Labeling of sialic acid-added glycoproteins based on proximity

Abstract

This specification describes systems and methods that enable profiling of local microenvironments across sialic acid-added proteomes through proximity labeling. In one embodiment, a conjugate having a composition and electronic structure for generating reactive labeling intermediates in the microenvironment of sialic acid-added cell surface glycoproteins is described herein. In some embodiments, the conjugate comprises a transition metal catalyst that couples to the cell surface glycoprotein. As further described herein, the transition metal catalyst may be coupled to the glycoprotein via a derivatized sialic acid linker.

Description

[Technical Field]

[0001] Related application data This application claims priority, pursuant to Article 8 of the Patent Cooperation Treaty, to U.S. Provisional Patent Application No. 63 / 419,519 filed on 26 October 2022, which is incorporated herein by whole reference.

[0002] field The present invention relates to proximity-based labeling, particularly compositions, systems, and methods for proximity-based labeling for profiling local microenvironments across sialic acid-added proteomes. [Background technology]

[0003] Glycosylation is one of the most common post-translational modifications (PTMs) to proteins, occurring in at least 50% of all known mammalian proteins and dramatically increasing the functional proteome. Glycosylation can alter both protein localization and function, and miscontrolled placement has been shown to contribute to a variety of disease phenotypes, such as cancer metastasis, viral immune evasion, viral entry, and inflammation. Glycoproteins also play a crucial role in the overall cell surface architecture, contributing to cell adhesion, cell signaling, viral docking, and intercellular interactions. Among the range of cell surface monosaccharides, sialic acid stands out as particularly influential on cellular function. This charged sugar is taken up by sialyltransferase and commonly modifies the ends of polysaccharide chains. During tumorigenesis, overexpression of sialyltransferase leads to hypersialylation, which then promotes tumor progression through two distinct paradigms: (1) sialic acid addition appears to inhibit apoptosis and allow cells to evade the immune system, and (2) sialoglycoconjugate sialyl Lewis X This promotes metastasis into nearby tissues through the spillage of cancer cells out of the bloodstream.

[0004] Despite these observations, the underlying biochemical mechanisms remain largely unknown, partly due to the lack of high-resolution tools to assess the functional role of sialic acid addition. [Overview of the Initiative]

[0005] In consideration of the aforementioned drawbacks, systems and methods for enabling profiling of local microenvironments across sialic acid-added proteomes through proximity labeling are described herein. In one embodiment, a conjugate having a composition and electronic structure for generating a reactive labeling intermediate in the microenvironment of sialic acid-added cell surface glycoproteins is described herein. In some embodiments, the conjugate comprises a transition metal catalyst for coupling to the cell surface glycoprotein. As further described herein, the transition metal catalyst may be coupled to the glycoprotein via a derivatized sialic acid linker. In some embodiments, the transition metal catalyst and the derivatized sialic acid linker are coupled via click chemistry. Suitable click chemistry moieties of the transition metal complex and / or derivatized sialic acid linker may be selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkynes, and azides. For example, sialic acid may be derivatized to include a click chemistry moiety suitable for coupling with the transition metal catalyst. Figure 1 illustrates sialic acid derivatized with azide functionality for reaction with the DBCO moiety of a transition metal catalyst. In some embodiments, the derivatized sialic acid linker is metabolically incorporated into cell surface glycoproteins and then reacts to capture a transition metal catalyst. Figure 1 illustrates an example, not limited to, of the metabolic incorporation of an azide-derivatized sialic acid linker into cell surface glycoproteins. In some embodiments, the transition metal catalyst may include a platinum group metal center. Furthermore, in some embodiments, the transition metal catalyst is of formula I: [ka] In the formula, M is a transition metal; A, D, E, G, Y, and Z are independently selected from C and N; R 3 ~R 7 each represents an optionally selected ring substituent from 1 to 4, and the optionally selected ring substituents from 1 to 4 are each independently alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, -C(O)O - -C(O)OR 8 and -R 9 OH, and is selected from the group consisting of; R 8 is selected from the group consisting of hydrogen and alkyl; R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocycle, and heteroarylene; L is an optionally selected linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 -OS(O2)R 9 thiol, biotin, oxyamine, and haloalkyl, and R 8 and R 9 are each independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl ester; X - is a counterion, and n is an integer from 0 to 20 is as provided in Formula I. The linking moiety L is optional, and thus may not be present in some embodiments of the transition metal catalyst.

[0006] The polarity of the transition metal complex is R 3 ~R 7Through the selection of R, it is also possible to tailor it to a specific cellular environment. In some embodiments, for example, R 3 ~R 7 One or more of these are selected to exhibit hydrophilic properties through their charged and / or polar chemical moieties. In such embodiments, the transition metal complex may exhibit hydrophilic properties suitable for placement in intercellular / extracellular environments. For example, the transition metal complex illustrated in Figure 6 incorporates charged and polar chemical moieties with respect to an aqueous intercellular environment. Alternatively, R 3 ~R 7 One or more of these are selected to exhibit hydrophobic, lipophilic, or nonpolar properties.

[0007] The transition metal catalyst may have an electronic structure for transferring energy to a protein label to produce a reactive intermediate. In some embodiments, the energy transfer is Dexter energy transfer or electron transfer. In some embodiments, the energy transfer to the protein label may arise from an excited state of the transition metal catalyst's electronic structure. The excited state of the catalyst may be, for example, a singlet excited state or a triplet excited state. The excited state of the catalyst may arise from one or more mechanisms, including energy absorption by the catalyst. In some embodiments, the catalyst is a photocatalyst, in which the excited state is induced by the absorption of one or more photons. In other embodiments, the catalyst may be brought into an excited state by interaction with one or more chemical species in the surrounding environment. Alternatively, the energy transfer to the protein label, including electron transfer, may arise from the ground state of the catalyst's electronic structure.

[0008] In another embodiment, a system is provided for profiling the microenvironment near a sialic acid-added proteome. In some embodiments, the system comprises a protein labeling agent and a conjugate comprising a transition metal catalyst coupled to a cell surface glycoprotein via a derivatized sialic acid linker, the transition metal catalyst having an electronic structure that enables energy transfer to the protein labeling agent, thereby providing a reactive intermediate. This reactive intermediate can be manipulated to label proteins or other biomolecules within a predetermined range of the conjugate. The predetermined range may be the diffusion range of the reactive intermediate.

