A Click Chemistry-Based Biorthogonal Approach for the Detection and Identification of Protein Lysine Malonylation for Osteoarthritis Research

Abstract

Lysine malonylation is a post-translational modification in which a malonyl group, characterized by a negatively charged carboxylate, is covalently attached to the ε-amino side chain of lysine, influencing protein structure and function. Our laboratory identified Mak upregulation in cartilage under aging and obesity, contributing to osteoarthritis (OA). Current antibody-based detection methods face limitations in identifying Mak targets. Here, we introduce an alkyne-functionalized probe, MA-diyne, which metabolically incorporates into proteins, enabling copper­(I) ion-catalyzed click reactions to conjugate labeled proteins with azide-based fluorescent dyes or affinity purification tags. In-gel fluorescence confirms MA-diyne incorporation into proteins across various cell types and species, including mouse chondrocytes, adipocytes, HEK293T cells, and Caenorhabditis elegans. Pull-down experiments identified known Mak proteins, such as GAPDH and Aldolase. The extent of MA-diyne modification was higher in Sirtuin 5-deficient cells, suggesting these modified proteins are Sirtuin 5 substrates. Pulse-chase experiments confirmed the dynamic nature of the protein malonylation. Quantitative proteomics identified 1136 proteins corresponding to 8903 peptides, with 429 proteins showing a 1-fold increase in the labeled group. Sirtuin 5 regulated 374 of these proteins. Pull down of newly identified proteins, such as β-actin and Stat3, was also done. This study highlights MA-diyne as a powerful chemical tool to investigate the molecular targets and functions of lysine malonylation under OA conditions.

1. Introduction

Post-translational modification of proteins refers to the biochemical covalent, enzymatic, and nonenzymatic addition of functional groups on proteins following their synthesis from ribosomes. These functional groups can be small electrophilic biochemical metabolites like phosphate, sugars, nucleotides, methyl group, and acetyl group, long chain/short chain acyl chains like palmitoyl, malonyl groups, radicals generated from redox reactions like S-nitrosylation (SNO) and S-glutathionylation, etc. Post-translational modifications induce changes in amino acid chemical properties such as deamination, deamidation, citrullination, and oxidation as well as protein autocatalysis that results in protein backbone cleavage. It offers complex and diverse functional roles to the existing proteome by regulating protein activity, structure, and conformation, its location, and molecular interactions. Post-translational covalent modification of proteins is fundamental to numerous cellular and biological functions. It plays a significant role in regulating normal cell physiology as well as the pathogenesis of diseases. Lysine malonylation (Mak) is a reversible protein post-translational modification wherein a malonyl group is added to the ε-amino group of the lysine residue in a protein. The addition of a negatively charged carboxylate group to the protein imparts significant changes in the protein’s structure and function. The reversible protein malonylation is theoretically regulated by acyltransferases and deacylases. The enzymes responsible for catalyzing the transfer of a malonyl group remain largely unidentified. However, recent work by Zang et al. has provided evidence that KAT2A is involved in histone malonylation. Meanwhile, Sirtuin 5 (Sirt5), a class III lysine deacetylases member, is known for its lysine demalonylation activity along with its lysine desuccinylation function. , Our lab has previously demonstrated that deficiency of Sirt5 in mouse primary chondrocytes increases the global protein MaK level and disrupts cellular metabolism. Identification of the protein substrates of Sirt5 is crucial for further elucidating the function of malonylation on the cellular metabolism and pinpointing the downstream targets. The antimalonyl lysine antibody has been used previously to enrich and identify several malonylated proteins. − Antibody-based enrichment is generally based on the development of different antibody domains specific to targeting antigens with specific PTM, followed by immunoaffinity pull-down of target peptides from the protein lysates. However, this strategy suffers from a lack of specificity by missing some of the target PTM site due to the presence of adjacent PTM present on the same sequence. To overcome this challenge, we took a chemical approach, utilizing an alkyne functionalized chemical probe to efficiently detect and quantify protein substrates of lysine malonylation through fluorescence visualization and further identify them using a quantitative proteomics approach (Figure B). An alkyne functionalized probe allows metabolic detection of protein substrates by utilizing the copper­(I) ion-catalyzed alkyne azide cycloaddition click chemistry to conjugate the labeled proteins with azide-fluorescent dyes or affinity purification tags. This approach has been widely utilized in identifying several other types of PTMs including lipidation like myristoylation, , succinylation, , acetylation, , and glycosylation. , Previously, Bao et al. developed a malonic acid-based chemical probe called MalAM-yne to detect lysine malonylation. In this study, we designed and synthesized a novel Meldrum’s acid-based probe, i.e., MA-diyne. Meldrum’s acid or isopropylidene malonate is a condensation product of malonic acid and acetone. We developed this new probe by functionalizing Meldrum acid or isopropylidene malonate with alkynylation under control conditions (Figure A). We hypothesize that Meldrum acid will be readily absorbed into the cells due to its cyclic structure and subsequently will be linearized to form a malonyl group by an unknown action of intracellular esterases, thus leading to effective detection and identification of the malonylated proteins.

1.

(A) Design, synthesis, and characterization of MA-diyne. (B) Schematic description of the workflow to detect and identify lysine malonylated proteins using MA-diyne.

