of each other a spacer group;

[0265] Z is a cleavable linker moiety; and

[0266] A is an affinity moiety, wherein the moiety X reacts with the one or more carbonyl functional group(s), the one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) to form one or more labelled protein(s);

[0267] (iii) isolating and identifying the one or more labelled protein(s).

[0268] In some embodiments, the invention provides a method for the isolation and identification of one or more protein(s), comprising the steps of:

[0269] (i) subjecting a sample comprising the one or more protein(s) to a chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain comprised in the one or more protein(s), to obtain one or more oxidized protein(s);

[0270] (ii) reacting the one or more oxidized protein(s) with a compound of Formula I:

[0271] X-S1-Z-S2-A wherein

[0272] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA);

[0273] Si and S2 are independently of each other a spacer group;

[0274] Z is a cleavable linker moiety comprising a functional group having a formula selected from the group consisting of: wherein Ri is selected from H or CH3; and

[0275] A is an affinity moiety comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s), wherein the moiety X reacts with the one or more carbonyl functional group(s), the one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) to form one or more labelled protein(s);

[0276] (iii) isolating and identifying the one or more labelled protein(s).

[0277] In some embodiments of the uses or methods, the isolation and identification of one or more protein(s) comprises:

[0278] (a) enriching the one or more labelled protein(s), wherein the one or more labelled protein(s) are contacted with a solid support and wherein the affinity moiety A specifically associates with or specifically reacts with a functional group attached or linked to the solid support to form one or more bond(s), optionally wherein the solid support is subjected to one or more washing steps after formation of the one or more covalent bond(s);

[0279] (b) releasing the one or more labelled protein(s) from the solid support, preferably by exposure to one or more oxidizing agent(s);

[0280] (c) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s); and

[0281] (d) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS).

[0282] In other embodiments of the uses or methods, the isolation and identification of one or more protein(s) comprises:

[0283] (a) enriching the one or more labelled protein(s), wherein the one or more labelled protein(s) are contacted with a solid support and wherein the affinity moiety A specifically associates with or specifically reacts with a functional group attached or linked to the solid support to form one or more bond(s), optionally wherein the solid support is subjected to one or more washing steps after formation of the one or more covalent bond(s);

[0284] (b) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s), optionally wherein the solid support is subjected to one or more washing steps after the fragmenting of the one or more labelled protein(s);

[0285] (c) releasing one or more peptide fragments from the solid support, preferably by exposure to one or more oxidizing agent(s); and

[0286] (d) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS).

[0287] In further embodiments of the uses or methods, the isolation and identification of one or more protein(s) comprises:

[0288] (a) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s);

[0289] (b) enriching one or more peptide fragments, wherein the one or more peptide fragments are contacted with a solid support and wherein the affinity moiety A specifically associates with or specifically reacts with a functional group attached or linked to the solid support to form one or more bond(s), optionally wherein the solid support is subjected to one or more washing steps after formation of the one or more covalent bond(s);

[0290] (c) releasing the one or more peptide fragments from the solid support, preferably by exposure to one or more oxidizing agent(s);

[0291] (d) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS).

[0292] In further embodiments of the uses or methods, the isolation and identification of one or more protein(s) comprises:

[0293] (a) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s); (c) optionally exposing the one or more peptide fragments to one or more oxidizing agent(s);

[0294] (d) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS).

[0295] In some embodiments of the uses or methods according to the invention, the one or more protein(s) is / are fragmented to obtain peptide fragments of the one or more labelled protein(s) prior to being subjected to a chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain. In these embodiments, the further steps of the uses or methods described herein are performed on the peptide fragments, instead of the one or more protein(s).

[0296] The compound of Formula I that is used in the uses or methods according to the invention can be a compound according to any of the embodiments described herein.

[0297] In some embodiments of the uses or methods according to the invention, the affinity moiety A specifically reacts with a functional group attached or linked to the solid support to form one or more covalent bond(s) through a cycloaddition reaction.

[0298] In some embodiments, releasing the one or more peptide fragments from the solid support, comprises exposure to one or more oxidizing agent(s). In some embodiments, the one or more oxidizing agent(s) are selected from one or more periodate(s) (e.g., periodate salt(s) and / or cyclic periodate(s)). In some embodiments, the one or more oxidizing agent(s) are selected from one or more cyclic periodate(s) and / or one or more periodate ions. In some embodiments, the oxidizing agent is sodium periodate.

[0299] In some embodiments of the inventive uses or methods, the chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain comprises reacting the one or more protein(s) with singlet oxygen (1C>2). Singlet oxygen can rapidly oxidize biomolecules, such as proteins or peptides (see, for example, Kuimova et al., J Am Chem, (2009), 131 :332-340; Muller et al., Nature Communications, (2021)12:7036). Amino acid residues that are efficiently oxidized by singlet oxygen include histidine, tryptophan, tyrosine, methionine or cysteine. The oxidation products for histidine and tryptophan are as described herein and include 2-oxo- histidine, histidine hydroperoxide, histidine endoperoxide and 2-hydroxy-tryptophan. In some embodiments, the singlet oxygen is generated by exposing a singlet oxygen generator (SOG) to light. As used herein, a singlet oxygen generator is a molecule or functional group that is capable of generating singlet oxygen upon irradiation with light.

[0300] Biocompatible SOGs are known in the art (see for example Di Mascio et al., Chem. Rev. 119, 2043-2086 (2019), Jacobson et al., Trends Cell Biol. 18, 443-450 (2008) or Yaraki et al., Nanomicro Lett. 2022; 14: 123). In some embodiments, the singlet oxygen is generated in presence of deuterium oxide. In some embodiments, the reaction rate of singlet-oxygen generation is regulated by exposure to light and / or deuterium oxide. In preferred embodiments, the reaction rate of singlet-oxygen generation is regulated by exposure to light. The reaction is performed using a wavelength that is suitable to excite the employed SOG. In some embodiments, the singlet oxygen is generated by exposing a singlet oxygen generator to light, wherein the light has a wavelength of between about 500 nm and about 700 nm, preferably between about 540 nm and about 640 nm, more preferably between about 560 nm and about 620 nm, even more preferably about 590 nm..

[0301] In some embodiments, the SOG is selected from the group consisting of thiorhodamine, a phenothiazinium dye (e.g., methylene blue, Nile blue or toluidine blue), a cyanine, a transition metal complex (e.g., bipyridine, bipyrazine or 2,2'-bipyrimidine complexes of Ru(ll), Os(ll), or Ir(lll)), a chalcogenopyrylium dye and derivatives thereof. In some embodiments, the SOG is NHS- thiorhodamine. In some embodiments, the SOG is azide-thiorodamine.

[0302] In some embodiments, the SOG is covalently coupled to an antibody, a small molecule, a peptide, a peptidomimetic, an aptamer or a viral particle. In some embodiments, the antibody, the small molecule, the peptide, the peptidomimetic, the aptamer or the viral particle is capable of specifically binding to the one or more protein(s) or to a target that is in close proximity to or in physical contact with the one or more protein(s). In preferred embodiments, the SOG is covalently coupled to an antibody that is capable of specifically binding to a target that is in physical contact with the one or more protein(s) and the reaction rate of singlet-oxygen production is regulated by exposure to light.

[0303] In some embodiments, the chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain takes place in a distance from the single oxygen generator of about 1-200 nm, about 1-100 nm, about 1-50 nm, about 1-20 nm, about 1-10 nm, or about 10-20 nm.

[0304] It is understood that in the context of the uses or methods according to the invention, the one or more protein(s) can be in solution, on the surface of a cell, within the cell, in a cell lysate and / or in protein mixtures. In some embodiments, the one or more protein(s) is located on a cell surface or within a cell, preferably in or on the plasma membrane or cell wall or in the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes, cytoplasm, vacuoles, cytoskeleton or chloroplasts. In some embodiments, the one or more protein(s) is located on a cell surface or within a cell. In some embodiments, the one or more protein(s) is located on a cell surface. In some embodiments, the one or more protein(s) is located within a cell.

[0305] The one or more protein(s) may be naturally occurring or may have been engineered or modified synthetically. In some embodiments, the one or more protein(s) is a membrane protein. In some embodiments, the one or more protein(s) is a cell surface protein. In preferred embodiments, the one or more protein(s) is a membrane protein present on the cell surface. Membrane proteins are generally associated to a plasma membrane and preferably have at least one extracellularly exposed amino acid.

[0306] The invention is not limited to particular types of proteins, i.e., various types of proteins may be used in the methods and uses disclosed herein. The one or more protein(s) may be a receptor, antibody, enzyme, hormone, regulatory factor, antigen, binding molecule, or the like.

[0307] In some embodiments of the uses or methods according to the invention, the cell is an animal cell, bacterial cell, yeast cell, insect cell or plant cell. In preferred embodiments, the cell is an animal cell. In more preferred embodiments, the animal cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell, a mouse cell, a hamster cell, a porcine cell, a rabbit cell, a dog cell, a cat cell, a rat cell or a non-human primate cell. In preferred embodiments, the cell is a mouse cell, a hamster cell or a human cell. In more preferred embodiments, the cell is a human cell.

[0308] The term “sample” or "biological sample", as used herein, refers to any solid or fluid sample obtained from, excreted by or secreted by a living cell or organism, including, but not limited to, tissue culture, bioreactors, human or animal tissue, plants, fruits, vegetables, single-celled microorganisms (such as bacteria and yeasts) and multicellular organisms. For example, a biological sample can be a biological fluid obtained from, e.g., blood, plasma, serum, urine, bile, seminal fluid, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g., a normal joint or a joint affected by disease such as a rheumatoid arthritis, osteoarthritis, gout or septic arthritis). A biological sample can also be, e.g., a sample obtained from any organ or tissue (including a biopsy or autopsy specimen), can comprise cells (whether primary cells or cultured cells), medium conditioned by any cell, tissue or organ, tissue culture.

[0309] The terms “protein” or “peptide” as used herein have the same meaning and refer to an amino acid polymer having any length of the sequence. These polymers may be a straight, branched or cyclic chain. An amino acid may be a naturally-occurring or non-naturally-occurring amino acid, or a variant amino acid. The term “peptide fragments” or “peptide fragment” refer to an amino acid polymer having a sequence length ranging from 1 amino acid residue to n-1 with respect to the full length of the reference protein of length n (i.e., the protein which the peptide fragment is derived from). Peptide fragments may for example be obtained by proteolytic digestion of the respective protein or peptide.

[0310] In some embodiments of the uses or methods according to the invention, fragmenting the one or more labelled protein(s) is done by proteolytic digestion. In some embodiments, the proteolytic digestion comprises the use of a protease selected from the group consisting of trypsin, chymotrypsin, ArgC, GluC, AspN, LysC, LysN, elastase and proteinase K and a combination thereof. In preferred embodiments, the proteolytic digestion comprises the use of trypsin and / or LysC.

[0311] The term “one or more oxidized protein(s)” refers to the one or more protein(s) obtained by subjecting a sample to a chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain comprised in the one or more protein(s).

[0312] The term “one or more labeled protein(s)” refers to the one or more protein(s) that has been covalently bound to a functional group or compound, wherein the functional group or the compound may serve as a detectable label. One or more labelled protein(s) is formed, when the moiety X of any one of the compounds of the invention reacts with the one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) comprised in an amino acid side chain comprised in the one or more protein(s).

[0313] In some embodiments, the identification of one or more individual labelled protein(s) and / or peptide fragments thereof is by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS), wherein the liquid-chromatography-mass spectrometry (LC-MS) comprises tandem mass spectrometry (MS / MS). Methods of mass spectrometry analysis are well known to those skilled in the art (see, for example, Yates, J. Mass Spect., (1998), 33:1-19; Kinter and Sherman, Protein Sequencing and Identification Using Tandem Mass Spectrometry, John Wiley and Sons, New York (2000); Aebersold and Goodlett, Chem. Rev., (2001), 101 :269-295).