[0009] The diffusion range of the reactive intermediate may be tailored to specific microenvironmental mapping (proximity-based labeling) considerations and may be limited to the nanometer scale. In some embodiments, for example, the diffusion range of the reactive intermediate may be less than 100 nm, less than 50 nm, less than 10 nm, less than 5 nm, less than 4 nm, less than 3 nm, or less than 2 nm before quenching in the surrounding environment. In some embodiments, the diffusion range may be 0.5 nm to 10 nm. Thus, the reactive intermediate will react with or crosslink with proteins or other biomolecules within its diffusion range, or, if no proteins or biomolecules are present, will be quenched by the surrounding environment. In this manner, the local environment can be mapped with high resolution by the coordinated force between the catalyst and the protein labeling agent. Furthermore, in some embodiments, the reactive intermediate is subjected to less than 5 ns, less than 4 ns, or less than 2 ns before quenching. 1 / 2 This can be shown. Reactive intermediates, for example, have a t of less than 1-5 ns. 1 / 2 This may be shown. In further embodiments, the diffusion range may be extended to 5 to 500 nm by extending the half-life of the reactive intermediate. For example, in some embodiments, the reactive intermediate may have a half-life of 1 to 100 μs or longer.

[0010] In some embodiments, the protein labeling agent may be diazirine. Triplet energy transfer from the excited state photocatalyst can promote diazirine to its triplet (T1) state. The diazirine triplet releases a free triplet carbene via the removal of N2, which undergoes spin equilibration on the picosecond time scale to its reactive singlet state (t 1 / 2 <1 ns), which either crosslinks with neighboring proteins or is quenched in an aqueous environment. In some embodiments, the extinction coefficient of the transition metal complex is 3 to 5 orders of magnitude larger than that of diazirine.

[0011] Any diazirine consistent with the technical principles discussed herein. Diazirine sensitization can be extended, for example, to various p- and m-substituted aryltrifluoromethyldiazirines, which possess payloads valuable for microscopy and proteomics applications, including free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups. Diazirine may be functionalized with a marker such as biotin. In some embodiments, the marker is desthiobiotin. The marker can assist in the identification of proteins labeled with the protein labeling agent. The marker can be useful, for example, in assay results through Western blot and / or other analytical techniques. Markers can include alkynes, azides, FLAG tags, fluorophores, and chloroalkane functionality in addition to biotin and desthiobiotin.

[0012] In a further embodiment where the transition metal catalyst is a photocatalyst, the protein labeling agent may be an azide. Triplet energy transfer from the excited state photocatalyst can promote nitrene formation from the azide. The reactive nitrene either crosslinks with neighboring proteins or is quenched in an aqueous environment. For nitrene formation, any azide operable to undergo energy transfer with the transition metal photocatalyst may be used. In some embodiments, the azide is an aryl azide.

[0013] In another aspect, a method for profiling the microenvironment in the vicinity of the sialylated proteome is provided. The method includes forming a conjugate comprising a transition metal complex coupled to a cell surface glycoprotein through a derivatized sialic acid linker, and activating a protein labeling agent to a reactive intermediate with a transition metal catalyst. The reactive intermediate couples to a protein or other biomolecule within a predetermined range of the conjugate. The transition metal catalyst, protein labeling agent, and reactive intermediate may have any of the compositions and / or properties described herein.

[0014] These and other embodiments are further described in the following detailed description.

Brief Description of the Drawings

[0015] [Figure 1] Exemplify the use of microenvironment mapping with the systems described herein, comprising a conjugate comprising a transition metal catalyst coupled to a cell surface glycoprotein through a derivatized sialic acid linker and a protein labeling agent, wherein the transition metal catalyst has an electronic structure that enables energy transfer to the protein labeling agent to provide a reactive intermediate. [Figure 2A] Figure 2A illustrates the workflow for the glycomapping experiment. The Ac4ManNAz incubation was carried out for 72 hours, but in this study, equivalent results were obtained in cell lines in a shorter time. The optimization of the Ir-DBCO incubation time is shown in the supplementary information of the appendix (Figure S3). [Figure 2B] Figure 2B provides a Western blot analysis of the whole cell lysate after the glycomapping experiment. [Figure 2C] Figure 2C provides an immunofluorescence analysis of the cells after the glycomapping experiment; red: streptavidin, blue: hoechst. [Figure 2D] Figure 2D is a Western blot of streptavidin-enriched lysates stained against nicastrin (upper lane) and CD55 (lower lane). [Figure 3A]Figure 3A illustrates a workflow for chemopromic discovery of the interactome of sialic acid-added glycoproteins based on TMT. Each experiment was performed in sets of three. [Figure 3B] Figure 3B provides quantitative chemopromic validation using a glucose map of HEK293T cells. All experiments used the same cutoff (>1.5Log2 (double change); >1.5-Log10 (p-value)). [Figure 4A] Figure 4A summarizes comparative proteomics experiments between primary cervical cells (PCCs) and HeLa cells. Top row: Sialic acid-added glycoproteins in PCCs (left) and HeLa cells (right). Bottom row: Interacting proteins in PCCs (left) and HeLa cells (right). Center: Venn diagrams of enriched proteins from each dataset. The same cutoff (>1.5Log2(double change); >1.5-Log10(p-value)) was used for the analysis of all datasets. [Figure 4B] Figure 4B shows the gene ontology (GO) analysis of the identified sialic acid-added glycoproteins (top) and their interacting factors (bottom). [Figure 4C] Figure 4C is a Venn diagram of concentrated solute carrier proteins (SLCs) that interact with sialic acid-added glycoproteins. [Figure 5A] Figure 5A illustrates a workflow for metabolomics analysis of HeLa cells. [Figure 5B] Figure 5B quantifies the metabolite levels of selected small molecules. The experiment was performed in sets of three. [Figure 5C] Figure 5C, left: GO analysis suggests that cation homeostasis is affected by sialic acid addition. Center: Enriched zinc transport factors in HeLa and PCC datasets. Right: Colorimetric zinc assay, showing significant changes in cellular zinc levels in response to desialic acid addition. P values ​​determined by unpaired t-tests. *P<0.05, **P<0.01. [Figure 6] We illustrate transition metal complexes that incorporate charged and / or polar chemical moieties for aqueous intercellular environments, according to several embodiments. [Figure 7] The diverse chemical species used in some embodiments of the compositions and methods described herein are illustrated below. [Modes for carrying out the invention]

[0016] The embodiments described herein may be more readily understood by referring to the following detailed description and examples, as well as any descriptions preceding or following them. However, the elements, apparatus and methods described herein are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the invention. Many modifications and adaptations will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

[0017] definition The term "alkyl," when used herein, alone or in combination, refers to a linear or branched saturated hydrocarbon group that is optionally substituted with one or more substituents. For example, alkyl groups are C1-C 30 Or C1~C 18 That's fine.

[0018] The term "alkenyl," when used herein, either alone or in combination, refers to a linear or branched hydrocarbon group having at least one intercarbon double bond and optionally substituted with one or more substituents.