2. Results and Discussion

2.1. Chemical Synthesis and Characterization

Bao et al. 2013 were the first to report a chemical probe with an alkyne handle on the malonate group named Mal-yne. However, they modified this probe to increase cell permeability by masking the carboxylate sides with two acetoxymethyl groups and called it MalAM-yne. In this report, we selected an alternative approach by adding an alkyne handle to Meldrum acid or isopropylidene malonate. , The high reactivity of this molecule is attributed to the methylene group present between the carbonyl groups, and this site was utilized to add the propargyl unit to Meldrum acid under controlled conditions. Thereby, we generated dipropargyl Meldrum acid or MA-diyne through the following chemical synthesis (Figure A). The synthesis involves dialkynylation on commercially available Meldrum acid. The reaction is performed under mild conditions such as DIPEA (diisopropyl ethylamine) with anhydrous DMF solvent at room temperature. The reaction is stirred for 24 h. After the work-up of the reaction, the product is taken for purification. The initial purification of the MA-diyne product is carried out with normal SiO₂, which leads to ring-opening of MA-diyne due to its acidity. Later, SiO₂ is neutralized with triethylamine, and then, purification is carried out. The pure product is characterized by ¹H NMR spectroscopy [¹H NMR (400 MHz): δ 1.84 (s, 6H), 2.18, (t, J = 5.2 Hz, 2H), 2.88 (d, J = 2.8 Hz, 4H) ppm. The respective assignments and full spectra of MA-diyne are given as the Supporting Information (Schemes SF1 and SF2). The synthesized probe showed two distinct quaternary carbons: (i) near two oxygen atoms and (ii) near two carbonyl groups. The same quaternary peaks were observed in the ¹³C NMR spectrum: one at 107 ppm, which is near oxygen atoms, and another at 53.43 ppm, which is near carbonyl groups. This confirms dipropargylation in the active methylene carbon of Meldrum’s acid. The ¹³C NMR spectrum peaks are depicted in Scheme SF3. Although mass spectrum analysis of dipropargyl Meldrum’s acid revealed no M+ peak for 220.07, we observed smaller fragments (sized 192.04, 148.05, 92.06, and 203.28) as shown in Scheme SF4. The reasons for the fragmentation of the parent molecule could be attributed to the labile nature of the lactone structure. However, the occurrence of smaller fragments indirectly confirms the expected compound. The previous method by Bao et al. 2013 involves a three-step strategy, where the first step is propargylation of diethyl malonate followed by hydrolysis to propargylated malonic acid with a yield of 30%. Then, propargylated malonic acid is further taken for masking the carboxylate groups with bromomethyl acetate under ambient temperature, resulting in the product formation of the Mal-yne probe with a yield of 77%. As a result, the reported method gives an overall yield of 23%. MA-diyne, on the other hand, was synthesized via a streamlined synthetic approach, employing dialkynylation of Meldrum’s acid, which is commercially available. This method proved both straightforward (a single-step process) and expeditious, achieving a maximum yield of 40%, markedly surpassing the 23% yield (overall) obtained with MalAM-yne. However, the yield limitation to 40% in the present method arises from the ring-opening reaction of Meldrum’s acid under the specified conditions. Furthermore, cyclic structures are seen with improved cell permeability due to their reduced conformational flexibility. − Therefore, we hypothesized that Meldrum acid due to its cyclic structure will be readily taken into the cell. MA-diyne demonstrated comparable labeling efficiency over a 0.5–1 h duration to MalAM-yne, without necessitating the protection or masking of Mal-yne’s carboxylate groups with two acetoxymethyl (AM) substituents (as required to form MalAM-yne). This suggests efficient cellular uptake. The enhanced permeability of MA-diyne may be ascribed to the stabilization of its carboxylate groups within a cyclic structure.