[0314] For high resolution polypeptide fragment separation, liquid chromatography ESI-MS / MS or automated LC-MS / MS, which utilizes capillary reverse phase chromatography as the separation method, can be used (Yates et al., Methods Mol. Biol. 112:553-569 (1999)). Data dependent collision-induced dissociation (CID) with dynamic exclusion or higher-energy collisional dissociation (HCD) with dynamic exclusion may be used (Goodlett et al., Anal. Chem. 72:1112-1118 (2000)).

[0315] For such an analysis, mass spectrometers are typically operated in the data dependent mode in which ion signals above a predetermined threshold automatically trigger the instrument to switch from MS to MS / MS mode for generating collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) spectra of peptides.

[0316] All MS / MS spectra are searched against a standard protein database using standard algorithms (e.g., DiaNN, SEQUEST, Mascot, Xltandem, OMSSA) and are typically filtered in order to limit the false-positive protein identification rate to below 1%.

[0317] In preferred embodiments, the uses and methods comprise mass spectrometric quantification of one or more individual labelled protein(s) and / or peptide fragments.

[0318] The concentration of one or more protein(s) (or peptide fragments thereof) in a first sample can be quantitatively compared to the concentration of the same one or more protein(s) (or peptide fragments thereof) in a second sample in order to detect specific enrichment of said protein in the first or second sample. For example, the first sample may have been exposed to light, thereby inducing SOG-mediated oxidation of the one or more protein(s), whereas the second sample has not been exposed to light, thus serving as a control sample.

[0319] Various mass spectrometry-based quantification methods can be used, including label-free methods, single reaction monitoring (SRM), stable isotope labeling with amino acids in cell culture (SILAC) (Nilsson et al. Nat. Methods (2010) vol. 7 (9) pp. 681-5), tandem mass tags (TMT), isobaric tags for relative and absolute quantification (iTRAQ) and the like. Advantageously, a compound according to the invention is used, wherein the compound comprises one or more isotopic label(s).

[0320] In some embodiments, the uses or methods according to the invention comprise determining whether the one or more protein(s) is in close proximity to or in physical contact with a particular target protein or protein of interest, e.g., a cell surface protein.

[0321] In any of the uses or methods according to the invention, the compound of Formula I may be a compound according to any one of the embodiments described herein.

[0322] The present invention further relates to a protein or peptide comprising at least one (e.g., one) amino acid side chain selected from the group consisting of

[0323]

[0324] (Wavy lines mark the bonds connecting the amino acid side chain to the protein or peptide backbone.)

[0325] The present invention further relates to a protein or peptide produced by the method of the invention, comprising at least one amino acid side chain selected from the group consisting of

[0326]

[0327] , and / or from the group consisting of

[0328] (Wavy lines mark the bonds connecting the amino acid side chain to the protein or peptide backbone.)

[0329] The invention is further illustrated by the non-limiting examples disclosed herein.

[0330] Items of the Invention

[0331] In view of the disclosure provided herein, it will be appreciated that the present invention also encompasses the following items.

[0332] 1. A compound of Formula I:

[0333] X-S1-Z-S2-A wherein

[0334] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine;

[0335] Si and S2 are independently of each other a spacer group;

[0336] Z is a cleavable linker moiety; and

[0337] A is an affinity moiety.

[0338] 2. A compound of Formula I:

[0339] X-S1-Z-S2-A wherein

[0340] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA);

[0341] Si and S2 are independently of each other a spacer group;

[0342] Z is a cleavable linker moiety comprising a functional group having a formula selected from the group consisting of: wherein R1 is selected from H or CH3; and

[0343] A is an affinity moiety comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s).

[0344] 3. The compound according to item 1 or item 2, wherein the amino acid side chain has been subjected to a chemical reaction to obtain an oxidized amino acid side chain, relative to the native amino acid side chain. 4. The compound according to any one of items 1 to 3, wherein the amino acid side chain comprises a carbonyl functional group, a hydroperoxide functional group and / or an endoperoxide functional group.

[0345] 5. The compound according to any one of items 1 to 4, wherein the amino acid is: (a) histidine and the amino acid side chain is selected from the group consisting of

[0346] (b) tryptophan and the amino acid side chain is selected from the group consisting of

[0347] (c) tyrosine and the amino acid side chain is selected from the group consisting of

[0348] (d) methionine and the amino acid side chain is

[0349] ; and / or

[0350] (f) cysteine and the amino acid side chain is selected from the group consisting of

[0351] 6. The compound according to any one of items 1 to 5, wherein X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1- methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA) or derivatives thereof, preferably wherein X is a hydrazide functional group or a derivative thereof.

[0352] 7. The compound according to any one of items 1 to 6, wherein X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1- methyl-4-aryl-urazole (MAUra), alkoxyamine or 3-ethylaminoaniline (3-EA) or derivatives thereof, preferably wherein X is a hydrazide functional group or a derivative thereof.

[0353] 8. The compound according to any one of items 1 to 7, wherein the cleavable linker moiety Z is cleavable by exposure to one or more oxidizing agent(s), preferably by exposure to one or more periodate salt(s) and / or cyclic periodate(s), more preferably by exposure to sodium periodate.

[0354] 9. The compound according to any one of items 1 to 8, wherein Z comprises a geminal diol or a secondary amino alcohol.

[0355] 10. The compound according to any one of items 1 , or 3 to 9, wherein Z comprises a functional group having a formula selected from the group consisting of: wherein Ri is selected from H or CH3.

[0356] 11 . The compound according to any one of items 1 to 10, wherein Z comprises a functional group having a formula selected from the group consisting of:

[0357] wherein R1 is selected from H or CH3.

[0358] 12. The compound according to any one of items 1 to 11 , wherein Z comprises a functional group having a formula selected from the group consisting of: wherein R1 is selected from H or CH3.

[0359] 13. The compound according to any one of items 1 or 3 to 12, wherein the affinity moiety A comprises a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s).

[0360] 14. The compound according to item 13, wherein the second functional group is covalently bound to a solid support, such as a bead, a filter, a plate, a membrane or a chromatographic resin, preferably wherein the second functional group is covalently bound to a bead or to a chromatographic resin.

[0361] 15. The compound according to any one of items 1 to 14, wherein A comprises a first functional group capable of reacting with a second functional group to form one or more covalent bond(s) through a cycloaddition reaction, optionally wherein the cycloaddition reaction is a [4+2] cycloaddition reaction.

[0362] 16. The compound according to any one of items 1 to 15, wherein A comprises a functional group selected from biotin, biocytin or derivatives thereof.

[0363] 17. The compound according to any one of items 1 to 16, wherein A comprises a functional group selected from tetrazine, methyltetrazine, trans-cyclooctene (TCO), azide (-N3), a terminal alkyne (-CCH) or derivatives thereof. 18. The compound according to any one of items 1 to 17, wherein Si and S2 are independently of each other (i) a single bond or (ii) a straight-chain or branched, substituted or unsubstituted C(1-24) hydrocarbon, wherein one or more, preferably non-adjacent, -CH2- groups independently from each other may be replaced by one or more bridging groups and / or an unsubstituted or substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl.

[0364] 19. The compound according to item 18, wherein the bridging group is selected from -CH(OH)-, -O-, -CO-, -CH2(CO)-, -SO-, -CH2(SO)-, -SO2-, -CH2(SO2)-, -COO-, -OCO-, -S-CO-, -CO-S-, -SOO-, -OSO-, -SOS-, -O-CO-O-, -OCH2-, -CH2O-, -NR3-, -NR3-CO-, -CO-NR3-, -NR3-CO- O-, -O-CO-NR3-, -NR3-CO-NR3-, -CH=CH-, -C=C-, -CH=CH-COO-, -OCO-CH=CH-, -CH=N- , -C(CH3)=N-, -N=N-, wherein each R3 independently represents H or C(1 -6)alkyl.

[0365] 20. The compound according to any one of items 1 to 19, wherein Si and S2 are each independently selected from a linear chain comprising one or more repeating units of Formula II:

[0366] -[Yl-(CH2)n]p- and / or Formula III:

[0367] -[Y2-(CH2)m-Y3]q-, or combinations thereof, wherein

[0368] Y1, Y2, Y3 are independently of each other a group selected from -O-, -CO-, COO-, -OCO-, - O-CO-O-, -OCH2-, -CH2O-, NR4-, -NR4-CO-, -CO-NR4-, wherein R4 represents H or C(1 -6)- alkyl, and n, m, p, and q are independently of each other an integer from 1 to 10.

[0369] 21 . The compound according to any one of items 1 to 20, wherein Si and S2 are each independently a linear chain comprising one or more repeating units of Formula II

[0370] -[Yl-(CH2)n]p-, wherein

[0371] Y1 is -O-, n is 2, and p is an integer from 1 to 10, preferably from 1 to 5, most preferably from 1 to 3. 22. The compound according to any one of items 1 to 21 , wherein the compound comprises one or more isotopic label(s), preferably wherein the one or more isotopic label(s) are each independently selected from180,13C,15N or2H.

[0372] 23. The compound according to item 22, wherein the one or more isotopic label(s) are comprised within the spacer group Si, the spacer group S2 and / or within the moiety X.

[0373] 24. The compound according to any one of items 1 to 23 having a structure selected from the group consisting of wherein

[0374] Si and S2 are independently of each other a spacer group;

[0375] Z is the cleavable linker moiety; and

[0376] A is the affinity moiety.

[0377] 25. The compound according to any one of items 1 to 23 having a structure selected from the group consisting of

[0378] wherein

[0379] Si and S2 are independently of each other a spacer group;

[0380] Z is the cleavable linker moiety comprising a functional group having a formula selected from the group consisting of: wherein Ri is selected from H or CH3; and

[0381] A is the affinity moiety comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s).

[0382] 26. The compound according to any one of items 1 to 23 having a structure according to formula wherein

[0383] Si and S2 are independently of each other a spacer group;

[0384] Z is the cleavable linker moiety; and

[0385] A is the affinity moiety. 27. The compound according to any one of items 1 to 23 having a structure according to formula wherein

[0386] Si and S2 are independently of each other a spacer group; Z is the cleavable linker moiety comprising a functional group having a formula selected from the group consisting of: wherein R1 is selected from H or CH3; and

[0387] A is the affinity moiety comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s).

[0388] 28. The compound according to any one of items 1-27 having a structure selected from the group consisting of

[0389] wherein

[0390] Si and S2 are independently of each other a spacer group;

[0391] X is the moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0392] A is the affinity moiety, and wherein R1 is selected from H or CHs.29. The compound according to any one of items 1-28 having a structure selected from the group consisting of wherein

[0393] Si and S2 are independently of each other a spacer group;

[0394] X is the moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X is_selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA); and

[0395] A is the affinity moiety_comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s), and wherein Ri is selected from H or CH3.

[0396] 30. The compound according to any one of items 1-29 having a structure selected from the group consisting of wherein

[0397] Si and S2 are independently of each other a spacer group;

[0398] X is the moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine; and

[0399] A is the affinity moiety, and wherein R1 is selected from H or CH3.

[0400] 31. The compound according to any one of items 1-29 having a structure selected from the group consisting of

[0401] wherein

[0402] Si and S2 are independently of each other a spacer group;

[0403] X is the moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X is.selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1 -methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA); and

[0404] A is the affinity moiety.comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s), and wherein R1 is selected from H or CH3.

[0405] 32. The compound according to any one of items 1 to 31 having a structure selected from the group consisting of wherein X is the moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine;

[0406] Si and S2 are independently of each other a spacer group;

[0407] Z is the cleavable linker moiety; and wherein

[0408] R2 is selected from H, CH3, or (CH2)2-COOH.