[0019] The term "alkynyl," when used herein, either alone or in combination, refers to a linear or branched hydrocarbon group having at least one carbon-carbon triple bond and optionally substituted with one or more substituents.

[0020] The term "aryl," when used herein, either alone or in combination, refers to an aromatic monocyclic or polycyclic system that is optionally substituted with one or more ring substituents.

[0021] The term "heteroaryl," when used herein, either alone or in combination, refers to an aromatic monocyclic or polycyclic system in which one or more ring atoms are elements other than carbon, such as nitrogen, boron, oxygen, and / or sulfur.

[0022] The term “heterocyclic” as used herein, either alone or in combination, refers to a monocyclic or polycyclic system in which one or more atoms of the cyclic system are elements other than carbon, such as boron, nitrogen, oxygen, and / or sulfur or phosphorus, and the cyclic system is optionally substituted with one or more ring substituents. Heterocyclic systems may include aromatic and / or non-aromatic rings containing one or more unsaturated rings.

[0023] The term "cycloalkyl," when used herein, either alone or in combination, refers to a non-aromatic monocyclic or polycyclic system that is optionally substituted with one or more ring substituents.

[0024] The term "heterocycloalkyl," as used herein, either alone or in combination, refers to a non-aromatic monocyclic or polycyclic system in which one or more atoms in the ring system are elements other than carbon, such as boron, nitrogen, oxygen, sulfur, or phosphorus, either alone or in combination, and the ring system is optionally substituted with one or more ring substituents.

[0025] The term "alkoxy," when used herein, either alone or in combination, refers to a partial RO- (wherein R is an alkyl, alkenyl, or aryl as defined above).

[0026] The term "halo," when used herein, alone or in combination, refers to an element of Group VIIA or Group 17 of the periodic table. Depending on the chemical environment, a halo can be neutral or anionic.

[0027] Terms not specifically defined herein have their common meanings in the art. [Examples]

[0028] Embodiments of this application are further illustrated in the following, less limited embodiments.

[0029] Iridium photocatalysts were developed for microenvironmental mapping to elucidate the interactome of sialic acid-added cell surface glycoproteins, as illustrated in Figure 1. Studies using iridium photocatalysts were initiated according to Figure 2A. After incubating HeLa cells with tetraacetyl-N-azidoacetylmannosamine (Ac4ManNAz), treatment with DBCO-iridium (Figure 7, S1) led to the uptake of the iridium photocatalyst onto glycoproteins. Irradiation in the presence of biotin-diaziline (Figure 7, S2) resulted in cell surface biotinylation, as observed by Western blotting (Figure 2B). Control reactions without azido sugar, DBCO-iridium reagent, or blue light irradiation showed minimal biotinylation. Importantly, immunoprecipitation on streptavidin beads showed strong enrichment of known sialic acid-added glycoprotein nicatrin (NCSTN) and complement degradation accelerator (CD55), providing certainty to our workflow (Figure 2C).

[0030] This method was compared to direct biotinylation of Ac4ManNAz using DBCO-biotin (S3), and sialome labeling was observed via Western blotting. These results showed that our catalytic labeling method allows for the installation of approximately 0.7 tags per catalyst (see ESI), which represents a significant improvement over existing PAL probes, which typically react with water (>95%) and exhibit minimal protein labeling. Finally, this protocol is also applicable to both HEK293T cells and primary cervical cells (PCCs) (Figure 2D), highlighting the versatility of the workflow. In all cases, confocal microscopy revealed strong biotinylation after treatment with either DBCO-iridium or DBCO-biotin (Figure 7, S3) and irradiation in the presence of biotin-diaziline. In the absence of azido-sugars, no labeling was observed in any case.

[0031] Encouraged by these results, we developed a tandem mass tag (TMT)-based quantitative chemoproteomics workflow to identify sialic acid-added cell surface glycoproteins and map their interactomes (Figure 3A). For each cell type, we performed comparative experiments using three different conditions: the first condition (Condition A) included SPAAC with DBCO-biotin, which resulted in biotinylation of sialic acid-added glycoproteins only; Condition B utilized SPAAC with DBCO-iridium for biotinylation of sialic acid-added glycoproteins and their cognitive interactomes via μMap; and finally, a control experiment was performed using DBCO-iridium in the absence of Ac4ManNAz. Taken together, these parameters enabled the identification of 1) sialic acid-added glycoproteins, 2) the interactomes of sialic acid-added glycoproteins and their vicinity, and 3) the selective identification of protein-interacting factors (glycomap) of the cell surface sialome. The inventors first investigated this chemopromics workflow for HEK293T cells (Figure 3B). Using condition A (vs. control), the inventors observed significant enrichment of 363 protein (>1.5 Log² (change); >1.5-Log²). 10 We found p-values, and 93% of these were known glycoproteins, including nicastriin (NCSTN), cadherin 2 (CDH2), small molecule cell adhesion glycoprotein (SMAGP), surface antigen classification 47 (CD47), basigin (BSG), surface antigen classification 166 (CD166), surface antigen classification 99 (CD99), and neuroplastin (NPTN). As expected from Figure 3A, condition B (vs. control) enriched both sialic acid-added glycoproteins and neighboring proteins, and therefore shared ~65% overlap with the proteins enriched in condition A (vs. control) (Figure S9). Analysis of the sialic acid interactome generated by the glycomap showed that 81% of the enriched proteins were membrane-bound, reflecting the accuracy of our labeling approach. Furthermore, several lysosomal proteins (21) were enriched, which likely occurred through the internalization of iridium-binding glycoproteins prior to proximity labeling.

[0032] In the initial analysis of the μMap dataset, we examined a known membrane-bound protein complex, gamma-secretase, to validate our methodology. This heterotetramer of membrane proteins (NCSTIN, APHIA, PSEN1, PEN-2) proteolytically degrades many endogenous membrane proteins, but NCSTN alone undergoes direct sialication. In our dataset, NCSTN was highly enriched under condition A (vs. control) (3.8 log2FC), while the non-sialication interaction factor APH1A was strongly enriched in the μMap (3.3 log2FC), demonstrating that the μMap workflow can differentiate between sialicated glycoproteins and their interaction factors.

[0033] With the workflow established, the next step was to investigate the excess sialic acid addition event in tumorigenesis. To investigate this, the inventors performed comparative glycomapping experiments on both primary cervical cancer cells (PCCs) and HeLa cervical adenocarcinoma cell lines (Figure 4A).