2.2. MA-Diyne Can Be Metabolically Incorporated into Cellular Proteins

Our first inquest was to analyze whether MA-diyne can be metabolically incorporated into proteins similarly to malonyl-CoA. Therefore, we incubated mouse primary chondrocytes with different concentrations (0–200 μM) of MA-diyne in complete medium for 6 h (a working stock of 100 mM was prepared by dissolving MA-diyne in DMSO; DMSO was taken as the control). The protein lysate was collected after harvesting the cells and was subjected to azide–alkyne click chemistry to conjugate the alkyne side group with IR680 azide dye. The clicked proteins were then resolved on SDS-PAGE and scanned using an in-gel IR fluorescence imager. The result showed that there was a dose-dependent increase of MA-diyne labeling of global proteins with the optimal concentration at less than 50 μM MA-diyne (Figure A). To analyze the time required for the metabolic labeling, a time-dependent experiment was conducted by incubating the primary chondrocytes with 100 μM of MA-diyne at different time points ranging from 0 to 12 h. MA-diyne was able to efficiently label the proteins in no more than 2 h (Figure B). Interestingly, we observed that MA-diyne showed more bands in gel fluorescence than in Western blot analysis of the same proteins from primary chondrocytes using a commercial anti-KMal antibody (Figure S1). The dose- and time-dependent experiment demonstrated the efficient labeling of proteins by MA-diyne. MA-diyne employs a strategy of metabolic labeling of proteins. Since malonyl-CoA can target both cysteine and lysine residues due to their nucleophilic properties, it is imperative to recognize that MA-diyne may label both amino acids. In fact, previous studies have shown that malonylation of a single cysteine residue is critical for the activity enzymes CPT1 (a pivotal enzyme facilitating the translocation of long-chain fatty acids from the cytoplasm to the mitochondrial matrix, modulated by its physiological inhibitor, malonyl-CoA) and DpgA (a bacterial type III polyketide synthase that decarboxylates and concatenates four malonyl-CoA units to yield 3,5-dihydroxyphenylacetyl-CoA). To elucidate the chemoselectivity of MA-diyne toward lysine, we conducted a labeling experiment in the presence and absence of hydroxylamine, a chemical to mask cysteine residues. As depicted in Figure S2A,B, the metabolic labeling exhibited minimal reduction when MA-diyne-labeled protein lysates were subjected to incubation with 1 M hydroxylamine at 95 °C for 5 min. This observation suggests that although MA-diyne can label both lysine and cysteine side chains on proteins, it can predominantly label lysine side chains with a markedly greater propensity than it can cysteine groups. Another possibility is that the lower abundance of cysteines than of lysines in the protein may result in only a minimal reduction in labeling when cysteines are masked by hydroxylamine. Further studies are warranted to investigate this possibility in more detail.

2.

Assessment of the ability of MA-diyne to metabolically label proteins. (A) Mouse primary chondrocytes were incubated with the indicated concentration of MA-diyne for 6 h. The cell lysates were then clicked with IR680-azide followed by in-gel fluorescence analysis. (B) Mouse primary chondrocytes were incubated with 100 μM MA-diyne for the indicated time points. Cell lysates were then clicked with IR680-azide and in-gel fluorescence analyses. β actin was used as a loading control. (C) Confocal microscopic fluorescence image depicting the ready uptake of the MA-diyne into the cells and subcellular localization of malonylated proteins in the primary chondrocytes. The primary chondrocytes grown on coverslips were labeled with 100 μM MA-diyne for 6 h and then subjected to click chemistry with Carboxyrhodamine 110 Azide (fluorescein isothiocyanate-FITC tag) after fixing and permeabilization. Scale bar: 50 μM. (D) Box plot showing the concentration of intracellular malonyl-CoA in the cells after incubation with different concentrations of MA-diyne for 4 h. n = 4. Data are presented as mean ± SEM. Three group comparisons were evaluated using two-way ANOVA. Significance is noted as ns p > 0.05, *p < 0.05, and **p < 0.01.

To confirm that the signal achieved in the primary chondrocytes after treatment with MA-diyne is due to metabolic labeling and not to nonspecific binding, we used qualitative fluorescence imaging to visualize the labeled proteins. The primary chondrocytes were cultured on coverslips overnight and treated with different doses of MA-diyne for 6 h. The cells were immediately fixed using ice-cold 4% paraformaldehyde and then permeabilized. The cells were then subjected to a click reaction using Carboxyrhodamine 110 Azide for 1 h. The cells on the coverslips were mounted on clean glass slides using a mounting reagent with DAPI. Control samples were generated by performing the same procedure without MA-diyne or Carboxyrhodamine 110 Azide. The slides were imaged using Nikon A1R confocal microscopy under 60× magnification (Figures C and S3). MA-diyne was found to be rapidly absorbed into the cells, which was demonstrated by the intensity of labeling only in the samples treated with MA-diyne followed by the click reaction with Carboxyrhodamine 110 Azide but not in the control samples incubated with Carboxyrhodamine 110 Azide only or samples treated with only MA-diyne (Figure S3). We observed widespread protein labeling in various cell compartments, prompting us to conduct quantitative proteomics. However, to ensure the labeling is due to the malonyl-CoA formed by the acyclization of Meldrum acid, we estimated the concentration of malonyl-CoA formed in the cells using an ELISA kit after incubating the cells with MA-diyne in different concentrations and compared it with that in control cells without MA-diyne. We observed a significant increase in the concentration of malonyl CoA at 100 μM. Here, it is noteworthy to state the limitation of the ELISA kit in estimating the malonyl CoA in the alkyne form. Nevertheless, as protein labeling by MA-diyne intensifies with prolonged incubation, the measured malonyl-CoA likely reflects the residual malonyl-CoA remaining after conjugation with proteins rather than the total malonyl-CoA produced from the linearization of MA-diyne.