[0409] 33. The compound according to any one of items 1 to 32 having a structure selected from the group consisting of wherein

[0410] X is the moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X is.selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA);

[0411] Si and S2 are independently of each other a spacer group;

[0412] Z is the cleavable linker moiety comprising a functional group having a formula selected from the group consisting of: wherein R1 is selected from H or CH3; and wherein

[0413] R2 is selected from H, CH3, or (CH2)2-COOH.

[0414] 34. The compound according to any one of the items 1-5 having a structure according to the formula

[0415]

[0416] 35. The compound according to any one of the items 1 -5 having a structure according to the formula 36. The compound according to any one of the items 1 -5 having a structure according to the formula

[0417] The compound according to any one of the items 1 -5 having a structure according to the formula

[0418]

[0419] 38. Use of the compound of any one of items 1 to 37 for the isolation of one or more protein(s).

[0420] 39. Use of the compound of any one of items 1 to 37 for the isolation and selective enrichment of one or more protein(s).

[0421] 40. Use of the compound of any one of items 1 to 37 for the isolation, selective enrichment and identification of one or more protein(s).

[0422] 41 . Use of the compound of any one of items 1 to 37 for the isolation, selective enrichment, identification and quantification of one or more protein(s).

[0423] 42. Use according to any one of items 38-41 , comprising:

[0424] (i) subjecting a sample comprising the one or more protein(s) to a chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain comprised in the one or more protein(s), to obtain one or more oxidized protein(s);

[0425] (ii) reacting the one or more oxidized protein(s) with a compound of Formula I:

[0426] X-S1-Z-S2-A wherein

[0427] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine;

[0428] Si and S2 are independently of each other a spacer group;

[0429] Z is a cleavable linker moiety; and

[0430] A is an affinity moiety, wherein the moiety X reacts with the one or more carbonyl functional group(s), the one or more hydroperoxide functional group(s) and / or the one or more endoperoxide functional group(s) to form one or more labelled protein(s);

[0431] (iii) isolating and identifying the one or more labelled protein(s).

[0432] 43. A method for the isolation and identification of one or more protein(s), comprising the steps of:

[0433] (i) subjecting a sample comprising the one or more protein(s) to a chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain comprised in the one or more protein(s), to obtain one or more oxidized protein(s);

[0434] (ii) reacting the one or more oxidized protein(s) with a compound of Formula I:

[0435] X-S1-Z-S2-A wherein

[0436] X is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine;

[0437] Si and S2 are independently of each other a spacer group;

[0438] Z is a cleavable linker moiety; and

[0439] A is an affinity moiety, wherein the moiety X reacts with the one or more carbonyl functional group(s), the one or more hydroperoxide functional group(s) and / or the one or more endoperoxide functional group(s) to form one or more labelled protein(s);

[0440] (iii) isolating and identifying the one or more labelled protein(s).

[0441] 44. The use according to item 42 or the method according to item 43, wherein the chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain comprises reacting the one or more protein(s) with singlet oxygen (1O2).

[0442] 45. The use according to any one of items 42 or 44 , or the method according to any one of items 43 or 44, wherein the singlet oxygen is generated by exposing a singlet oxygen generator (SOG) to light. 46. The use or the method according to item 45, wherein the SOG is selected from the group consisting of thiorhodamine, a phenothiazinium dye (e.g., methylene blue, Nile blue or toluidine blue), a cyanine, a transition metal complex (e.g., bipyridine, bipyrazine or 2,2'- bipyrimidine complexes of Ru(ll), Os(ll), or Ir(lll)), a chalcogenopyrylium dye and derivatives thereof.

[0443] 47. The use or the method according to item 45 or item 46, wherein the SOG is covalently coupled to an antibody, a small molecule, a peptide, a peptidomimetic, an aptamer or a viral particle.

[0444] 48. The use or the method according to item 47, wherein the antibody, the small molecule, the peptide, the peptidomimetic, the aptamer or the viral particle is capable of specifically binding to the one or more protein(s) or to a target that is in close proximity to or in physical contact with the one or more protein(s).

[0445] 49. The use according to any one of items 42 or 44-48, or the method according to any one of items 43 to 48, wherein the isolation and identification of one or more protein(s) comprises:

[0446] (a) enriching the one or more labelled protein(s), wherein the one or more labelled protein(s) are contacted with a solid support and wherein the affinity moiety A specifically associates with or specifically reacts with a functional group attached or linked to the solid support to form one or more bond(s), optionally wherein the solid support is subjected to one or more washing steps after formation of the one or more covalent bond(s);

[0447] (b) releasing the one or more labelled protein(s) from the solid support, preferably by exposure to one or more oxidizing agent(s);

[0448] (c) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s); and

[0449] (d) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS).

[0450] 50. The use according to any one of items 42 or 44-48, or the method according to any one of items 43 to 48, wherein the isolation and identification of one or more protein(s) comprises:

[0451] (a) enriching the one or more labelled protein(s), wherein the one or more labelled protein(s) are contacted with a solid support and wherein the affinity moiety A specifically associates with or specifically reacts with a functional group attached or linked to the solid support to form one or more bond(s), optionally wherein the solid support is subjected to one or more washing steps after formation of the one or more covalent bond(s);

[0452] (b) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s), optionally wherein the solid support is subjected to one or more washing steps after the fragmenting of the one or more labelled protein(s);

[0453] (c) releasing one or more peptide fragments from the solid support, preferably by exposure to one or more oxidizing agent(s); and

[0454] (d) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS).

[0455] 51 . The use according to any one of items 42 or 44-48, or the method according to any one of items 43 to 48, wherein the isolation and identification of one or more protein(s) comprises:

[0456] (a) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s);

[0457] (b) enriching one or more peptide fragments, wherein the one or more peptide fragments are contacted with a solid support and wherein the affinity moiety A specifically associates with or specifically reacts with a functional group attached or linked to the solid support to form one or more bond(s), optionally wherein the solid support is subjected to one or more washing steps after formation of the one or more covalent bond(s);

[0458] (c) releasing the one or more peptide fragments from the solid support, preferably by exposure to one or more oxidizing agent(s);

[0459] (d) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS).

[0460] 52. The use according to any one of items 42 or 44-49, or the method according to any one of items 43 to 48, wherein the isolation and identification of one or more protein(s) comprises:

[0461] (a) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s);

[0462] (c) optionally exposing the one or more peptide fragments to one or more oxidizing agent(s); (d) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS).

[0463] 53. The use or the method according to any one of items 49 to 52, wherein the affinity moiety A specifically reacts with a functional group attached or linked to the solid support to form one or more covalent bond(s) through a cycloaddition reaction.

[0464] 54. The use according to any one of items 42 or 44-53, or the method according to any one of items 43-53, wherein the identification of one or more individual labelled protein(s) and / or peptide fragments thereof is by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS), wherein the liquid-chromatography-mass spectrometry (LC-MS) comprises tandem mass spectrometry (MS / MS).

[0465] 55. The use according to any one of items 42 or 44-54, or the method according to any one of items 43-54, wherein the one or more protein(s) is located on a cell surface or within a cell.

[0466] 56. The use according to any one of items 42 or 44-55, or the method according to any one of items 43-55, wherein the one or more protein(s) is located in or on the plasma membrane or cell wall or in the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes, cytoplasm, vacuoles, cytoskeleton or chloroplasts.

[0467] 57. The use or the method according to items 55 or 56, wherein the cell is an animal cell, bacterial cell, yeast cell, insect cell or plant cell, preferably wherein the cell is an animal cell, more preferably wherein the animal cell is a mammalian cell.

[0468] 58. The use or the method according to item 57, wherein the mammalian cell is a human cell, a mouse cell, a hamster cell, a porcine cell, a rabbit cell, a dog cell, a cat cell, a rat cell or a non-human primate cell, preferably a human cell.

[0469] 59. The use according to any one of items 38-42 or 44-58 , or the method according to any one of items 43 to 58, wherein the one or more protein(s) is a membrane protein, preferably a membrane protein present on the cell surface.

[0470] 60. The use according to any one of items 38-42 or 44-59, or the method according to any one of items 43 to 59, wherein the use or the method comprises determining whether the one or more protein(s) is in close proximity to or in physical contact with a particular target protein or protein of interest.

[0471] 61 . The use according to any one of items 42 or 44-60, or the method according to any one of items 43 to 60, wherein the compound of Formula I is a compound according to any one of items 1 to 37. 62. A protein or peptide comprising at least one amino acid side chain selected from the group consisting of

[0472] 63. A protein or peptide produced by the method of any one of claims 43 to 62, comprising at least one amino acid side chain selected from the group consisting of

[0473]

[0474] , and / or from the group consisting of

[0475] Examples

[0476] Example 1 : Synthesis of HIT-taq (cmTaq comprising a hydrazide functional group)

[0477] Synthesis was carried out following the steps described below.

[0478] Step A:

[0479] Compound 1 (2.0 g, 13.60 mmol, 1.0 eq.), zinc trifluoromethanesulfonate (2.47 g, 6.80 mmol, 0.5 eq.), hydrazine monohydrate (17 mL, 50 eq.) and acetonitrile (7.1 mL, 10.0 eq.) were mixed under nitrogen atmosphere and stirred at 60°C for 13 h. After such time, the reaction mixture was cooled down to room temperature, 5 mL of an aqueous solution of sodium nitrite (1 .88 g, 271 .9 mmol, 20.0 eq.) was slowly added to the mixture. Afterward, 1 M HCI solution was slowly added until pH = 3. The mixture was extracted with EtOAc (3x150 mL) and the combined organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Column chromatography of the residue on silica gel with DCM / MeOH / AcOH (100:1 :0.1 , v / v / v) gave compound 2 (2.35 g, 80%).

[0480] Step B:

[0481] Fluoren-9-ylmethylchloroformate (57.8 g, 223.3 mmol, 1.1 eq.), in 1 ,4-dioxane (500 mL), was added to compound 3 (50.0 g, 203.0 mmol, 1.0 eq.) and NaHCCh, (85.3 g, 1.02 mol, 5 eq.) in H2O (500 mL) and the mixture was stirred for 16 h at r.t. before being partitioned between DCM (1000 mL) and aqueous HCI (10 M, 150 mL). The organic phase was separated, washed with water and dried over sodium sulfate, filtered, and concentrated under reduced pressure to give compound 4 (85.5 g, 90%). Step C:

[0482] DIPEA (63.65 g, 492.6 mmol, 2.7 eq.) was added to a solution of compound 4 (85.5 g, 182.4 mmol, 1 eq.), tert-butyl hydrazinecarboxylate (26.3 g, 200.7 mmol, 1.1 eq.) and HATU (76.3 g, 200.7 mol, 1.1 eq.) in DMF (500 mL) and the mixture was stirred at room temperature for 16 h. After completion of the reaction, the mixture was poured into cold water and then extracted with EtOAc (1500 mL). Combined organic phases were washed with aqueous NaHSCU (1 M, 2x300 mL), NaHCCh solution (saturated, 3x100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give compound 5 (63.8 g, 60%). Step D:

[0483] Diethylamine (300 mL) was added to a solution of compound 5 (63.8 g, 109.9 mmol) in methanol (500 mL) and the mixture was stirred at room temperature overnight. The reaction mixture was diluted with EtOAc (1000 mL) and extracted with aqueous NaHSO4 (1 M, 2x400 mL). The combined water layers were basified with aqueous NaOH (2 M) to pH 12, followed by extraction with EtOAc (2x300 mL). The combined organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give compound 6 (15.5 g, 39%). Step E:

[0484] To a solution of compound 6 (15.5 g, 43.0 mmol, 1 eq.) in isopropyl alcohol (200 mL) a solution of 2-{[(2S)-oxiran-2-yl]methyl}-2,3-dihydro-1 H-isoindole-1 ,3-dione (7.94 g, 39.1 mmol, 1 eq.) in isopropyl alcohol (200 mL) was added dropwise at 0°C. The mixture was stirred for 1 hr at 0°C and then at 85°C for 10 hr. After completion of the reaction, the mixture was evaporated in vacuo at 45°C to give compound 7 (21 .5 g, 97%).