[0034] Consistent with previous observations of upregulation of sialic acid addition, chemoproteomics analysis revealed significantly higher sialic acid addition in HeLa cells (447 enriched proteins) compared to PCCs (223 enriched proteins) (Figure 4A). This increase in sialome in HeLa cells resulted in a greater number of interacting proteins (166 enriched proteins in HeLa cells vs. 63 enriched proteins in PCCs).

[0035] Next, broad gene ontology (GO) analysis was performed to categorize the enriched sialic acid-added proteins and their interacting factors (Figure 4B). Functional enrichment from both cell types was consistent with the known roles of the identified glycoproteins: cell adhesion, host cell entry, and regulation of migration, death, and defense. Furthermore, there were significant differences between primary and cancerous cervical cells (≧10 Log). 10(p-value change) Testing under GO conditions verified that sialic acid-modified glycoproteins in cancer cells are associated with typical oncological phenotypes, including cell morphogenesis, intercellular adhesion, extracellular matrix organization, and tubular morphogenesis.

[0036] Interestingly, when comparing the roles of the identified sialic acid-interacting proteins, conditions related to small molecule transport were clearly enriched in HeLa cells (organic ion transport, small molecule transport, vitamin transport), and we focused in particular on many enriched solute carrier proteins (SLCs) in this dataset (Figure 4C). In particular, interactions between SLCs and sialic acid-added proteins related to ethanolamine, carnitine, and zinc transport were all significantly enriched in HeLa cells than in PCCs.

[0037] To explore the potential consequences of these interactions, the inventors investigated the metabolic results of enzymatically depleting them (Figure 5A). Treatment of HeLa cells with sialidase (VC-Sia) isolated from Vibrio Cholerae efficiently cleaved α2,3-, α2,6-, or α2,8-linked sialic acids to cell surface glycans, allowing for regulation of a wide range of sialic acid addition states. The inventors incubated HeLa cells in or without VC-Sia, and then performed metabolomics quantification based on mass spectrometry on cell metabolite extracts. While most metabolite levels were minimally affected by sialidase treatment, the inventors found that levels of ethanolamine derivatives, including cytidine diphosphate ethanolamine (CDP-Etn), phosphate ethanolamine (P-Etn), and cytidine diphosphate choline (CDP-choline), were significantly increased in sialidase-treated cells (Figure 5B). Although the solute carrier protein choline-like transporter 1 (SLC44A1), which is involved in ethanolamine transport, is not known to be glycosylated, our dataset suggests that its function is not regulated by nearby sialic acid-added glycoproteins. Based on these results, we hypothesize that cell surface sialic acid can present a negatively charged surface around membrane-bound transporters, which may subsequently influence the transport of ions, including metabolites.

[0038] Similarly, we also investigated the effect of sialic acid addition on zinc uptake (Figure 5C). Zinc is transported across the cell membrane by a series of solute carrier proteins of the SLC39 family, four of which have been shown to undergo sialic acid addition in our HeLa dataset (SLC39A6, SLC39A8, SLC39A10, and SLC39A14), and one (SLC39A1) is suggested to interact with sialic acid-added glycoproteins. Zinc is an essential micronutrient that plays a significant role in cellular function, and its transport is dysregulated in many cancers.

[0039] Using a colorimetric assay to determine zinc levels in untreated and sialidase-treated HeLa cells, we found that zinc levels were significantly higher in cells treated with VC-Sia. These data suggest that cell surface sialic acid addition and / or interaction with sialic acid-added glycoproteins play a role in regulating cellular zinc concentration.

[0040] In conclusion, hypersialic acid addition in cancer has attracted considerable interest from the academic and medical sectors in recent years. However, tools for understanding the biochemical consequences of hypersialic acid addition remain limited. Herein, we describe a novel proximity labeling platform for identifying sialic acid-added cell surface glycoproteins and their interacting factors. This highly sensitive and accurate method is robust and compatible with a diverse range of cell lines, including primary cells. Our comparative proteomics studies between primary and cancerous cervical cell lines demonstrate a significant association between sialic acid addition and solute carrier proteins, suggesting a novel role for sialic acid addition. Metabolomics data suggest that these interactions control the function of specific solute carriers. Taken together, our platform represents a powerful novel approach to sialic acid interactome profiling and provides a system-level tool for elucidating the biochemical consequences of hypersialic acid addition.

[0041] material All buffers and materials were used as received from commercial sources. Tetraacetyl-N-azidoacetylmannosamine (Ac4ManNAz) (900917), bovine serum albumin (BSA) (A7906), and Eppendorf Protein LoBind tubes (Z666505) were purchased from Millipore Sigma (St. Louis, MO). DBCO-sulfoconjugated biotin (DBCO-biotin) (BP-22296) was purchased from Broadpharm (San Diego, CA). Biotin-(peg)3-diazirine (biotin-diazirine) and [Ir(dCO2HdFCF3ppy)2(bpy-dbco)(DBCO-iridium) were synthesized as previously described. 1,2RIPA buffer (89900), 1X DPBS (14190144), Pierce BCA protein assay kit (23227), and iBright Prestained protein ladder (LC5615) were purchased from Thermo Scientific (Rockford, IL). TBST (IBB-581X) was purchased from Boston BioProducts (Ashland, MA). 12% Criterion TGX precast gel (5671044) and 4x Laemmli sample buffer (161-0747) were purchased from BioRad (Hercules, CA). Poly-L-lysine solution was purchased from Sigma-Aldrich (St. Louis, MO). Paraformaldehyde (16% solution) was obtained from Thermo Fisher Scientific (Rockford, IL). Streptabidin-Alexa Fluor 488 was obtained from BioLegend (San Diego, CA). Standard tissue culture plates were obtained from Thermo Fisher Scientific (Waltham, MA). DPBS (Gibco, #14190250), DMEM high glucose (Gibco, #31053036), DMEM high glucose, phenol red-free (Gibco, #31053028), fetal bovine serum (Gibco, #10437-028), penicillin-streptomycin (Gibco, #15070063), trypsin-EDTA (Gibco, #25300054), trypsin protease MS (Pierce, #PI90057), and RIPA buffer (Thermo, #89900) were obtained from Thermo Fisher Scientific. PMSF (Sigma Aldrich, #78830) and a completely EDTA-free protease inhibitor (Roche, #11873580001) were obtained from Sigma Aldrich. Streptoavidin magnetic beads were obtained from Thermo Fisher Scientific (Pierce, #88816).Trifluoroacetic acid (Optima grade), acetonitrile (Optima grade), water (Optima grade), and acetic acid (Optima grade) were obtained from Thermo Fisher Scientific. Triethylammonium bicarbonate (1M Sigma Aldrich, #90360), 50% hydroxylamine solution (Sigma Aldrich, #438227), ammonium bicarbonate (LiChropur, Merck, #5438350), and iodoacetamide (Sigma Aldrich, #I1149) were obtained from Sigma Aldrich. The TMT10plex kit (Thermo), urea (Pierce, Sequanal, #29700), and DTT (Thermo, #R0862) were obtained from Thermo Fischer Scientific.