2.3. MA-Diyne Was Dynamically Removed from the Proteins, Representing the Reversible Nature of Lysine Malonylation

Lysine modifications like succinylation, glutarylation, and malonylation are reversible forms of protein post-translational modifications. ,, Although the enzyme responsible for adding a malonyl group to the proteins is still unknown, Sirtuin 5 (Sirt5) has been well-documented as the enzyme responsible for removing this modification. , To examine whether the labeling by MA-diyne is also reversible, we conducted a pulse-chase experiment. The wild type and Sirt5 knockdown primary chondrocytes were first incubated with 200 μM MA-diyne for 1 h. The cells were then washed and chased with 200 μM Meldrum acid for 0–5 h. The cells were harvested at different time points, and the protein lysates were conjugated with IR680-azide dye. The in-gel fluorescence imaging reveals that the labeling signal by MA-diyne in wild-type primary chondrocytes started fading after 0.5 h of Meldrum acid incubation, indicating the removal of MA-diyne labeling from the proteins (Figures A and S4). In comparison, in Sirt5 knockdown cells, the labeling signal with MA-diyne became more intense after 0.5 h than the baseline level, and there is less removal of MA-diyne from the labeled proteins than from the wild-type cells (Figures B and S4). We also noticed that the labeling by MA-diyne was not modulated by the endogenous levels of malonyl-CoA, evidenced by no difference between wild type cells and cells deficient of acetyl-CoA carboxylase (ACC1), an enzyme that produces malonyl-CoA (Figure S5). These results suggested that the metabolically modified proteins by MA-diyne could be substrates of Sirt5 demalonylase and the process is reversible.

3.

Pulse-chase experiment to determine the dynamic nature of lysine malonylation. Wild-type mouse primary chondrocytes (A) and Sirt5 KD primary chondrocytes (B) were labeled with 200 μM MA-diyne for 1 h and then pulse chased with 200 μM Meldrum acid (precursor). The lysates were collected at the indicated time points followed by a click reaction with IR680-azide and in gel fluorescence analysis. β actin was used as a loading control. The same blot was probed with an anti-Sirt5 antibody to quantify the knockdown efficiency.

2.4. MA-Diyne Successfully Identified Putative Malonylated Proteins

Our previous studies have demonstrated the important role of Sirt5 in chondrocyte metabolism by regulating the malonylation of metabolic enzymes. − We have also used proteomics to identify the malonylome in chondrocytes using the traditional Kmal PTMscan antibody-based enrichment method. We reported the enrichment of 1000 peptides corresponding to 469 proteins. Herein this report, we applied a chemoproteomics-based approach to enrich malonylated proteins from primary chondrocytes using MA-diyne. Wild type and Sirt5 knockdown primary chondrocytes were labeled with MA-diyne (100 μM) for 6 h, and then, the protein lysates were conjugated to biotin azide through an azide–alkyne copper cycloaddition reaction. The biotin-conjugated proteins were then immunoprecipitated using avidin agarose beads. The beads were thoroughly washed with HPLC water to prevent detergent or protease inhibitor contamination in the LC–MS, followed by the addition of a 9 M urea wash. The beads were then subjected to reductive alkylation with dithiothreitol (DTT) and iodoacetamide (IAA), on-bead trypsin digestion, and desalting. The enriched peptides were then subjected to bottom-up quantitative proteomics on an Orbitrap Astral Instrument. A total of 1136 proteins corresponding to 8903 peptides across all samples (Supporting Information excel 1) were identified, which was 2.4 times more than the proteins we identified before using Kmal PTMscan antibody enrichment. 430 proteins were seen to show a more than 1-fold increase in the probe group in comparison to the control group with more than 6 unique peptides (Supporting Information excel 2). This indicated that MA-diyne could enrich malonylated proteins. The enrichment of proteins in the control group accounts for the endogenously biotinylated proteins that might have been enriched with avidin beads. Furthermore, 387 out of the 430 proteins were found to have a more than 1-fold increase in the Sirt5 knockdown + MA-diyne group in comparison to the wild type + MA-diyne group (Supporting Information excel 2). This indicates that Ma-diyne successfully enriched proteins that are regulated by Sirt5 demalonylase enzyme. It is possible that MA-diyne may nonspecifically bind to other protein residues due to the structural difference of Meldrum acid (precursor of MA-diyne) from malonic acid. Therefore, we compared the 8903 peptides to those modified sites identified by Kmal PTMscan antibody enrichment in our previous study. Interestingly, some of the peptides sequences identified in MA-diyne enrichment matched with the sequence identified in the LC–MS/MS data acquired after enrichment with the Kmal PTMscan antibody (Table ). All these observations indicate that MA-diyne can efficiently detect and identify lysine malonylated proteins, though we still could completely exclude the possibility of nonspecific binding. Since our data lack site-specific identification of malonylation, our lab is currently exploring alternative proteomic approaches to identify malonylated sites on proteins detected by MA-diyne in MA-diyne-treated primary chondrocytes. Further, these proteins will be pulled down using enrichment tags and analyzed by LC–MS/MS for the signature satellite peaks.