[0485] Step F: B0C2O (9.16 g, 41.96 mmol, 1.1 eq.) was added dropwise to a solution of 7 (21.5 g, 38.14 mmol, 1.0 eq.) in methanol (250 mL). The mixture was stirred for 10 hr at 20°C to give a solution of 8 which was used in the next step without isolation.

[0486] Step G:

[0487]

[0488] Hydrazine monohydrate (9.55 mL, 190.58 mmol, 5.0 eq.) was added to the solution of compound 8 from step F. The mixture was stirred for 10 hr at 65°C. After completion of the reaction, the mixture was filtered and the filtrate was evaporated in vacuo at 60°C to give compound 9 (10.4 g, 51%). Step H:

[0489] Compound 2 (2.35 g, 10.87 mmol, 1 eq.) was dissolved in THF (80 mL). DIPEA (2.8 g, 21.74 mmol, 2.0 eq.) was added dropwise at 0°C followed by EDCI hydrochloride (2.5 g, 13.04 mmol, 1.2 eq.) and 1 H-1 ,2,3-benzotriazol-1-ol (1.76 g, 13.04 mmol, 1.2 eq.). The mixture was stirred for 15 min at 0°C, then compound 9 (5.8 g, 10.87 mmol, 1.0 eq.) was added and the reaction was left for overnight at r.t. After completion of the reaction, the mixture was evaporated in vacuo at 45°C. The residue was dissolved in EtOAc (100 mL) and the organic phase was washed with NaHSCU water solution (1 M, 2x30 mL), NaHCOs water solution (saturated, 3x30 mL). The organic phase was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (hexane / MTBE = 9 / 1 to 0 / 1) to give compound 10 (1.7 g, 21%).

[0490] Step I:

[0491]

[0492] HIT-tag

[0493] Compound 10 (1.7 g, 2.32 mmol) was dissolved in 1 ,4-dioxane (5 mL). Hydrogen chloride 4.0 M in 1 ,4-dioxane (10 mL) was added. The mixture was stirred for 10 hr at 20°C. The formed precipitate was filtered, washed with diethyl ether and dried in vacuo to give a compound of the invention (the HIT-tag) (1 .0 g, 80%) as trihydrochloride.

[0494] Example 2: Synthesis of a cm Tag comprising a hydrazide functional group and PEG-3 spacer

[0495] Step A:

[0496] Oxalyl chloride (2.92 g, 23.16 mmol, 1 .96 mL, 2.0 equiv) was added dropwise to a stirred solution of 4-(6-methyl-1 ,2,4,5-tetrazin-3-yl)benzoic acid (2.5 g, 11.58 mmol) and DMF (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 5 hours. The resulting solution was concentrated under reduced pressure. The residue was dissolved in DCM (25 mL). The obtained solution was added dropwise to a mixture of tert-butyl 3-[2-[2-(2-aminoethoxy) ethoxy]ethoxy]propanoate (3.37 g, 12.16 mmol) and triethylamine (1.76 g, 17.37 mmol) in DCM (30 mL) at 0 °C. The reaction mixture was stirred at room temperature for 5 hours. After completion of the reaction (LCMS control), the mixture was diluted with water (20 mL). The organic layer was washed with 1 M NaHSO4(20 mL), saturated aqueous NaHCO3(20 mL), and brine (20 mL), dried over Na2SO4, and filtered. The filtrate was evaporated under reduced pressure to afford tert-butyl 3- [2-[2-[2-[4-(6-methyl-1 ,2,4,5-tetrazin-3-yl)phenyl]formamidoethoxy]ethoxy]ethoxy]propanoate (4.0 g, 8.41 mmol, 72.6% yield), which was used in the next step without further purification.

[0497] Step B:

[0498] To a solution of tert-butyl 3-[2-[2-[2-[4-(6-methyl-1 ,2,4,5-tetrazin-3-yl)phenyl]formamidoethoxy] ethoxy]ethoxy]propanoate (4.0 g, 8.41 mmol) in DCM (20 mL), 4 M HCI in dioxane (40 mL) was added at 0 °C. The reaction mixture was stirred at room temperature for 5 hours and concentrated under reduced pressure. The residue was triturated with Et2O, and the precipitate was filtered and dried under reduced pressure to afford 3-[2-[2-[2-[4-(6-methyl-1 ,2,4,5-tetrazin-3-yl)phenyl] formamidoethoxy]ethoxy]ethoxy]propanoic acid (2.5 g, 5.96 mmol, 70.9% yield).

[0499] Step C:

[0500] 4 5

[0501] Ethylbis(isopropyl)amine (41.37 g, 320.35 mmol, 55.76 mL, 2.5 equiv) was added dropwise to a solution of (2S)-2-[(tert-butoxy)carbonyl]amino-6-([(9H-fluoren-9-yl)methoxy]carbonylamino) hexanoic acid (60.0 g, 128.14 mmol), (tert-butoxy)carbohydrazide (20.31 g, 153.77 mmol), and [(dimethylamino)(3H-[1 ,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]dimethylazanium hexafluorophosphate (58.45 g, 153.77 mmol) in DMF (600 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. After reaction completion (LCMS control), the mixture was poured into ice water (1200 mL), and the resulting precipitate was filtered and washed with water (2 x 500 mL). The precipitate was dissolved in EtOAc (1500 mL), dried over Na2SO4, and concentrated under reduced pressure to afford tert-butyl N-[(1S)-1-N’-[(tert-butoxy)carbonyl] hydrazinecarbonyl]-5-([(9H-fluoren-9-yl)methoxy]carbonylamino)pentylcarbamate (70.0 g, 120.13 mmol, 93.8% yield), which was used in the next step without further purification.

[0502] Step D:

[0503] 5 6

[0504] Diethylamine (4.2 mol, 439.02 mL, 35.0 equiv) was added dropwise to a solution of tert-butyl N- [(1S)-1-N’-[(tert-butoxy)carbonyl]hydrazinecarbonyl]-5-([(9H-fluoren-9-yl)methoxy]carbonylamino) pentylcarbamate (70.0 g, 120.13 mmol) in methanol (700 mL) at 0 °C. The reaction mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure, and the residue was purified by flash chromatography to afford tert-butyl N-[(2S)-6-amino-1-([(tert- butoxy)carbonyl]amino)amino]-1-oxohexan-2-ylcarbamate (15.0 g, 41.61 mmol, 34.6% yield).

[0505] Step E:

[0506] A mixture of tert-butyl N-[(2S)-6-amino-1-([(tert-butoxy)carbonyl]amino)amino]-1-oxohexan-2- ylcarbamate (15.0 g, 41.64 mmol) and 2-[(2S)-oxiran-2-yl]methyl-2,3-dihydro-1 H-isoindole-1 ,3-dione (6.76 g, 33.31 mmol) in iPrOH (200 mL) was refluxed for 16 hours. After reaction completion (LCMS control), the mixture was concentrated under reduced pressure to afford crude tert-butyl N-[(1S)-1- N’-[(tert-butoxy)carbonyl]hydrazinecarbonyl]-5-[(2R)-3-(1 ,3-dioxoisoindolin-2-yl)-2- hydroxypropyl]aminopentylcarbamate (20.0 g, 40% purity, 34.1% yield), which was used in the next step without further purification.

[0507] Step F:

[0508]

[0509] To a stirred solution of crude tert-butyl N-[(1 S)-1-N’-[(tert-butoxy)carbonyl]hydrazinecarbonyl]-5- [(2R)-3-(1 ,3-dioxoisoindolin-2-yl)-2-hydroxypropyl]aminopentylcarbamate (20.05 g, 40.0% purity, 14.23 mmol) in DCM (200 mL) were added di-tert-butyl dicarbonate (3.1 g, 14.23 mmol, 3.27 mL, 1 .0 equiv) and triethylamine (2.16 g, 21 .34 mmol, 2.97 mL, 1 .5 equiv). The reaction mixture was stirred at room temperature for 16 hours. The mixture was diluted with water (100 mL), and the aqueous layer was extracted with DCM (100 mL). The combined organic layers were washed with 0.5 M NaHSO4, brine, dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (DCM:MTBE, 1 :1) to afford tert-butyl N-[(5S)-5-[(tert-butoxy)carbonyl]amino]-5-N’-[(tert-butoxy)carbonyl] hydrazinecarbonylpentyl]-N- [(2S)-3-(1 ,3-dioxoisoindolin-2-yl)-2-hydroxypropyl]carbamate (6.0 g, 9.04 mmol, 63.5% yield).

[0510] Step G:

[0511] A solution of tert-butyl N-[(5S)-5-[(tert-butoxy)carbonyl]amino]-5-N’-[(tert-butoxy)carbonyl] hydrazinecarbonylpentyl]-N-[(2S)-3-(1 ,3-dioxoisoindolin-2-yl)-2-hydroxypropyl]carbamate (6.0 g, 9.05 mmol) and hydrazine hydrate (1.13 g, 22.61 mmol) in MeOH (60 mL) was stirred at 60 °C for 16 hours. The mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure. The residue was diluted with DCM (100 mL), filtered again, and the filtrate was washed with water (2 x 20 mL). The organic phase was dried over Na2SO4and concentrated to give tert-butyl N-[(2S)-3-amino-2-hydroxypropyl]-N-[(5S)-5-[(tert-butoxy)carbonyl]amino]-6-([(tert- butoxy)carbonyl]amino)amino-6-oxohexylcarbamate (3.8 g, 7.12 mmol, 78.7% yield).

[0512] Step H:

[0513] Oxalyl chloride (1.53 g, 12.18 mmol) was added dropwise to a stirred solution of 3-[2-[2-[2-[4-(6- methyl-1 ,2,4,5-tetrazin-3-yl)phenyl]formamidoethoxy]ethoxy]ethoxy]propanoic acid (2.55 g, 6.09 mmol) and DMF (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 5 hours. The solution was concentrated under reduced pressure. The residue was dissolved in DCM (25 mL). The resulting solution was added dropwise to a mixture of tert-butyl N- [(2S)-3-amino-2-hydroxypropyl]-N-[(5S)-5-[(tert-butoxy)carbonyl]amino]-6-([(tert-butoxy) carbonyl]amino)amino-6-oxohexylcarbamate (3.41 g, 6.39 mmol) and ethylbis(isopropyl)amine (1.18 g, 9.13 mmol, 1 .59 mL, 1 .5 equiv) in THF (70 mL) at 0 °C. The mixture was stirred at room temperature for 5 hours. After reaction completion (LCMS control), the mixture was diluted with DCM (150 mL) and water (50 mL). The organic layer was washed with 1 M NaHSO4(40 mL), saturated aqueous NaHCO3(40 mL), and brine (40 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (MTBE:DCM, 4:1) to afford tert-butyl N-[(5S)-5-[(tert-butoxy)carbonyl]amino]-5-N’-[(tert- butoxy)carbonyl]hydrazinecarbonylpentyl]-N-[(2S)-2-hydroxy-3-[3-[2-[2-[2-[4-(6-methyl-1 ,2,4,5- tetrazin-3-yl)phenyl]formamidoethoxy]ethoxy]ethoxy]propanamido]propyl]carbamate (4.1 g, 4.38 mmol, 72% yield).