[0042] cell line HEK293 (CRL321) and HeLa cells (CCL2) were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle medium (DMEM) high glucose (Gibco, #31053036) supplemented with 10% fetal bovine serum (Gibco, #10437-028) and 1% penicillin streptomycin (Gibco, #15070063) at 37°C and in 5% CO2 air in 10 cm plates.

[0043] Primary cervical epithelial cells (ATCC, #PCS-480-011) were cultured in the recommended medium (ATCC, #PCS-480-032) supplemented with the recommended proliferation kit (ATCC, #PCS-480-042), according to the protocol provided by ATCC.

[0044] antibody Anti-NCSTN (rabbit, polyclonal): Invitrogen (#PA5-17735) Anti-CD55 (rabbit, polyclonal): Invitrogen (#PA5-29657) Anti-actin (mouse, monoclonal): Cell Signaling Technologies (#3700S)

[0045] Glucose mapping experiment A typical experimental workflow is illustrated in Figure 2A.

[0046] Optimization of sugar mapping conditions HEK293T cells (approximately 0.4 x 10⁻¹⁰) 6Cells were incubated in 2 mL of complete DMEM in a 6-well plate at 37°C for 72 hours, either in or without Ac4ManNAz (100 μM). Cells were washed with DPBS (3 x 1 mL) and then incubated in 2 mL of complete DMEM containing DBCO-iridium (2.5-10.0 μM) at 37°C for 3-24 hours. Cells were washed with DPBS (3 x 1 mL) and irradiated in 200 μL of DMEM (phenol red-free) containing biotin-diazirine (250 μM) under a biophotoreactor (blue LED) for 20 minutes at room temperature. Cells were washed with DPBS (3 x 1 mL), scraped into 1 mL of DPBS, and transferred to a 1.5 mL Eppendorf tube. Cells were pelleted at 400 x G for 5 minutes and resuspended in Ripa lysis buffer (500 μL). Cells were lysed by sonication (bioruptor) at 4°C for 10 minutes (20 cycles, 100% power on for 15 seconds, off for 15 seconds). Protein concentration was standardized by BCA assay, and the lysates were analyzed by Western blotting (10 μg protein per lane, 12% gel, 150 V). The gel was transferred to an NC membrane using iBlot 2. After transfer, the membrane was stained with total protein stain, washed with washing solution (3 x 5 seconds), and imaged in a 700 nm channel using a Li-Cor Odyssey CLx scanner. The membrane was then immersed in Odyssey blocking buffer (Li-Cor, 927-50000) and incubated at room temperature for 1 hour. The blocking solution was decanted, and 10 mL of fresh blocking buffer containing 0.5 μL of IRDye 800CW streptavidin (Li-Cor, 926-32230) was added. This mixture was rocked for 60 minutes. After decanting the buffer and washing the membrane with 1X TBST (4x5 minutes) and water (3x5 seconds), it was imaged through a Li-Cor Odyssey CLx scanner in an 800 nm channel. Labeling efficiency was evaluated by concentration measurement by comparison with experiments performed without any Ac4ManNAz.

[0047] Western blot analysis HeLa cells (approximately 0.4 x 10⁻¹⁰) 6Cells were incubated in a 6-well plate in complete DMEM (2 mL) containing Ac4ManNAz (100 μM) at 37°C for 72 hours. The cells were washed with DPBS (3 x 1 mL) and then incubated in complete DMEM (2 mL) containing DBCO-iridium (5 μM) or DBCO-biotin (5 μM) at 37°C for 24 hours. The cells were washed with DPBS (3 x 1 mL) and irradiated in a biophotoreactor (blue LED) at room temperature for 20 minutes in DMEM (phenol red-free, 200 μL) containing biotin-diazirine (250 μM). The cells were washed with DPBS (3 x 1 mL), scraped into DPBS (1 mL), and transferred to a 1.5 mL Eppendorf tube. The cells were pelleted at 400 x G for 5 minutes and resuspended in Ripa lysis buffer (500 μL). Cells were lysed by sonication (bioruptor) at 4°C for 10 minutes (20 cycles, 15 seconds on at 100% power, 15 seconds off). Protein concentration was standardized by BCA assay, and the lysates were analyzed by Western blotting (20 μg protein per lane, 12% gel, 150V). The gel was transferred to an NC membrane using iBlot 2. After transfer, the membrane was immersed in Odyssey blocking buffer (Li-Cor, 927-50000) and incubated at room temperature for 1 hour. The blocking buffer was replaced with fresh blocking buffer (10 mL) containing anti-actin antibody (10 μL), and the membrane was locked for 1 hour. The buffer was decanted, and the membrane was washed with 1X TBST (4 x 5 minutes) and water (3 x 5 seconds). The blocking solution was decanted, and 10 mL of fresh blocking buffer containing 1 μL of IRDye 680RD goat anti-mouse IgG secondary antibody (Li-Cor, 926-68070) and 1 μL of IRDye 800CW streptavidin (Li-Cor, 926-32230) was added. This mixture was locked for 60 minutes. The buffer was decanted, and the membrane was washed with 1X TBST (4x5 minutes) and water (3x5 seconds), and then imaged through a Li-Cor Odyssey CLx scanner in 700 nm and 800 nm channels.

[0048] Confocal microscopy Cells (HEK293T, HeLa, or primary cervical epithelial cells) (approximately 2 x 10⁻¹⁰) 4 Cells were incubated at 37°C for 48 hours in polylysine-coated 8-well chamber slides in complete DMEM (200 μL) containing Ac4ManNAz (100 μM). Cells were washed with DPBS (3 x 200 μL) and then incubated at 37°C for 24 hours in complete DMEM (200 μL) containing DBCO-iridium (5 μM) or DBCO-biotin (5 μM). Cells were washed with DPBS (3 x 200 μL) and irradiated in a biophotoreactor (blue LED) for 20 minutes at room temperature in DMEM (phenol red-free, 200 μL) containing biotin-diaziline (250 μM). Cells were washed with DPBS (3 x 200 μL) and fixed in pre-warmed 4% paraformaldehyde (200 μL) for 30 minutes at room temperature. Cells were washed with DPBS (2 x 200 μL) and blocked with 3% BSA in DPBS (200 μL) at room temperature for 1 hour. The blocking buffer was replaced with fresh 3% BSA in DPBS (200 μL) containing Hoechst (1:100) and streptavidin-AlexaFluor 555 conjugate (1:1000). Cells were stained in the dark at room temperature for 1 hour and then stored in the dark at 4°C. Cells were imaged at 20x magnification on a NIKON A1R-SI microscope (Nikon Instruments, Inc., Melville, NY). Images were processed with Fiji-ImageJ. The images shown are representative of numerous cross-sectional images taken during each session.