1. List of Manually Validated Peptides Sequences with Modified Malonylated Sites.

Protein description	Accession number	Peptide sequence identified with Kmal enrichment	Corresponding peptides enriched in MA-diyne treatment
L-Lactate dehydrogenase A chain	P06151	IVSSKDYCVTANSK*	IVSSKDYCVTANSK, DYCVTANSK
Glucose-6-phosphate Isomerase	P06745	ELQAAGK*SPEDLEK	ELQAAGKSPEDLEK
Annexin A1	P10107	K*ALLALAK	KALLALAK
Annexin A2	P07356	ASM#K*GLGTDEDSLIEIICSR ELYDAGVK*R TK*GVDEVTIVNILTNR	TPAQYDASELKASM#K, TPAQYDASELK, GLGTDEDSLIEIICSR ELYDAGVKR TKGVDEVTIVNILTNR
Calmodulin-1	P0DP26; P0DP27; P0DP28	EAFSLFDK*DGDGTITTK	EAFSLFDK, DGDGTITTK
Protein S100-A4	P07091	ELPSFLGK*R	ELPSFLGK
Actin, cytoplasmic 1	P60710; P63260	K*DLYANTVLSGGTTM#YPGIADR	KDLYANTVLSGGTTM#YPGIADR
Src substrate cortactin	Q60598	SAVGHEYQSK*LSK	SAVGHEYQSK
Moesin	P26041	AK*FYPEDVSEELIQDITQR	AKFYPEDVSEELIQDITQR
Vinculin	Q64727	NLGPGM#TKM#AK*	NLGPGMTK
Glyceraldehyde-3-phosphate dehydrogenase	P16858	VIHDNFGIVEGLM#TTVHAITATQK*TVDGPSGK	VIHDNFGIVEGLM#TTVHAITATQK
Glyceraldehyde-3-phosphate dehydrogenase	P16858	LVINGK*PITIFQERDPTNIK	LVINGKPITIFQERDPTNIK
Glyceraldehyde-3-phosphatedehydrogenase	P16858	TVDGPSGK*LWR	TVDGPSGKLWR
Peptidyl-prolyl cis–trans isomerase A	P17742	SIYGEK*FEDENFILK	SIYGEKFEDENFILK
Pyruvate kinase PKM	P52480	CCSGAIIVLTK*SGR	CCSGAIIVLTK
Myosin regulatory light chain 12B	Q3THE2	K*GNFNYIEFTR	GNFNYIEFTR
High mobility group protein B1	P63158	K*HPDASVNFSEFSK	KHPDASVNFSEFSK
Elongation factor 1-alpha 1	P10126	SGK*KLEDGPK	THINIVVIGHVDSGK, KLEDGPK
Elongation factor 2	P58252	EDLYLK*PIQR	EDLYLKPIQR

We then conducted a pull-down experiment to validate further that MA-diyne could enrich malonylated proteins. The eluted protein from the avidin beads in a separate experiment were resolved on the SDS-PAGE followed by immunoblotting against antibodies for some already known malonylated proteins, ALDOA (fructose-bisphosphate aldolase A) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The detection of GAPDH and ALDOA by Western blot analysis of the eluted proteins from the beads (Figure A) confirmed that MA-diyne can indeed enrich malonylated proteins. We could also pull down other novel proteins in our proteomics list like Stat3 and β actin that have not been reported before (Figure B). Furthermore, we also experimented to validate the chemoselectivity of MA-diyne by introducing a single amino acid mutation in the GAPDH gene at position 184 by replacing lysine amino acid with glutamate amino acid. The primary chondrocytes were transfected with wild type GAPDH as well as GAPDH K184E plasmids, and then, the cells were incubated with MA-diyne and the protein lysates were tagged with biotin azide via a click reaction followed by avidin beads enrichment. The eluted proteins from the avidin beads were subjected to Western blot and probed for the anti-GAPDH antibody. Figure C demonstrates that MA-diyne was unable to label GAPDH with single amino acid mutation of K184E in comparison to wild GAPDH, suggesting the chemoselectivity of MA-diyne to the lysine residue.

4.

Validation of lysine malonylated protein targets of MA-diyne. (A) Pull down of known malonylated proteins such as GAPDH and Aldolase A after enrichment using avidin beads. n = 2. (B) Pull down of newly identified proteins after MA-diyne enrichment. n = 2. (C) Pull-down of MA-diyne labeled GAPDH protein after overexpressing GAPDH wild type and GAPDH K184E plasmids in primary chondrocytes. Primary chondrocytes were overexpressed with GAPDH wild type and GAPDH K184E plasmids for 48 h and then labeled with MA-diyne. The protein lysates were clicked with biotin azide, and avidin beads were used to enrich the biotinylated proteins. The elute proteins from the avidin beads were resolved on SDS Gel and then transferred on a PVDF membrane. The blot was probed for anti-GAPDH antibodies. (D) Pie chart depicting the percentage of malonylated proteins found in different subcellular compartments by GO analysis. (E) Distribution of enriched proteins based on their biological process by GO analysis, (F) KEGG pathway analysis of the enriched proteins. p value < 0.05.

Gene ontology (GO) and pathway analysis of the identified proteins revealed that the malonylated proteins are localized differentially in various subcellular compartments including the cytoplasm, plasma membranes, endoplasmic reticulum, nucleus, mitochondria, and Golgi apparatus of the primary chondrocytes (Figure D and Supplementary excel 3). These findings align with similar subcellular localization patterns of malonylated proteins observed in other cell types, such as plants, prokaryotes, parasites, and mammalian cells. GO analysis based on the biological processes revealed that the identified malonylated proteins belonged to processes related to musculoskeletal tissues like collagen biosynthesis and ossification. Additionally, it was revealed that malonylated proteins are highly involved in glucose, amino acid, and pyruvate metabolic and lipid transport processes. The identified proteins are also enriched in biological processes like ribose phosphate metabolism, ATP production, TCA cycle, glycolysis, and oxidative phosphorylation (Figure E and Supplementary excel 4). These findings are consistent with previous reports on the involvement of lysine malonylation in regulation of metabolic disorders like type 2 diabetes, cardiovascular diseases, osteoarthritis, , etc. We also observed the role of malonylated proteins in the mechanisms related to the quality control of proteins such as response to endoplasmic reticulum stress, autophagy, and proteasome-mediated protein processing (Figure E and Supplementary excel 5). KEGG analysis of the identified malonylated proteins revealed regulation of several signaling pathways like VEGF, Wnt signaling, cGMP-PKG, PI3K-AKT, estrogen signaling, EGFR tyrosine kinase inhibitor resistance, growth hormone synthesis and secretion, phospholipase D signaling pathways, etc (Figure F and Supplementary excel 5).