[0514] Step I:

[0515] cmTag with a hydrazide functional group and PEG-3 spacer

[0516] Tert-butyl N-[(5S)-5-[(tert-butoxy)carbonyl]amino]-5-N’-[(tert-butoxy)carbonyl]hydrazinecarbonyl pentyl]-N-[(2S)-2-hydroxy-3-[3-[2-[2-[2-[4-(6-methyl-1 ,2,4,5-tetrazin-3-yl)phenyl]formamidoethoxy] ethoxy]ethoxy]propanamido]propyl]carbamate (2.5 g, 2.67 mmol) was dissolved in DCM (30 mL). Then, 4 M HCI in dioxane (40 mL) was added dropwise at 0 °C. The mixture was stirred at room temperature for 16 hours. The resulting precipitate was collected by filtration, washed with MTBE, and purified by HPLC (0-25% H20 / ACN / 0.003% HCI; flow: 30 mL / min; column: Chromatorex 18 SMB100-5T 100x19 mm, 5 pm) to afford N-[(2R)-3-[(5S)-5-amino-5-(hydrazinecarbonyl)pentyl] amino-2-hydroxypropyl]-3-[2-[2-[2-[4-(6-methyl-1 ,2,4,5-tetrazin-3-yl)phenyl]formamidoethoxy] ethoxy]ethoxy]propanamide trihydrochloride (268.8 mg, 361 .24 pmol, 13.5% yield) as a pink solid.

[0517] Example 3: Synthesis of a cmTag comprising an aniline moiety

[0518] Step A: To a stirred solution of compound 1 (80.0 g, 170.86 mmol) and tert-butyl N-(3-aminophenyl) carbamate (39.12 g, 187.94 mmol) in anhydrous DMF (800 mL) at 0 °C, DIPEA (59.58 g, 461 .32 mmol) and HATU (71 .44 g, 187.94 mmol) were added slowly. The mixture was stirred at room temperature for 2 h and then quenched with saturated aqueous sodium hydrogen sulfate (400 mL). The aqueous layer was extracted with ethyl acetate (3 x 400 mL), and the combined organic layers were washed with brine (5 x 200 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 2 (92 g, 72% purity, 100.55 mmol, 58.8% yield).

[0519] Step B:

[0520] 2 3

[0521] To a stirred solution of compound 2 (92.0 g, 72% purity, 100.55 mmol) in anhydrous MeOH (1.5 L) at 0 °C, diethylamine (367.45 g, 5.03 mol, 524.93 mL, 50.0 equiv) was added slowly. The mixture was stirred at room temperature overnight, then concentrated under reduced pressure. The residue was purified by flash column chromatography to afford compound 3 (22.0 g, 90.0% purity, 50.4 mmol, 50.1% yield) as a white solid.

[0522] Step C:

[0523] To a stirred solution of compound 3 (22.01 g, 50.46 mmol) in anhydrous MeOH (250 mL) at 0 °C, benzaldehyde (5.35 g, 50.46 mmol, 5.12 mL, 1.0 equiv) was added slowly. The mixture was stirred at room temperature for 20 h, then concentrated under reduced pressure to afford compound 4 (25.0 g, 47.65 mmol, 94.4% yield).

[0524] Step D:

[0525]

[0526] To a stirred solution of compound 4 (25.0 g, 47.65 mmol) in anhydrous MeOH (250 mL) at 0 °C, sodium borohydride (1.81 g, 47.65 mmol) was added in small portions. The mixture was stirred at room temperature for 2 h, then quenched with saturated aqueous ammonium chloride (100 mL). The aqueous layer was extracted with ethyl acetate (3 x 250 mL), and the combined organic layers were washed with water (200 mL), dried over sodium sulfate, and concentrated under reduced pressure to afford compound 5 (24.0 g, 45.57 mmol, 95.6% yield).

[0527] Step E: To a stirred solution of compound 5 (24.0 g, 45.57 mmol) and compound 6 (9.25 g, 45.57 mmol) in anhydrous iPrOH (1 .2 L) at 0 °C, DIPEA (29.43 g, 227.85 mmol, 39.66 mL, 5.0 equiv) was added slowly. The mixture was stirred at 110 °C for 2 days, then concentrated under reduced pressure. The residue was purified by flash column chromatography to afford compound 7 (18.0 g, 24.66 mmol, 54.1% yield) as a white solid. Step F:

[0528]

[0529] To a stirred solution of compound 7 (17.99 g, 24.65 mmol) in MeOH (200 mL), di-tert-butyl dicarbonate (8.07 g, 36.98 mmol, 8.5 mL, 1 .5 equiv) and Pd / C (10%, 1 .8 g) were added under a nitrogen atmosphere. The mixture was stirred under hydrogen atmosphere (balloon) at room temperature for 16 h. An additional portion of Pd / C (10%) was added, and stirring was continued under hydrogen for 2 more days. The reaction mixture was filtered, washed with MeOH (3 x 100 mL), and concentrated under reduced pressure to afford compound 8 (15.0 g, 20.27 mmol, 82.2% yield).

[0530] Step G: Hydrazine hydrate (2.0 mL, 62.5 mmol) was added dropwise to a solution of compound 8 (15.0 g, 20.27 mmol) in MeOH (300 mL). The reaction mixture was heated to 60 °C and stirred overnight. Water (100 mL) was added, and the mixture was extracted with DCM (3 x 200 mL). The combined organic layers were dried over sodium sulfate, concentrated, and purified by flash column chromatography to afford compound 9 (8.0 g, 13.12 mmol, 64.7% yield).

[0531] Step H:

[0532] Compound 9 (7.99 g, 13.1 mmol) was dissolved in DCM (160 mL), and TEA (1.59 g, 15.72 mmol, 2.19 mL, 1.2 equiv) was added at 0 °C. After 15 min of stirring at 0 °C, 4-(6-methyl-1 ,2,4,5-tetrazin- 3-yl)benzoyl chloride (3.07 g, 13.1 mmol) was added. The mixture was stirred at 20 °C for 10 h. Water (50 mL) was added, and the mixture was extracted with DCM (3 x 100 mL). The organic layers were dried over sodium sulfate, concentrated, and purified by flash chromatography to afford compound 10 (3.4 g, 4.21 mmol, 32.1 % yield).

[0533] Step I: cmTag with an aniline moiety

[0534] Compound 10 (3.4 g, 4.21 mmol) was dissolved in DCM (35 mL), and 4 M hydrogen chloride in dioxane (35 mL) was added. The mixture was stirred at 20 °C for 18 h. The resulting solid was filtered, washed with diethyl ether, and dried under reduced pressure to yield the cmTag of the invention comprising an aniline moiety (1.0 g, 1.62 mmol, 38.5% yield). Example 4: Synthesis of a cmTaq comprising an alkoxyamine moiety

[0535] Step A:

[0536] 1 2

[0537] To a solution of (2S)-2-[(tert-butoxy)carbonyl]amino-6-([(9H-fluoren-9-yl)methoxy]carbonylamino) hexanoic acid (69.99 g, 149.49 mmol) in DMF (700 mL) were added [(dimethylamino)(3H- [1 ,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]dimethylazanium hexafluorophosphate (62.5 g, 164.44 mmol) and ethylbis(propan-2-yl)amine (48.27 g, 373.72 mmol, 65.05 mL, 2.5 equiv). The mixture was stirred at room temperature for 15 minutes, after which tert-butyl N-(3- aminopropoxy)carbamate (28.42 g, 149.49 mmol) was added in one portion. The reaction was stirred for 16 hours at room temperature, then poured into ice water. The precipitate was collected by filtration, washed with water (x3), and dried under vacuum to yield tert-butyl N-[(1S)-1-[3-([(tert- butoxy)carbonyl]aminooxy)propyl]carbamoyl-5-([(9H-fluoren-9-yl)methoxy]carbonylamino) pentyl]carbamate (89.0 g, 138.9 mmol, 92.9% yield) as a white powder, used in the next step without further purification.

[0538] Step B:

[0539] 2 3

[0540] To a solution of the product from Step A (89.0 g, 138.9 mmol) in methanol (1000 mL), diethylamine (507.59 g, 6.94 mol, 725.13 mL, 50.0 equiv) was added. The mixture was stirred at room temperature for 16 hours, concentrated, and purified by flash chromatography (DCM:MeOH, 100:1 to 2:1). Tert-butyl N-[(1S)-5-amino-1-[3-([(tert-butoxy)carbonyl]aminooxy)propyl]carbamoylpentyl] carbamate (32.0 g, 76.46 mmol, 55% yield) was obtained as a light-yellow gum.

[0541] Step C:

[0542]

[0543] To a solution of the product from Step B (16.0 g, 38.23 mmol) in methanol (300 mL), 4- methoxybenzaldehyde (5.2 g, 38.23 mmol) was added. The mixture was stirred for 16 hours at room temperature, then sodium borohydride (1.6 g, 42.05 mmol) was added portionwise over 15 minutes. Stirring continued for 16 hours, after which the reaction was quenched with saturated NH4CI and concentrated. The residue was diluted with EtOAc (500 mL) and water (500 mL). The organic phase was separated, dried over sodium sulfate, and evaporated to give tert-butyl N-[(1 S)-1-[3-([(tert- butoxy)carbonyl]aminooxy)propyl]carbamoyl-5-[(4-methoxyphenyl)methyl]aminopentyl]carbamate (16.0 g, 29.7 mmol, 77.7% yield) as an orange gum. Step D:

[0544] To a solution of the product from Step C (16.0 g, 29.7 mmol) in i-PrOH (700 mL), 2-[(2S)-oxiran-2- yl]methyl-2,3-dihydro-1 H-isoindole-1 ,3-dione (6.03 g, 29.7 mmol) was added. The mixture was heated at 80 °C for 16 hours, then cooled, concentrated, and purified by flash chromatography (DCM:MeOH, 100:1 to 3:1). Tert-butyl N-[(1 S)-1-[3-([(tert-butoxy)carbonyl]aminooxy)propyl] carbamoyl-5-[(2R)-3-(1 ,3-dioxoisoindolin-2-yl)-2-hydroxypropyl][(4-methoxyphenyl) methyl]aminopentyl]carbamate (15.5 g, 20.89 mmol, 70.3% yield) was obtained as a yellow gum.

[0545] Step E:

[0546]

[0547] To a solution of the product from Step D (15.5 g, 20.89 mmol) in MeOH (300 mL), 10% Pd / C (1.5 g), di-tert-butyl dicarbonate (6.84 g, 31 .34 mmol, 1.5 equiv), and triethylamine (5.28 g, 52.23 mmol) were added. The mixture was degassed and stirred under hydrogen at room temperature for 3 days. After completion, the catalyst was removed by filtration, and the filtrate was concentrated and purified by flash chromatography (DCM:MeOH, 100:1 to 3:1) to yield tert-butyl N-[(5S)-5-[(tert- butoxy)carbonyl]amino-5-[3-([(tert-butoxy)carbonyl]aminooxy)propyl]carbamoylpentyl]-N-[(2S)-3- (1 ,3-dioxoisoindolin-2-yl)-2-hydroxypropyl]carbamate (4.5 g, 6.23 mmol, 29.8% yield) as a colorless gum. Step F:

[0548] 7 8

[0549] Hydrazine hydrate (468.0 mg, 9.35 mmol, 1.5 equiv) was added to a solution of the product from Step E (4.5 g, 6.23 mmol) in MeOH (100 mL). The reaction mixture was heated at 60 °C for 16 hours. The resulting precipitate was filtered off, and the mixture was concentrated. The residue was extracted with EtOAc (100 mL) and water (100 mL), washed with water (x1), dried over sodium sulfate, and evaporated to give tert-butyl N-[(2S)-3-amino-2-hydroxypropyl]-N-[(5S)-5-[(tert- butoxy)carbonyl]amino-5-[3-([(tert-butoxy)carbonyl]aminooxy)propyl]carbamoylpentyl]carbamate (2.8 g, 4.73 mmol, 75.9% yield).