[0049] streptavidin immunoprecipitation HeLa cells (approximately 5 x 10) 6Cells were incubated in a 10cm plate in 10mL of complete DMEM containing Ac4ManNAz (100μM) at 37°C for 72 hours. The cells were washed with DPBS (3x5mL) and then incubated in 5mL of complete DMEM containing DBCO-iridium (5μM) at 37°C for 24 hours. The cells were washed with DPBS (3x5mL) and irradiated in a biophotoreactor (blue LED) at room temperature for 20 minutes in 5mL of DMEM (phenol red-free) containing biotin-diazirine (250μM). The cells were washed with DPBS (3x5mL), scraped into 5mL of DPBS, and transferred to a 15mL conical tube. The cells were pelleted at 400xG for 5 minutes and resuspended in 1mL of Ripa lysis buffer containing a protease inhibitor cocktail. The cells were lysed by sonication (bioruptor) at 4°C for 10 minutes (20 cycles, 15 seconds on at 100% power, 15 seconds off).

[0050] Protein concentrations were standardized by BCA assay. Lysate (0.75 mg protein / experiment) was added to Pierce streptavidin beads (80 μL), and the beads were inverted at 4°C for 16 hours. The beads were washed with 1% SDS (3 x 500 μL, 5 minutes per wash), 1 M NaCl (3 x 500 μL), and 10% EtOH (3 x 500 μL). The protein was then eluted with elution buffer / laemmli (3:1, 40 μL) and boiled at 95°C for 15 minutes. The mother liquor was separated while still hot and analyzed by Western blotting, and compared to the lysate input (10 μg of protein per input lane) (12%, 150V).

[0051] The gel was transferred to the NC membrane using iBlot 2. After transfer, the membrane was immersed in Odyssey blocking buffer (Li-Cor, 927-50000) and incubated at room temperature for 1 hour. The blocking buffer was replaced with 10 mL of fresh blocking buffer containing 10 μL of anti-CD55 antibody, and the membrane was locked at 4°C for 16 hours. The buffer was decanted, and the membrane was washed with 1X TBST (4 x 5 minutes) and water (3 x 5 seconds). The blocking solution was decanted, and 10 mL of fresh blocking buffer containing 1 μL of IRDye 800CW goat anti-rabbit IgG secondary antibody (Li-Cor, 926-32211) was added. This mixture was locked at room temperature for 60 minutes. After decanting with buffer and washing the membrane with 1X TBST (4x5 minutes) and water (3x5 seconds), it was imaged through a Li-Cor Odyssey CLx scanner in an 800nm ​​channel.

[0052] For subsequent staining, the membrane was detached using Restore PLUS Western blot detachment buffer (Thermo Fisher Scientific, 46430) at room temperature for 30 minutes. The membrane was blocked and stained with anti-NCSTN antibody (1:1000) as described above.

[0053] Proteomics Workflow HeLa cells (approximately 5 x 10) 6Cells were incubated in a 10cm plate in 10mL of complete DMEM containing Ac4ManNAz (100μM) at 37°C for 72 hours. The cells were washed with DPBS (3x5mL) and then incubated in 5mL of complete DMEM containing DBCO-iridium (5μM) at 37°C for 24 hours. The cells were washed with DPBS (3x5mL) and irradiated in a biophotoreactor (blue LED) at room temperature for 20 minutes in 5mL of DMEM (phenol red-free) containing biotin-diazirine (250μM). The cells were washed with DPBS (3x5mL), scraped into 5mL of DPBS, and transferred to a 15mL conical tube. The cells were pelleted at 400xG for 5 minutes and resuspended in 1mL of Ripa lysis buffer containing a protease inhibitor cocktail. The cells were lysed by sonication (bioruptor) at 4°C for 10 minutes (20 cycles, 15 seconds on at 100% power, 15 seconds off).

[0054] Protein concentrations were standardized by a BCA assay. Lysate (2.0 mg protein / experiment) was added to Pierce streptavidin beads (200 μL), and the beads were inverted at 4°C for 16 hours. The beads were washed with 1% SDS (3 x 500 μL, 5 minutes per wash), 1 M NaCl (3 x 500 μL), and 10% EtOH (3 x 500 μL). The beads were resuspended in RIPA buffer (500 μL) and transferred to fresh 1.5 mL Lo-bind tubes.

[0055] The supernatant was removed, and the beads were washed with DPBS (3 x 500 μL) and NH4HCO3 (100 mM) (3 x 500 μL). The beads were resuspended in 6 M urea (500 μL) in DPBS, and 200 mM DTT (25 μL) in 25 mM NH4HCO3 was added. The beads were inverted at 55°C for 30 minutes. Subsequently, 500 mM iodoacetamide (30 μL) in 25 mM NH4HCO3 was added, and the beads were inverted in the dark at room temperature for 30 minutes. The supernatant was removed, and the beads were washed with DPBS (3 x 500 μL) and TEAB (50 mM) (3 x 500 μL). The beads were resuspended in TEAB (500 μL), transferred to a new Protein LoBind tube, pelletized, and the supernatant was removed.

[0056] The beads were resuspended in 50 mM TEAB (40 μL), trypsin (1 mg / mL in 50 mM acetic acid; 1.2 μL) was added, and the beads were inverted overnight at 37°C. After 16 hours, an additional 0.8 μL of trypsin was added, and the beads were inverted for another hour at 37°C. Subsequently, the beads were pelletized. Meanwhile, TMT10plex labeling reagent (0.8 mg) (Thermo) was equilibrated to room temperature, diluted in anhydrous acetonitrile (Optima grade; 41 μL, vortexed for 5 minutes), and the contents were collected by centrifugation.

[0057] Next, each set of trypsylated peptides was added to the corresponding TMT label (41 μL in MeCN, with 40 μL in TEAB added). The beads were then washed with an additional 20 μL of TEAB to collect the remaining peptides. The labeling reaction was allowed to proceed at room temperature for 2 hours. The samples were then quenched with 5% hydroxylamine (8 μL) and incubated at room temperature for 15 minutes. The samples were pooled in new Protein LoBind tubes and quenched with TFA (16 μL, Optima). The samples were stored at -80°C until proteomics was performed. The samples were desalted, fractionated, and electrophoresed.