2.5. MA-Diyne Can Detect Lysine Malonylation in a Wide Range of Cell Types In Vitro

To assess the applicability of the MA-diyne probe in detecting malonylated proteins in different types of cells, we analyzed the metabolic labeling profile of MA-diyne in subcutaneous primary adipocytes (Figure A) and HEK 293T cell lines (Figure B). Both cells exhibited robust MA-diyne signaling compared to the control, suggesting successful metabolic incorporation of MA-diyne into cellular proteins. In addition, we assessed whether MA-diyne can be used to metabolically label proteins of C. elegans. We incubated live C. elegans with 1 mM MA-diyne for 6 h with constant shaking and then lysed the worms to extract proteins. The proteins were then conjugated with the IR680 azide. In-gel fluorescence analysis revealed that MA-diyne resulted in a robust labeling of malonylated proteins (Figure C). The route for the metabolic labeling is unclear, but it is assumed that MA-diyne could have penetrated C.elegans cells via either the esophageal route or epidermal absorption/passive diffusion.

5.

MA-diyne can detect lysine malonylated proteins in other cells like primary subcutaneous adipocytes, HEK293T cells, and C. elegans, n = 2.

3. Conclusions

In summary, we have developed a novel chemical probe MA-diyne for identifying and quantifying the protein malonylation in primary chondrocytes and several other types of cells. This probe was synthesized by adding a propargyl group to the Meldrum acid for utilizing the conventional chemoproteomic approach to pull down the malonylated proteins. MA-diyne was observed to be readily uptaken into the cells, and after getting acyclized to malonyl-CoA by the action of intracellular esterases, it then metabolically labeled the proteins. It was also observed that labeling by MA-diyne was dynamic and regulated by the demalonylase enzyme, Sirt5. Moreover, the labeling by MA-diyne was observed to be nonenzymatic and not dependent on the endogenous levels of malonyl CoA. Quantitative proteomics could identify a significantly larger number of proteins than our previous attempt with the Kmal PTMscan antibody. Moreover, this further enables us to validate the enzymatic function of several identified metabolic proteins and their role in the progression of osteoarthritis.

4. Methods

4.1. Preparation of MA-Diyne

To a stirred solution of Meldrum acid (1.0 g, 6.94 mmol, 1.0 equiv) in DMF (10 mL) was added DIPEA (6.04 mL, 34.7 mmol, 5.0 equiv) followed by propargyl bromide (0.79 mL, 10.41 mmol, 1.5 equiv) at room temperature under an inert atmosphere. The resulting reaction mixture was allowed to stir at room temperature for 24 h. The reaction mixture was then diluted with cold water (100 mL) and extracted with EtOAc (2 × 50 mL). The combined organic extracts were washed with brine solution (100 mL), dried over anhydrous Na2SO₄, filtered, and concentrated under reduced pressure to obtain the crude. The crude was purified by flash column chromatography [eluent: 1% MeOH in DCM] to afford dialkynylated Meldrum acid (460 mg, 40% yield) as an off-white solid.

4.2. Isolation of Primary Chondrocytes and Culture

Primary mouse chondrocytes were isolated from the articular cartilage of the knee joints obtained from the ∼7 days-old C57BL-6 mice according to the previously mentioned protocol. Briefly, the articular cartilage from the tibia and femur part was extracted from the knee joints of ∼7 day-old C57BL-6 mice and dipped in a serum-free Dulbecco’s modified Eagle’s medium (DMEM) (Life Technology, 10567014) solution at room temperature. After thorough washes with 1× phosphate-buffered saline (PBS), cartilage tissues were incubated in 3 mg/mL Collagenase D (Roche) in serum-free DMEM solution for 45 min twice and then transferred to overnight incubation in 0.5 mg/mL Collagenase D in DMEM solution supplemented with 3% Liberase TL (Sigma). The next day, tissues were homogenized by using pipettes to release and suspend the cells multiple times. Finally, the homogenate was filtered through a 40 μm strainer to remove large debris and resuspended in fresh DMEM supplemented with 10% FBS and 1% pen–strep solution. The suspension of cells was then plated in 60 mm cell culture dishes and allowed to expand as passage zero cells at 37 °C and 5% CO₂.

Other cell lines such as primary mouse adipocytes and HEK293T were also cultured in DMEM supplemented with 10% FBS and 1% pen–strep solution.