[0550] Step G:

[0551] Oxalyl chloride (716.06 mg, 5.69 mmol, 480.0 pL, 1 .2 equiv) was added dropwise to a solution of 4- (6-methyl-1 ,2,4,5-tetrazin-3-yl)benzoic acid (1 .02 g, 4.74 mmol) in DCM (50 mL) at 0 °C. After addition of 2 drops of DMF, the mixture was stirred at room temperature for 3 hours, then concentrated and co-evaporated with toluene (x2). The resulting acid chloride in DCM (50 mL) was added dropwise to a solution of the product from Step F (2.8 g, 4.74 mmol) and triethylamine (718.74 mg, 7.11 mmol, 990.0 pL) in DCM (100 mL) at 0 °C. The mixture was stirred at room temperature for 16 hours and extracted twice each with NaHCO3and 1 M NaHSO4. The organic phase was dried over sodium sulfate and evaporated to afford tert-butyl N-[(2S)-3-amino-2- hydroxypropyl]-N-[(5S)-5-[(tert-butoxy)carbonyl]amino-5-[3-([(tert-butoxy)carbonyl] aminooxy)propyl]carbamoylpentyl]carbamate (2.4 g, 4.06 mmol, 85.6% yield) as a pink solid.

[0552] Step H: cmTag with an alkoxyamine moiety

[0553] Tert-butyl N-[(5S)-5-[(tert-butoxy)carbonyl]amino-5-[3-([(tert-butoxy)carbonyl]aminooxy) propyl]carbamoylpentyl]-N-[(2S)-2-hydroxy-3-[4-(6-methyl-1 ,2,4,5-tetrazin-3- yl)phenyl]formamidopropyl]carbamate (2.3 g, 2.91 mmol) was dissolved in DCM (100 mL), and 4 M dioxane HCI (40 mL) was added dropwise at 0 °C. The reaction was stirred at room temperature for 16 hours. The precipitate was filtered, washed with cold MeCN, and purified by HPLC to afford (2S)- 2-amino-N-[3-(aminooxy)propyl]-6-[(2R)-2-hydroxy-3-[4-(6-methyl-1 ,2,4,5-tetrazin-3- yl)phenyl]formamidopropyl]aminohexanamide trihydrochloride (600.0 mg, 1.0 mmol, 34.4% yield) as a pink solid.

[0554] Step I:

[0555] PhthN

[0556] To a refluxing solution of 2-(3-bromopropyl)-2,3-dihydro-1H-isoindole-1 ,3-dione (100.0 g, 374.55 mmol) and tert-butyl N-hydroxycarbamate (54.83 g, 412.0 mmol) in toluene (2000 mL), 2H,3H,4H,6H,7H,8H,9H,10H-pyrimido[1 ,2-a]azepine (62.68 g, 412.0 mmol, 61.57 mL, 1.1 equiv) was added dropwise. The mixture was stirred for 1 hour at reflux, then concentrated. The residue was redissolved in DCM (1000 mL) and washed 4x with 5% aqueous citric acid. The organic layer was dried over sodium sulfate and evaporated to yield tert-butyl N-[3-(1 ,3-dioxoisoindolin-2- yl)propoxy]carbamate (105.0 g, 327.78 mmol, 87.5% yield).

[0557] Step J:

[0558] Hydrazine hydrate (18.06 g, 360.85 mmol, 18.06 mL, 1.1 equiv) was added to a solution of tert-butyl N-[3-(1 ,3-dioxoisoindolin-2-yl)propoxy]carbamate (105.02 g, 328.05 mmol) in EtOH (1000 mL). The mixture was heated to 60 °C and stirred for 16 hours. The resulting precipitate was filtered off and the filtrate was evaporated. Tert-butyl N-[3-(1 ,3-dioxoisoindolin-2-yl)propoxy]carbamate (66.0 g, 206.03 mmol, 62.8% yield) was obtained and used directly in the next step without further purification.

[0559] Example 5: Synthesis of the biocvtin-isoseramox-hvdrazide (BIH) tag

[0560]

[0561] Intermediate 6 tert-butyl N-[(2S)-3-amino-2-hydroxypropyl]-N-[(5S)-5-[(tert-butoxy)carbonyl]amino-6- ([(tert-butoxy)carbonyl]aminoamino)-6-oxohexyl]carbamate was as described in Example 2. Step F:

[0562] To a mixture of 5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid (15.0 g, 61.45 mmol) and N,N'-dicyclohexylcarbodiimide (13.94 g, 67.6 mmol) in DMF (100 mL) was added N-hydroxysuccinimide (7.42 g, 64.53 mmol). The reaction mixture was stirred at room temperature for 16 hours. It was then filtered, and the filtrate was concentrated under reduced pressure. The residue was triturated with Et2O (100 mL). The resulting precipitate was collected by filtration, washed with Et2O (50 mL), and dried under vacuum to give 2,5-dioxopyrrolidin-1-yl 5- [(3aS,4S,6aR)-2-oxohexahydro-1 H-thieno[3,4-d]imidazol-4-yl]pentanoate (10.0 g, 29.29 mmol, 47.7% yield).

[0563] Step G:

[0564] To a solution of 2,5-dioxopyrrolidin-1-yl 5-[(3aS,4S,6aR)-2-oxohexahydro-1 H-thieno[3,4-d]imidazol- 4-yl]pentanoate (10.0 g, 29.32 mmol) and (2S)-6-amino-2-[(tert-butoxy)carbonyl]aminohexanoic acid (7.94 g, 32.25 mmol) in THF (100 mL) was added sodium bicarbonate (7.39 g, 87.95 mmol). The mixture was stirred at room temperature for 16 hours. It was then filtered, and the filtrate was evaporated under reduced pressure. The residue was diluted with water (100 mL) and acidified to pH 3 using 2 M HCI. The resulting precipitate was filtered, washed several times with water, and dried under vacuum to afford (2S)-6-[5-[(3aS,4S,6aR)-2-oxohexahydro-1 H-thieno[3,4-d]imidazol-4- yl]pentanamido]-2-[(tert-butoxy)carbonyl]aminohexanoic acid (5.0 g, 10.58 mmol, 36.1% yield).

[0565] Step H:

[0566] Ethylbis(propan-2-yl)amine (1 .45 g, 11 .25 mmol) was added dropwise to a mixture of (2S)-6-[5- [(3aS,4S,6aR)-2-oxohexahydro-1 H-thieno[3,4-d]imidazol-4-yl]pentanamido]-2-[(tert- butoxy)carbonyl]aminohexanoic acid (1.77 g, 3.75 mmol), tert-butyl N-[(2S)-3-amino-2- hydroxypropyl]-N-[(5S)-5-[(tert-butoxy)carbonyl]amino-6-([(tert-butoxy)carbonyl]aminoamino)-6- oxohexyl]carbamate (2.0 g, 3.75 mmol), and [(dimethylamino)(3H-[1 ,2,3]triazolo[4,5-b]pyridin-3- yloxy)methylidene]dimethylazanium; hexafluorophosphate (1.71 g, 4.5 mmol) in DMF (20 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. After confirming completion by LC-MS, the mixture was diluted with water (50 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with 1 M NaHSO4(20 mL), saturated aqueous NaHCO3(20 mL), and brine (20 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by flash chromatography (DCM:MeOH = 10:1) to yield tert-butyl N-[(2S)-3-[(2S)-6-[5-[(3aS,4S,6aR)-2- oxohexahydro-1 H-thieno[3,4-d]imidazol-4-yl]pentanamido]-2-[(tert-butoxy)carbonyl] aminohexanamido]-2-hydroxypropyl]-N-[(5S)-5-[(tert-butoxy)carbonyl]amino-5-N'-[(tert- butoxy)carbonyl]hydrazinecarbonylpentyl]carbamate (2.5 g, 2.53 mmol, 67.5% yield).

[0567] Step I:

[0568] Tert-butyl N-[(2S)-3-[(2S)-6-[5-[(3aS,4S,6aR)-2-oxohexahydro-1 H-thieno[3,4-d]imidazol-4- yl]pentanamido]-2-[(tert-butoxy)carbonyl]aminohexanamido]-2-hydroxypropyl]-N-[(5S)-5-[(tert- butoxy)carbonyl]amino-5-N'-[(tert-butoxy)carbonyl]hydrazinecarbonylpentyl]carbamate (2.5 g, 2.53 mmol) was dissolved in DCM (10 mL), and 4 M HCI in dioxane (20 mL) was added dropwise at 0 °C. The mixture was stirred at room temperature for 16 hours. The resulting precipitate was collected by filtration, washed with MTBE, and purified by HPLC (90-90-65-0%, 0-2-6-7 min, H20 / ACN / 0.003% HCI, flow: 30 mL / min; column: Uptisphere Strategy HILIC-HIA, 100 x 21.2 mm, 5 pm) to yield (2S)-6-[5-[(3aS,4S,6aR)-2-oxohexahydro-1 H-thieno[3,4-d]imidazol-4-yl]pentanamido]- 2-amino-N-[(2R)-3-[(5S)-5-amino-5-(hydrazinecarbonyl)pentyl]amino-2-hydroxypropyl]hexanamide tetrahydrochloride (738.4 mg, 1.01 mmol, 39.8% yield) as a white solid. The procedure was repeated several times.

[0569] Example 6: Enrichment and oxidative release of a spike-in protein

[0570] For SDS-PAGE (Fig. 1 B), the spike-in protein is covalently coupled to NHS-thiorhodamine SOG (Atto-Tec AD Thio12-31) by incubating 100 pg of transferrin (Sigma T4132-100 mg) with NHS-SOG in a coupling buffer (10 mM sodium bicarbonate in PBS, pH 8.3) at a 1 :5 molar ratio. The NHS- modified protein is purified using 7-kDa ZebaSpin Columns (Thermo 89882) as per manufacturer instructions, and eluted in D2O-based PBS. The protein-SOG construct is then illuminated for 15 minutes at 4°C using four Precision LED spotlights at 590 nm with a 190 mm working distance. A solution of 1 mM cleavable crosslinking reagent (HIT-tag provided in Example 1) and 50 mM 2- (Aminomethyl)imidazole dihydrochloride (AMI) are subsequently added, followed by a 2-hour incubation at room temperature (RT). Free crosslinking reagent is removed via 7-kDa ZebaSpin Columns per manufacturer’s instructions. The modified protein is then bound to pre-equilibrated TCO agarose (Vectorlabs CCT-1198) or Streptavidin (Thermo 53116) beads in PBS or in the presence of 1 mg unlabelled cell lysate for 1 hour at room temperature on an end-to-end rotator. Beads are washed in Mobicol spin columns with 8M urea in PBS (pH 7.4) and PBS to remove unbound proteins. For oxidative cleavage of the crosslinking reagent, the beads are treated with 2 mM sodium periodate in the dark for 15 minutes. Aliquots for SDS-PAGE analysis are collected throughout the enrichment process, heated to 99°C for 10 minutes in loading buffer (1x Laemmli buffer with beta-mercaptoethanol), and loaded onto NuPAGE 12-well gels (NP0322BOX). Electrophoresis is conducted at a constant voltage of 120 V for 2 hours, followed by silver staining (ThermoFisher, 24612) as per manufacturer instructions.