[0058] Labeling efficiency Based on the Western blot results shown in Figure 2B, the labeling efficiency may be calculated via a sugar map using a concentration assay. In this calculation, it is assumed that the SPAAC efficiency of DBCO-iridium is comparable to that of DBCO-biotin. Using a concentration assay on streptavidin-stained Western blots (MW range from 45 kDa to the top of the blot), the inventors calculate approximately one tag per catalyst. The complete calculations are listed below: Concentration measurement readings for DBCO-iridium: 108,442 (total for all experiments) Concentration measurement reading DBCO-Iridium control: 6,164 Experiment / Control: 17.6 Concentration measurement readings for DBCO-biotin: 158,335 (total experiments) Concentration measurement reading DBCO-biotin control: 5,937 Experiment / Control: 26.7 Comparison: 17.6 / 26.7 = 0.7 tags per catalyst

[0059] Metabolomics experiments Metabolite extraction HeLa cells were grown in 5 mL of complete DMEM supplemented with 10% FBS for 72 hours in clear 10 cm plates (x6) in or without neuraminidase (20 U / mL). After 72 hours, the cells were washed with DPBS (3 x 5 mL) and incubated with TrypLE (1 mL) at 37°C for 15 minutes. To wash the plates, 4 mL of DPBS was used to transfer the cells to 15 mL conical tubes. The cells were pelleted at 500 x G for 4 minutes and resuspended in 1 mL of DPBS. The cells were counted, and 500,000 cells / experiment were transferred to 1.5 mL Eppendorf tubes. The cells were pelleted (500 x G for 4 minutes), the supernatant was removed, and the cells were lysed in ice-cold 80% MeOH (60 μL) at 0°C for 30 minutes. The cell lysates were clarified by centrifugation at 20,000xg for 25 minutes, transferred to fresh 0.5 mL Eppendorf tubes, and stored at -80°C until mass spectrometry was performed.

[0060] LC-MS HPLC-grade water, methanol, and acetonitrile were obtained from Thermo Fisher Scientific. Supernatant samples were melted at room temperature and maintained at 4°C in an autosampler. Samples were analyzed using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) coupled with a Vanquish UHPLC system. LC separation was achieved using an XBridge BEH amide column (2.1 mm × 150 mm, 2.5 μm particle size, 130 Å pore size; Waters, Milford, MA, USA) with a gradient of solvent A (20 mM ammonium acetate + 20 mM ammonium hydroxide in 95:5 water / acetonitrile) and solvent B (acetonitrile). The flow rate was 150 μl / min. The gradients were as follows: 0 min, 90%B; 2 min, 90%B; 3 min, 75%; 7 min, 75%B; 8 min, 70%; 9 min, 70%B; 10 min, 50%B; 12 min, 50%B; 13 min, 25%B; 14 min, 25%B; 16 min, 0%B; 20.5 min, 0%B; 21 min, 90%B; 25 min, 90%B. The column temperature was 25°C and the injection volume was 5 μL. The mass spectrometer was operated in full scan mode, with positive and negative modes performed separately, including m / z 70-1000. The resolution was 140,000 at m / z 200, the AGC target was 5E6, and the maximum injection time was 200 ms.

[0061] Data Analysis We performed data analysis using El-Maven and identified metabolites in our internal library using authentic standards. 3 Next, the metabolite signal intensities were further processed using Excel (median correction) and Graphpad Prism (transformation, t-test, and volcano plot generation).

[0062] Colorimetric analysis zinc assay The zinc assay was purchased from Abcam (ab102507), and the experiment was conducted according to the following method, adapted from the supplier's instructions for use.

[0063] HeLa cells were grown in complete medium (DMEM, supplemented with 10% FBS) for 96 hours in clear 10 cm plates, either in the presence (x3) or absence (x3) of VC-sialidase (20 mU / mL medium). The medium was changed every 24 hours, and fresh sialidase was added at these times. After 96 hours, the medium was removed and DPBS (5 mL) was added. The cells were scraped off, transferred to a 15 mL conical tube, and pelletized at 400 x G for 5 minutes. The supernatant was removed, and the cells were lysed in 50 μL of EDTA-free lysis buffer and lysed by sonication (bioruptor) at 4°C for 10 minutes.

[0064] The lysates were clarified at 18,000xg for 15 minutes and analyzed by BCA assay. 30 μL of cell lysates were transferred to a 1.5 mL Eppendorf tube containing 30 μL of 7% TCA to precipitate the proteins. The mixture was clarified at 18,000xg for 5 minutes, and 50 μL of the resulting solution was used for a zinc detection assay.

[0065] Zinc concentration (nmol zinc / mg in protein-free soluble form): 1 (Untreated): 1.7 nmol / mg 2 (Untreated): 2.0 nmol / mg 3 (Untreated): 1.8 nmol / mg 4 (Sia treatment): 1.4 nmol / mg 5 (Sia treatment): 1.4 nmol / mg 6 (Sia treatment): 1.4 nmol / mg

[0066] Comparison of zinc levels in PCCs and HeLa cells HeLa cells (x3) and PCC cells (x3) were grown in a clear 10 cm plate in complete medium for primary cervical cells (ATCC, PCS-480-032). The medium was removed and 5 mL of DPBS was added. The cells were scraped off, transferred to a 15 mL conical tube, and pelletized at 400 x G for 5 minutes. The supernatant was removed, and the cells were lysed in 50 μL of EDTA-free lysis buffer and lysed by sonication (bioruptor) at 4°C for 10 minutes.

[0067] The lysates were clarified at 18,000xg for 15 minutes and analyzed by BCA assay. 30 μL of cell lysates were transferred to a 1.5 mL Eppendorf tube containing 30 μL of 7% TCA to precipitate the proteins. The mixture was clarified at 18,000xg for 5 minutes, and 50 μL of the resulting solution was used for a zinc detection assay.

[0068] Zinc concentration (nmol zinc / mg in protein-free soluble form): 1(PCC): 2.7 nmol / mg 2(PCC): 2.7 nmol / mg 3(PCC): 3.0 nmol / mg 4(HeLa):2.3 nmol / mg 5(HeLa):2.5 nmol / mg 6(HeLa):2.3 nmol / mg

[0069] Various embodiments of the present invention are described in relation to the realization of various objectives of the present invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Many modifications and adaptations will be readily apparent to those skilled in the art without departing from the spirit and scope of the present invention.

Claims

1. Transition metal catalysts that couple to cell surface glycoproteins A conjugate that includes this.

2. The conjugate according to claim 1, wherein the transition metal catalyst is coupled to the glycoprotein via a derivatized sialic acid linker.

3. The conjugate according to claim 2, wherein the transition metal catalyst and the derivatized sialic acid linker are coupled through click chemistry.