4.3. Metabolic Labeling and Cell Lysis

The cells were treated with MA-diyne as prescribed. The same volume of DMSO was used as a negative control. After the stipulated incubation time for metabolic labeling, cells were washed with ice-cold 1× PBS three times and scraped using a plastic scraper in 1× PBS. The cells were collected in a 1.5 mL centrifuge tube and centrifuged at 3000 rpm. The cell pellet thus obtained was lysed using ice-cold lysis buffer (1% NP 40, 150 mM NaCl, 50 mM N-(2-hydroxyethyl)­piperazine-N′-ethanesulfonic acid (HEPES), 2 mM MgCl₂, 20% Glycerol pH 7.4, EDTA free protease inhibitor cocktail) for 15 min on ice, followed by centrifugation at 16,000g for 15 min at 4 °C. The supernatant was collected, and protein concentration was estimated using a BCA protein estimation kit (Pierce). The cell lysates were then diluted using dilution buffer (150 mM NaCl, 50 mM HEPES, 2 mM MgCl2, 20% glycerol pH 7.4, EDTA-free protease inhibitor cocktail) to give the final concentration of 1.5 mg/mL for the click reaction.

4.4. Copper­(I)-Catalyzed Alkyne–Azide Cycloaddition Reaction (CuAAC)

The procedure for the CuAAC click chemistry reaction with protein lysates was performed as described in Yang et al. 2010 with slight modification. Briefly, a click reaction master mixture for the required volume of protein lysates was prepared by mixing the reagents in the following order: 100 μM IR680 azide dye (click chemistry tools) for in-gel fluorescence/100 μM Biotin azide (Click chemistry tools) for streptavidin enrichment and Western blotting, 100 μM tris­((1-benzyl-4-triazolyl)­methyl)­amine (TBTA) (Click chemistry tools), 1 mM tris­(carboxyethyl)­phosphine (TCEP) (Sigma), and 1 mM CuSO₄. The protein lysates were incubated with the click reaction master mix for 1.5 h at room temperature.

4.5. In-Gel Fluorescence Imaging of Proteins

After the protein was incubated with the click reaction mixture for 1.5 h, 4 volumes of ice-cold acetone were used to stop/quench the reaction. The proteins were then incubated at −20 °C overnight to precipitate the proteins. The protein acetone solution was centrifuged at 6000g for 5 min at 4 °C. The obtained pellet was washed with ice-cold methanol once and then air-dried for 10 min. The protein pellets were solubilized in 1× loading buffer with 100 mM DTT, heated at 95 °C for 10 min, and then resolved in 10% SDS PAGE gel. The labeled proteins were visualized by scanning the gel under the Odyssey CLx Imaging System (excitation maxima-672 nm, emission maxima- 694 nm).

4.6. Pulse-Chase Experiment

Wild-type primary chondrocytes and Sirt5KO cells were isolated from the wild-type or the Sirt5 conditional knockout (Sirt5-CKO-Aggrecan-Cre^ERT2; Sirt5^flox/flox) mice, respectively. The primary chondrocyte cells (wild type and SirtKO) were treated with 4-hydroxy tamoxifen for 48 h to induce the cre-lox system. The cells were labeled with 200 μM MA-diyne for 1 h. The medium was removed, and the cells were washed thrice with 1× PBS to ensure removal of residual MA-diyne. Fresh medium supplemented with Meldrum acid (200 μM) was added to the cells. The cells were extracted at different time points (0–5 h). The cells were then subjected to protein lysis as described earlier, followed by the click reaction with IR680 azide.

4.7. Labeling the Cells for Confocal Imaging

The cells were cultured on sterile coverslips in 6-well culture dishes. The cells were treated with different concentrations of MA-diyne for 6 h and washed with 1× PBS. The cells were then fixed using ice-cold 4% paraformaldehyde solution for 20 min at 4 °C. The cells on coverslips were then washed thoroughly with ice-cold 1× PBS several times and permeabilized using 0.25% triton X-100 prepared in 1× PBS for 25 min at room temperature. The cells were later washed with 1× PBS, and then, 5% bovine serum albumin blocking buffer was added to the wells and left for shaking at room temperature for another 30 min. The cells were then incubated with the click reaction mix containing 100 μM Carboxyrhodamine 110 Azide, 100 μM TBTA, 1 mM TCEP, and 1 mM CuSO₄ in PBS for 1 h. Finally, cells were washed with 1× PBS to remove the remaining staining, and coverslips were mounted on clean glass slides with the DAPI-containing mounting reagent. The image was collected in a Nikon A1R confocal microscope at 60× magnification.

4.8. Measurement of Malonyl CoA in Cell Supernatant Using a Malonyl CoA ELISA Kit

The cells were treated with MA-diyne for 2–4 h at different concentrations. After the incubation, cells were trypsinized and resuspended in 1× PBS at a concentration of 1 × 10⁶ cells/mL. The cells were lyzed by repeated freeze–thaw cycles and centrifuged at 2000–3000 rpm for 20 min at 4 °C. The supernatant was collected to estimate the malonyl CoA concentration using the mouse malonyl CoA ELISA kit (MyBioSource). The instructions followed for the experiment were as mentioned in the manufacturer’s protocol.