[0571] Example 7: Generation and MS analysis of highflyer peptides

[0572] A synthetic peptide containing one amino acid amenable for singlet oxygen modification (LLGHVEGLK, 0.5 mM) was mixed with 0.1 mM azide-thiorhodamine SOG (Atto-Tec AD Thio12- 105) in 10 mM MES / 50% acetonitrile buffer and illuminated for 10 minutes at 4°C using four Precision LED spotlights at 590 nm with a 190 mm working distance. Immediately after illumination, 1 mM cleavable crosslinking reagent (HIT-tag provided in Example 1) and 10 mM 2- (Aminomethyl)imidazole dihydrochloride (AMI) are added, followed by a 2-hour incubation at 40°C in the dark. The modified peptide reaction is then bound to pre-equilibrated TCO agarose beads (Vectorlabs CCT-1198) for 20 min at room temperature on an end-to-end rotator. Beads are washed with 10 mM MES 1 20% acetonitrile and PBS to remove unbound peptides. Oxidative cleavage was performed by incubating bead-bound peptides with 2 mM sodium periodate for 5 min at RT in the dark followed by a 20 mM sodium sulfite incubation for 1 h at RT. The resulting peptides are purified via C18 columns (BioPureSPN MINI, The Nest Group HUMS18V) per manufacturer instructions and subjected to mass spectrometric analysis using an nLC 1200 (Thermo Scientific) QExactive (Thermo Scientific) system in data-dependent acquisition (DDA) mode, with mass resolutions of 60,000 and 30,000 for precursor and fragment ions, respectively. The run employed an MS2 AGC target of 1 e6 and a maximum injection time of 120 ms, with dynamic exclusion set to 10 seconds, using a gradient from 5% to 40% B over 30 minutes. Resulting spectra were analyzed using Freestyle software v1.8 SP1 (Thermo Scientific) (Fig. 2).

[0573] Example 8: Spike-In Protein Preparation for Mass Spectrometry (MS)

[0574] The spike-in protein is first modified through singlet oxygen-mediated reactions and then labeled with a cleavable crosslinking reagent (a HIT-tag provided in Example 1 , or a derivative thereof comprising a biotin affinity moiety instead of the methyltetrazin moiety). Specifically, a solution of 75 pM transferrin (Sigma T4132-100 mg) in D2O-based PBS is combined with 100 pM thiorhodamine SOG- azide (Atto-Tec AD Thiol 2-105) and 1 % DMSO, then illuminated for 15 minutes at 4°C using four Precision LED spotlights at 590 nm from a 190 mm working distance. Following this, a 1 mM cleavable crosslinking reagent and 10 mM 2-(Aminomethyl)imidazole dihydrochloride (AMI) are added, and the mixture is incubated at room temperature for 2 hours. Unreacted SOG-azide and crosslinker are removed using 7-kDa ZebaSpin Columns (Thermo 89882) according to the manufacturer’s instructions.

[0575] The modified protein is subsequently bound to pre-equilibrated TCO agarose (Vectorlabs CCT- 1198) or Streptavidin (Thermo 53116) beads, with binding performed for 1 hour at room temperature on an end-to-end rotator. The beads are washed sequentially in Mobicol spin columns (MoBiTec M105035F) with 8 M urea in 50 mM ammonium bicarbonate (pH 8.0) and then with 50 mM ammonium bicarbonate. Bead-bound protein undergoes overnight digestion at 37°C with sequencing-grade trypsin (Promega V511 C) at an enzyme-to-protein ratio of 1 :100. Non-bound tryptic peptides are removed, and the beads are washed three times with PBS. For oxidative cleavage of the crosslinking reagent, the beads are treated with 2 mM sodium periodate in the dark for 15 minutes, followed by a 1-hour incubation with 20 mM sodium sulfite. The resulting peptides are purified using C18 columns (BioPureSPN MINI, The Nest Group HUMS18V) per the manufacturer’s instructions and subjected to mass spectrometric analysis on an nLC 1200 (Thermo Scientific) QExactive (Thermo Scientific) system in data-dependent acquisition (DDA) mode, with mass resolutions of 60,000 and 30,000 for precursor and fragment ions, respectively. The run employed an MS2 AGC target of 1e6 and a maximum injection time of 120 ms, with dynamic exclusion set to 10 seconds, using a linear gradient from 5% to 40% B over 30 minutes (buffer A: 99.9% H2O, 0.1% formic acid; buffer B: 99.9% acetonitrile, 0.1% formic acid). Data analysis was performed using Fragpipe (v19.1 ) and MSFragger (v3.7) with a database that included the spike-in protein and common contaminants. Initially, an open search was conducted to identify mass modifications and their localizations, followed by diagnostic ion mining through mass offset search to detect reporter ions and remainder peptides. Finally, a hybrid search was performed, incorporating identified variable and labile modifications, to identify crosslinker reagent-modified peptides with a minimum required signature ion intensity of 1% (Fig. 3C).

[0576] Example 9: Labeling and enrichment of cell surface proteins using a cleavable crosslinking reagent

[0577] To prepare antibody-SOG conjugates, 2.5 pg of antibody (Mouse Isotype control antibody, Invitrogen 10400C; anti-human IgM antibody, Invitrogen 14-9998-82) per sample was purified using 7-kDa ZebaSpin Desalting Columns (Thermo Scientific, 89882) according to the manufacturer's protocol, and eluted in 10 mM sodium bicarbonate in PBS at pH 8.3. The antibody was conjugated to NHS thiorhodamine SOG (Atto-Tec AD Thio12-31) at a 1 :5 molar ratio (antibody:SOG), purified using 7-kDa ZebaSpin columns, and immediately used in further experiments. Ramos B lymphoma cells (20 x 1 o6) were treated with 10 pg of SOG-conjugated antibody (final concentration 40 pg / mL) for 30 minutes at 4°C in the dark. 15 min into incubation, aliquots of 5 x 1 o6cells were distributed onto a precooled Curiox laminar wash plate and allowed to settle for 15 min. Cells were washed with PBS on the Curiox HT2000 Laminar wash (12 cycles, 5 pL / sec) and afterwards resuspended in photo-oxidation buffer (10 mM cleavable crosslinking reagent [hydrazide-isoseramox-biocytin HIB or alternatively referred to as BIH, biocytin-isoseramox-hydrazide] in D2O-based PBS, pH 7.4), and illuminated for 5 minutes at a 19 cm distance using four 590 nm Precision LED spotlights. 2- (aminomethyl)imidazole dihydrochloride in PBS, pH 7.4 was added to each cell suspension to a final concentration of 50 mM and incubated in the dark for 60 minutes at 4°C and 300 RPM shaking for the first 45 min. Cells were washed with PBS on the Curiox HT2000 Laminar wash (12 cycles, 5 pL / sec) and afterwards harvested into 1.5 mL LoBind Eppendorf tubes. Cells were spun down (250 ref, 2min), the supernatant was removed and the cell pellets were snap-frozen in liquid nitrogen and stored at -20 °C until further use. For lysis, pellets were thawed on ice and resuspended in 80 pL Trifluoroacetic acid (Fisher Chemical, T-3255-PB05) and incubated at room temperature for approximately 3 min. Afterwards 800 pL of 2M Tris base was added to each sample and samples were heated to 95 °C for 5 min. Next, 50 pL Pierce™ Streptavidin Plus UltraLink™ Resin (Thermo Scientific Cat: 53117) was added to each sample for protein binding. Sample tubes were rotated for 60 min at room temperature. The beads were washed sequentially in Mobicol spin columns (MoBiTec M105035F) with 7M urea in PBS (pH 8.0) and then with PBS and 50 mM Sodium bicarbonate. Bead-bound protein underwent overnight digestion at 37°C in 1 M Urea with 0.8 pg sequencing-grade trypsin (Promega V511C) and 1 pg Lysyl endopeptidase (Fujifilm 121-05063). Non-bound tryptic peptides are removed, and the beads are washed twice with PBS. The tryptic fractions were acidified to 1% formic acid and stored at -20 °C. Next, beads were washed with 7M Urea and PBS. For oxidative cleavage of the crosslinking reagent, the beads are treated with 2 mM sodium periodate in the dark for 15 minutes. Eluate fractions were collected directly into Evotips (Evosep EV2013). Tryptic and eluate fractions were desalted using Evotips according to manufacturer recommendations. Samples were subsequently measured using the Evosep One Platform with a 15 cm Endurance column (Evosep: EV1106) on the proprietary Evosep 44 min gradient. Samples were measured using a Q Exactive HF-X mass spectrometer (Thermo Scientific) in DDA mode with HCD28. Raw files were converted to the mzml format using MSConvert and searched in Fragpipe V19.1 against the human proteome. 160.08 m / z and 174.12 m / z were used as variable modifications on histidine and 174.12 m / z was additionally used for labile search with 161.14 m / z as diagnostic ion and 13.98 m / z as remainder mass on histidine. Results were quantified using FragPipe analyst and plots were created in R 4.1 using the package ggplot2 (Fig. 4B and 4C).

[0578] Example 10: Selective oxidative release of a HIT-TCO-AF488 construct from surface proteins

[0579] 2.5 pg of antibody (Mouse Isotype control antibody, Invitrogen 10400C; anti-human CD45 antibody, Invitrogen 14-0459-82) per sample was purified using 7-kDa ZebaSpin Desalting Columns (Thermo Scientific, 89882) according to the manufacturer's protocol, and eluted in 10 mM sodium bicarbonate in PBS at pH 8.3. Each antibody was conjugated to NHS thiorhodamine SOG (Atto-Tec AD Thiol 2-31) at a 1 :5 molar ratio (antibody:SOG), purified using 7-kDa ZebaSpin columns, and immediately used in experiments. Ramos B lymphoma cells (5 x 106) were treated with 2.5 pg of SOG-conjugated antibody (final concentration 5 pg / mL) for 30 minutes at 4°C in the dark. Following incubation, cells were washed with PBS, resuspended in photo-oxidation buffer (0.5 mM HIT-Tag in D2O-based PBS, pH 7.4), and illuminated for 5 minutes at a 19 cm distance using four 590 nm Precision LED spotlights. Cells were pelleted by centrifugation, resuspended in chilled labeling buffer (10 mM 2-(aminomethyl)imidazole dihydrochloride in PBS, pH 7.4), and incubated in the dark for 50 minutes at 4°C. Cells were washed extensively with PBS, stained with 1 pM AZDye488 TCO (Vectorlabs CCT-1356) for 20 minutes. Oxidative release on cells was performed by incubating cells in 6.5 mM sodium periodate for 10 min and after washing, cells were analyzed on an Accuri C6 Flow Cytometer (BD Biosciences) using FlowJo software (v10.07) (Fig. 5).

[0580] Example 11 : The peptide- and protein-centric detection of mass-shifts and reporter ions

[0581] Mass-shifts and reporter ions (also referred herein as diagnostic ions or signature ions) for different embodiments of cleavable crosslinking reagents of the invention (referred to in the following as cmTags) were confirmed as follows, in exemplary peptide- and protein-centric detection workflows.

[0582] Peptide-centric discovery: A histidine-containing peptide was tagged with cmTags in the presence of free SOG-azide and captured using magnetic beads functionalized with TCO groups and the beads washed using high acetonitrile conditions. The tagged peptides were oxidatively cleaved and measured in an Orbitrap QE Plus HF mass spectrometer. MS2 spectra were matched according to a predefined list of plausible mass shifts and reporter ions (Fig. 7(A)).

[0583] Protein-centric discovery. A mixture of Aldolase, Apo-transferrin and Catalase was tagged with cmTags in the presence of free SOG-azide and captured on magnetic TCO-beads. The beads were washed and digested overnight with trypsin. The tryptic fraction was collected and the still bound peptides released from the beads with sodium periodate. Both fractions were measured using DDA and the data was subject to open search and labile search in Fragpipe 19.1 to determine cmTag specific mass shifts (Fig. 7(B)).