4. The aforementioned transition metal complex is given by formula: 【Chemistry 1】 In the formula, M is a transition metal; A, D, E, G, Y, and Z are independently selected from C and N; R 3 ~R 7 Each of the following ring substituents represents an optional ring substituent from 1 to 4, and each of the optional ring substituents from 1 to 4 is independently alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, or -C(O)O - , -C(O)OR 8 , and -R 9 Selected from the group consisting of OH, R 8 R is selected from the group consisting of hydrogen and alkyl, 9 is alkyl; R 1 The group is selected from the group consisting of directly bonded alkylenes, alkenylenes, cycloalkylenes, cycloalkenylenes, arylenes, heteroalkylenes, heteroalkenylenes, heterocycloenes, and heteroarylenes; L is an optional linking portion selected from the group consisting of amides, esters, sulfonamides, sulfonates, carbamates, and ureas; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl, and R 8 and R 9 are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl ester; X - is a counterion and n is an integer from 0 to 20 The conjugate according to claim 1, wherein the conjugate is as described above.

5. R 2 The conjugate according to claim 4, wherein the click chemistry portion is selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkynes, and azides.

6. The conjugate according to claim 1, wherein M is a platinum group metal.

7. The conjugate according to claim 6, wherein M is iridium.

8. The conjugate according to claim 1, wherein the transition metal catalyst is a photocatalyst.

9. The conjugate according to claim 1, wherein the transition metal catalyst has an electronic structure for energy transfer to a protein labeling agent.

10. The conjugate according to claim 9, wherein the energy transfer is Dexter energy transfer.

11. A system for profiling the microenvironment near the sialic acid-added proteome; Protein labeling agent, A conjugate comprising a transition metal catalyst coupled to a cell surface glycoprotein via a derivatized sialic acid linker, wherein the transition metal catalyst has an electronic structure that enables energy transfer to the protein labeling agent and provides a reactive intermediate, and The system including the above.

12. The system according to claim 11, wherein the reactive intermediate can be operated to label proteins within a predetermined range of the conjugate.

13. The system according to claim 11, wherein the transition metal catalyst and the derivatized sialic acid linker are coupled by click chemistry.

14. The aforementioned transition metal complex is given by formula: 【Chemistry 2】 In the formula, M is a transition metal; A, D, E, G, Y, and Z are independently selected from C and N; R 3 ~R 7 Each of the following ring substituents represents an optional ring substituent from 1 to 4, and each of the optional ring substituents from 1 to 4 is independently alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, or -C(O)O - , -C(O)OR 8 , and -R 9 Selected from the group consisting of OH, R 8 R is selected from the group consisting of hydrogen and alkyl, 9 is alkyl; R 1 The group is selected from the group consisting of directly bonded alkylenes, alkenylenes, cycloalkylenes, cycloalkenylenes, arylenes, heteroalkylenes, heteroalkenylenes, heterocycloenes, and heteroarylenes; L is an optional linking portion selected from the group consisting of amides, esters, sulfonamides, sulfonates, carbamates, and ureas; R 2 Alkynes, amines, protective amines, azides, hydrazides, aryls, heteroaryls, cycloalkyls, cycloalkenyls, cycloalkylyls, heterocyclyls, hydroxyls, carboxyls, halos, alkoxys, maleimides, -C(O)H, -C(O)OR 8 , -OS(O 2 )R 9 , thiols, biotin, oxyamines, and haloalkyls, R 8 and R 9 X is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; X - is the pair ion, and n is an integer between 0 and 20. The system according to claim 11, wherein the system is as described above.

15. R 2 The system according to claim 14, wherein the click chemistry portion is selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkynes, and azides.

16. The system according to claim 14, wherein M is a platinum group metal.

17. The system according to claim 16, wherein M is iridium.

18. The system according to claim 11, wherein the transition metal catalyst is a photocatalyst.

19. The system according to claim 11, wherein the energy transfer is Dexter energy transfer.

20. The system according to claim 11, wherein the protein labeling agent is diazirine.

21. The system according to claim 20, wherein the diazirine contains a molecular marker.

22. A method for profiling the microenvironment near a sialic acid-added proteome: The steps include forming a conjugate containing a transition metal catalyst that is coupled to a cell surface glycoprotein via a derivatized sialic acid linker, The protein labeling agent is activated into a reactive intermediate using the transition metal catalyst, The steps include coupling the reactive intermediate to a protein within a predetermined range of the conjugate, and The method, including the method described above.

23. The method according to claim 22, wherein the step of activating the protein labeling agent in the reactive intermediate includes energy transfer from the transition metal catalyst to the protein labeling agent.

24. The method according to claim 22, wherein the protein labeling agent is diazirine.

25. The method according to claim 24, wherein the diazirine is functionalized with a marker.

26. The method according to claim 22, wherein the predetermined range is 2 to 100 nm.

27. The method according to claim 22, wherein the reactive intermediate is quenched outside the predetermined range, and binding to biomolecules outside the predetermined range is eliminated.

28. The method according to claim 22, further comprising detecting or analyzing the protein coupled to the reactive intermediate.

29. The aforementioned transition metal complex is given by formula: 【Transformation 3】 In the formula, M is a transition metal; A, D, E, G, Y, and Z are independently selected from C and N; R 3 ~R 7 Each of the following ring substituents represents an optional ring substituent from 1 to 4, and each of the optional ring substituents from 1 to 4 is independently alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, or -C(O)O - , -C(O)OR 8 , and -R 9 Selected from the group consisting of OH, R 8 R is selected from the group consisting of hydrogen and alkyl, 9 is alkyl; R 1 The group is selected from the group consisting of directly bonded alkylenes, alkenylenes, cycloalkylenes, cycloalkenylenes, arylenes, heteroalkylenes, heteroalkenylenes, heterocycloenes, and heteroarylenes; L is an optional linking portion selected from the group consisting of amides, esters, sulfonamides, sulfonates, carbamates, and ureas; R 2 Alkynes, amines, protective amines, azides, hydrazides, aryls, heteroaryls, cycloalkyls, cycloalkenyls, cycloalkylyls, heterocyclyls, hydroxyls, carboxyls, halos, alkoxys, maleimides, -C(O)H, -C(O)OR 8 , -OS(O 2 )R 9 , thiols, biotin, oxyamines, and haloalkyls, R 8 and R 9 X is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; X - is the pair ion, and n is an integer between 0 and 20. The method according to claim 22.

30. R 2 The method according to claim 29, wherein the click chemistry portion is selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkynes, and azides.

31. The method according to claim 29, wherein M is a platinum group metal.

32. The method according to claim 31, wherein M is iridium.

33. The method according to claim 22, wherein the transition metal catalyst is a photocatalyst.

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