4.9. Pull down of Labeled Proteins after Streptavidin Enrichment

The cells were labeled with MA-diyne as instructed above, and then, the protein lysates were conjugated with biotin azide via the click reaction. The reaction was quenched using 4 volumes of ice-cold acetone, and the solution was incubated at −20 °C overnight to precipitate the proteins. The proteins were pelleted at 3000g for 5 min and washed with methanol thrice. The protein pellet was air-dried for 10 min and solubilized in 100 μL of solubilizing buffer (4% SDS, 20 mM EDTA, and 20% glycerol in 1× PBS) by vortexing and gentle heating. The solution was diluted with 1× PBS to decrease the SDS concentration to 0.5%. 100 μL of prewashed (three times 0.2% SDS prepared in 1× PBS) avidin agar beads was added to the above protein solution and kept for incubation for 1.5 h at room temperature on a gyrator rocker. The solution was centrifuged at 3000 rpm for 2 min, and the supernatant was discarded. The beads were washed thoroughly as follows: 5 times with 10 mL of 0.2% SDS in 1× PBS, 5 times with 10 mL of 1× PBS, and finally 5 times with distilled water. The beads were boiled in equal volume of 100 μL of elution buffer (loading buffer + 100 mM DTT) at 95 °C for 5 min, and the elute was loaded on the SDS PAGE gel. The proteins were then transferred to the nitrocellulose membrane and probed against antistreptavidin as well as the primary antibody of choice.

4.10. Sample Preparation for LC–MS/MS

The avidin beads bound to the biotinylated proteins were washed as instructed above and stored at −80 °C before being sent for the LC–MS/MS at Cell Signaling Technology, Inc. Proteomics facility. The beads were washed again with HPLC water to prevent detergent or protease inhibitor contamination in the mass spectrometer. The beads were incubated with 9 M urea for 10 min at room temperature. The cysteines were then subjected to reductive alkylation with dithiothreitol (DTT) and iodoacetamide (IAA). Finally, the whole solution was diluted with 3 volumes of 20 mM HEPES so that urea concentration was reduced to less than 2 M. The beads were then subjected to on-bead trypsin digestion overnight followed by the addition of 1% trifluoroacetyl (final concentration). The peptides were then desalted using a stage tip and eluted with 50% acetonitrile. Peptide concentration was measured using a BCA assay, and samples were dried. The enriched peptides were then subjected to bottom-up quantitative proteomics on an Orbitrap Astral Instrument. Peptides were loaded directly onto an Aurora Ultimate TS column (25 cm × 75 μm ID, 1.7 μm C18) packed with the C18 reversed-phase resin. The column was developed with a 45 min linear gradient of acetonitrile at 400 nL/min. “A” buffer = 3% acetonitrile, 0.1% formic acid in water. “B” buffer = 80% acetonitrile, 0.1% formic acid in water. Full MS parameter settings are available upon request.

4.11. Identification of Target Proteins

MS/MS spectra were evaluated by using Spectronaut software from Biognosys. Hybrid DIA searches were performed by using factory default settings. Searches were performed against the most recent update of the Uniprot Mus musculus database. Tryptic cleavage was required with up to 4 missed cleavage sites per peptide allowed. Carbamidomethyl modification on cysteine was kept as static modification, while oxidized methionine and N-terminal acetylation were set as variable modifications with a maximum of 5 modifications/peptide. All of the results were filtered to a 1% false discovery rate at the precursor peptide level.

4.12. Bioinformatics

Gene ontology analysis (https://predictprotein.org/) was used to identify the subcellular localization and biological processes of the proteins enriched by MA-diyne. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was also conducted to evaluate the pathways involved (https://www.genome.jp/kegg/pathway).

Ethical approval for the animal experiments reported here was obtained from the Institutional Animal Care and Use Committee (IACUC) at Ohio University, and the IACUC protocol number is 20-H-014.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomedchemau.4c00151.

• 1H NMR spectral data of the MA-diyne probe, 1H NMR full spectrum of the MA-diyne probe, ¹³C NMR spectrum of the MA-diyne probe, mass spectrometry analysis of the MA-diyne probe, summary of obtained fragments in mass spectrometry analysis, an image of Western blot analysis probed for the anti-MaK antibody on the proteins obtained from primary chondrocytes treated with or without ND-630, metabolic labeling of proteins to validate the chemoselectivity of MA-diyne, confocal microscopic fluorescence image of the cells treated with different concentrations of MA-diyne after the click reaction with Carboxyrhodamine 110 Azide, Relative intensity of MA-diyne labeling in wild type and Sirt5 KD cells normalized to β actin after the pulse-chase experiment with Meldrum acid at different time points, and image for in-gel fluorescence depicting similar labeling when the endogenous levels of malonyl CoA were changed (PDF)

• PTMscan total proteome results (XLSX)

• Proteins in MA-yne vs control and Sirt5KO + probe (XLSX)

• Proteins identified (XLSX)

• Gene ontology analysis (XLSX)

• KEGG analysis (XLSX)

⊥.

A.B. and P.N. contributed equally to the work. CRediT: Anupama Binoy conceptualization, data curation, formal analysis, investigation, methodology, visualization, writing - original draft; Pandurangan Nanjan data curation, investigation, software, validation, writing - original draft; Kavya Chellamuthu data curation, methodology; Huanhuan Liu investigation, writing - review & editing.

The authors declare no competing financial interest.