[0584] Figure 8 shows the cmTag variants used in this Example and open search results for tryptic and oxidative release fractions of four different cmTag variants and a digest control. The different variants comprise (1) a hydrazide functional group (a HIT-tag provided in Example 1); (2) an aniline moiety (a cmTag provided in Example 3); (3) a hydrazide functional group and PEG-3 spacer (a cmTag provided in Example 2 which is a derivative of the HIT-tag provided in Example 1 further comprising (-[O-(CH2)2]3-)-spacer); (4) an alkoxyamine moiety (a cmTag provided in Example 4). The bars show the number of PSMs identified with a specific mass shift in the different samples. Results were filtered for mass shifts that were specific to the oxidative release fractions of one cmTag variant family. The results are shown in Figures 9A-9C for a hydrazide reactive cmTag variants (a HIT-tag provided in Examples 1 and its derivative provided in Example 2), Figures 10A- 10D for an alkoxyamine reactive cmTag variant (a cmTag provided in Example 4) and Figures 11A- 11C for an aniline reactive cmTag variant (a cmTag provided in Example 3). Example 12: Comparative analysis of proximity labeling achieved by a cleavable and non- cleavable linker

[0585] In this example, non-cleavable linker biocytin-hydrazide (BH) and cleavable linker biocytin- isoseramox-hydrazide (BIH tag, as provided in Example 5) were directly compared within an identical proximity-labeling workflow. Both BH and BIH were used for proximity labeling of IgM (as target) on Ramos B cells, and each condition was analyzed in quadruplicate. Protein abundances were quantified relative to an IgG isotype control. All samples were processed using the same experimental protocol and analyzed on a Q-Exactive HF-X mass spectrometer operated in DDA mode. The results for the BH and BIH linkers are shown in Figures 6A and 6B, respectively.

[0586] In contrast to the non-cleavable BH linker, the cleavable BIH linker enabled the direct identification of peptides carrying the characteristic signature mass shift. In Figure 6B, proteins that were identified through unique peptides carrying this signature mass shift are indicated by triangles (proximal proteins) and squares (target protein(s)).

[0587] A proportional Venn diagram (Fig. 6C) summarizes the overlap of significantly enriched, surfaceome-annotated proteins obtained by using either the cleavable or non-cleavable biocytinhydrazide linkers in the proximity-labeling and enrichment workflow described above. Use of the cleavable BIH linker resulted in the detection of additional targets that were not identified with the non-cleavable BH linker.

Claims

1. Claims1. A compound of Formula I:X-S1-Z-S2-A whereinX is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA);Si and S2 are independently of each other a spacer group;Z is a cleavable linker moiety comprising a functional group having a formula selected from the group consisting of:wherein R1 is selected from H or CH3; andA is an affinity moiety comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s).

2. The compound according to claim 1 , wherein the amino acid side chain (i) has been subjected to a chemical reaction to obtain an oxidized amino acid side chain, relative to the native amino acid side chain and / or (ii) comprises a carbonyl functional group, a hydroperoxide functional group and / or an endoperoxide functional group, optionally wherein the amino acid is:(a) histidine and the amino acid side chain is selected from the group consisting of(b) tryptophan and the amino acid side chain is selected from the group consisting ofPage 98 of 110(c) tyrosine and the amino acid side chain is selected from the group consisting of(d) methionine and the amino acid side chain is; and / or(f) cysteine and the amino acid side chain is selected from the group consisting of3. The compound according to claim 1 or claim 2, wherein X is a hydrazide functional group.

4. The compound according to any one of claims 1 to 3, wherein the cleavable linker moiety Z is cleavable by exposure to one or more oxidizing agent(s), preferably by exposure to one or more periodate salt(s) and / or cyclic periodate(s), more preferably by exposure to sodium periodate.

5. The compound according to any one of claims 1 to 4, wherein Z comprises a functional group having a formula selected from the group consisting of:Page 99 of 110wherein Ri is selected from H or CH3.

6. The compound according to any one of claims 1 to 5, wherein the affinity moiety A comprises:(a) a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s), wherein the second functional group is covalently bound to a solid support, such as a bead, a filter, a plate, a membrane or a chromatographic resin, preferably wherein the second functional group is covalently bound to a bead or to a chromatographic resin;(b) a first functional group capable of reacting with a second functional group to form one or more covalent bond(s) through a cycloaddition reaction, optionally wherein the cycloaddition reaction is a [4+2] cycloaddition reaction; and / or(c) a functional group selected from biotin, biocytin, tetrazine, methyltetrazine, transcyclooctene (TCO), azide (-N3), or a terminal alkyne (-CCH).

7. The compound according to any one of claims 1 to 6, wherein Si and S2 are independently of each other (i) a single bond or (ii) a straight-chain or branched, substituted or unsubstituted C(1-24) hydrocarbon, wherein one or more, preferably non-adjacent, -CH2- groups independently from each other may be replaced by one or more bridging groups and / or an unsubstituted or substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl, optionally wherein the bridging group is selected from -CH(OH)-, -O-, -CO-, -CH2(CO)-, - SO-, -CH2(SO)-, -SO2-, -CH2(SO2)-, -COO-, -OCO-, -S-CO-, -CO-S-, -SOO-, -OSO-, -SOS-, -O-CO-O-, -OCH2-, -CH2O-, -NR3-, -NR3-CO-, -CO-NR3-, -NR3-CO-O-, -O-CO-NR3-, -NR3- CO-NR3-, -CH=CH-, -C=C-, -CH=CH-COO-, -OCO-CH=CH-, -CH=N-, -C(CH3)=N-, -N=N-, wherein each R3 independently represents H or C(1 -6)alkyl, optionally wherein Si and S2 are each independently selected from a linear chain comprising one or more repeating units of Formula II:-[Yl-(CH2)n]p-Page 100 of 110and / or Formula III:-[Y2-(CH2)m-Y3]q-, or combinations thereof, whereinYi, Y2, Y3are independently of each other a group selected from -O-, -CO-, COO-, -OCO-, - O-CO-O-, -OCH2-, -CH2O-, NR4-, -NR4-CO-, -CO-NR4-, wherein R4 represents H or C(1 -6)- alkyl, and n, m, p, and q are independently of each other an integer from 1 to 10, preferably wherein Si and S2are each independently a linear chain comprising one or more repeating units of Formula II-[Yl-(CH2)n]p-, whereinY1 is -O-, n is 2, and p is an integer from 1 to 10, preferably from 1 to 5, most preferably from 1 to 3.

8. The compound according to any one of claims 1 to 7, wherein the compound comprises one or more isotopic label(s), preferably wherein the one or more isotopic label(s) are each independently selected from180,13C,15N or2H, optionally wherein the one or more isotopic label(s) are comprised within the spacer group Si, the spacer group S2and / or within the moiety X.

9. The compound according to any one of claims 1 to 8 having a structure selected from the group consisting ofI.Page 101 of 110whereinSi and S2 are independently of each other a spacer group;Z is the cleavable linker moiety comprising a functional group having a formula selected from the group consisting of:wherein R1 is selected from H or CH3; andA is the affinity moiety comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s);II.Page 102 of 110whereinSi and S2 are independently of each other a spacer group;X is the moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA); andA is the affinity moiety.comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s), and wherein R1 is selected from H or CH3; and / orIII.whereinX is the moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein X is selected from the group consisting of an aromatic hydrazinePage 103 of 110functional group, a hydrazide functional group, 1 -methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3-ethylaminoaniline (3-EA);Si and S2 are independently of each other a spacer group;Z is the cleavable linker moiety comprising a functional group having a formula selected from the group consisting of:wherein R1 is selected from H or CH3; and whereinR2 is selected from H, CH3, or (CH2)2-COOH.

10. The compound according to claim 1 having a structure according to the formula selected from the group consisting of:Page 104 of 11011 . Use of the compound of any one of claims 1 to 10 for the isolation, selective enrichment, identification and quantification of one or more protein(s).

12. A method for the isolation and identification of one or more protein(s), comprising the steps of:(i) subjecting a sample comprising the one or more protein(s) to a chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain comprised in the one or more protein(s), to obtain one or more oxidized protein(s);(ii) reacting the one or more oxidized protein(s) with a compound of Formula I:X-S1-Z-S2-A whereinX is a moiety reactive with an amino acid side chain, preferably wherein the amino acid is histidine, tryptophan, tyrosine, methionine or cysteine, wherein is selected from the group consisting of an aromatic hydrazine functional group, a hydrazide functional group, 1-methyl-4-aryl-urazole (MAUra), alkoxyamine, aniline or 3- ethylaminoaniline (3-EA);Page 105 of 110Si and S2 are independently of each other a spacer group;Z is a cleavable linker moiety comprising a functional group having a formula selected from the group consisting of:wherein R1 is selected from H or CH3; andA is an affinity moiety_comprising a first functional group capable of reacting with a second functional group attached or linked to a solid support to form one or more bond(s), wherein the moiety X reacts with the one or more carbonyl functional group(s), the one or more hydroperoxide functional group(s) and / or the one or more endoperoxide functional group(s) to form one or more labelled protein(s);(iii) isolating and identifying the one or more labelled protein(s).

13. The method according to claim 12, wherein the chemical reaction to oxidatively generate one or more carbonyl functional group(s), one or more hydroperoxide functional group(s) and / or one or more endoperoxide functional group(s) in an amino acid side chain comprises reacting the one or more protein(s) with singlet oxygen (102) , optionally wherein the singlet oxygen is generated by exposing a singlet oxygen generator (SOG) to light, optionally wherein the SOG is(a) selected from the group consisting of thiorhodamine, a phenothiazinium dye (e.g., methylene blue, Nile blue or toluidine blue), a cyanine, a transition metal complex (e.g., bipyridine, bipyrazine or 2,2'-bipyrimidine complexes of Ru(ll), Os(ll), or Ir(lll)) and a chalcogenopyrylium dye; and / or(b) covalently coupled to an antibody, a small molecule, a peptide, a peptidomimetic, an aptamer or a viral particle, optionally wherein the antibody, the small molecule, the peptide, the peptidomimetic, the aptamer or the viral particle is capable of specifically binding to the one or more protein(s) or to a target that is in close proximity to or in physical contact with the one or more protein(s).

14. The method according to claim 12 or claim 13, wherein the isolation and identification of one or more protein(s) comprises:Page 106 of 110I. (a) enriching the one or more labelled protein(s), wherein the one or more labelled protein(s) are contacted with a solid support and wherein the affinity moiety A specifically associates with or specifically reacts with a functional group attached or linked to the solid support to form one or more bond(s), optionally wherein the solid support is subjected to one or more washing steps after formation of the one or more covalent bond(s);(b) releasing the one or more labelled protein(s) from the solid support, preferably by exposure to one or more oxidizing agent(s);(c) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s); and(d) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS);II. (a) enriching the one or more labelled protein(s), wherein the one or more labelled protein(s) are contacted with a solid support and wherein the affinity moiety A specifically associates with or specifically reacts with a functional group attached or linked to the solid support to form one or more bond(s), optionally wherein the solid support is subjected to one or more washing steps after formation of the one or more covalent bond(s);(b) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s), optionally wherein the solid support is subjected to one or more washing steps after the fragmenting of the one or more labelled protein(s);(c) releasing one or more peptide fragments from the solid support, preferably by exposure to one or more oxidizing agent(s); and(d) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS);III. (a) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s);(b) enriching one or more peptide fragments, wherein the one or more peptide fragments are contacted with a solid support and wherein the affinity moiety APage 107 of 110specifically associates with or specifically reacts with a functional group attached or linked to the solid support to form one or more bond(s), optionally wherein the solid support is subjected to one or more washing steps after formation of the one or more covalent bond(s);(c) releasing the one or more peptide fragments from the solid support, preferably by exposure to one or more oxidizing agent(s); and(d) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS); orIV. (a) fragmenting the one or more labelled protein(s) to obtain peptide fragments of the one or more labelled protein(s);(b) optionally exposing the one or more peptide fragments to one or more oxidizing agent(s); and(c) identifying one or more individual labelled protein(s) and / or peptide fragments thereof, preferably by peptide analysis by liquid-chromatography-mass spectrometry (LC-MS).

15. A protein or peptide produced by the method of any one of claims 12 to 14, comprising at least one amino acid side chain selected from the group consisting of, and / or from the group consisting ofPage 108 of 110Page 109 of 110

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