Metabolic Labeling Of Cells Research Articles

Unnatural lipids for simultaneous mRNA delivery and metabolic cell labeling.

Lipids have demonstrated tremendous promise for mRNA delivery, as evidenced by the success of Covid-19 mRNA vaccines. However, existing lipids are mostly used as delivery vehicles and lack the ability to monitor and further modulate the target cells. Here, for the first time, we report a class of unnatural lipids (azido-DOTAP) that can efficiently deliver mRNAs into cells and meanwhile metabolically label cells with unique chemical tags (e.g., azido groups). The azido tags expressed on the cell membrane enable the monitoring of transfected cells, and can mediate subsequent conjugation of cargos via efficient click chemistry for further modulation of transfected cells. We further demonstrate that the dual-functional unnatural lipid is applicable to different types of cells including dendritic cells, the prominent type of antigen presenting cells, potentially opening a new avenue to developing enhanced mRNA vaccines.

Diacylglycerol kinase-ε is S-palmitoylated on cysteine in the cytoplasmic end of its N-terminal transmembrane fragment

Diacylglycerol kinase-ε (DGKε) catalyzes phosphorylation of diacylglycerol to phosphatidic acid with a unique specificity toward 1-stearoyl-2-arachidonoyl-sn-glycerol, which is a backbone of phosphatidylinositol (PI). Owing to this specificity, DGKε is involved in the PI cycle maintaining the cellular level of phosphorylated PI derivatives of signaling activity and was also found crucial for lipid metabolism. DGKε dysfunction is linked with the development of atypical hemolytic uremic syndrome (aHUS) and possibly other human diseases. Despite the DGKε significance, data on its regulation by cotranslational and/or post-translational modifications are scarce. Here, we report that DGKε is S-palmitoylated at Cys38/40 (mouse/human DGKε) located in the cytoplasmic end of its N-terminal putative transmembrane fragment. The S-palmitoylation of DGKε was revealed by metabolic labeling of cells with a palmitic acid analogue followed by click chemistry and with acyl-biotin and acyl-polyethylene glycol exchange assays. The S-acyltransferases zDHHC7 (zinc finger DHHC domain containing) and zDHHC17 and the zDHHC6/16 tandem were found to catalyze DGKε S-palmitoylation, which also increased the DGKε abundance. Mouse DGKε-Myc ectopically expressed in human embryonic kidney 293 cells localized to the endoplasmic reticulum where zDHHC6/16 reside and in small amounts also to the Golgi apparatus where zDHHC7 and zDHHC17 are present. The Cys38Ala substitution upregulated, whereas hyperpalmitoylation of wild-type DGKε reduced the kinase activity, indicating an inhibitory effect of the Cys38 S-palmitoylation. In addition, the substitution of neighboring Pro31 with Ala also diminished the activity of DGKε. Taken together, our data indicate that S-palmitoylation can fine-tune DGKε activity in distinct cellular compartments, possibly by affecting the distance between the kinase and its substrate in a membrane.

A Super-SILAC Approach for Profiling Histone Posttranslational Modifications.

Histone posttranslational modifications (PTMs) play an important role in the regulation of gene expression and have been implicated in a multitude of physiological and pathological processes. During the last decade, mass spectrometry (MS) has emerged as the most accurate and versatile tool to quantitate histone PTMs. Stable-isotope labeling by amino acids in cell culture (SILAC) is an MS-based quantitation strategy involving metabolic labeling of cells, which has been applied to global protein profiling as well as histone PTM analysis. The classical SILAC approach is associated with reduced experimental variability and high quantitation accuracy, but provides limited multiplexing capabilities and can be applied only to actively dividing cells, thus excluding clinical samples. Both limitations are overcome by an evolution of classical SILAC involving the use of a mix of heavy-labeled cell lines as a spike-in standard, known as "super-SILAC". In this chapter, we will provide a detailed description of the optimized protocol used in our laboratory to generate a histone-focused super-SILAC mix and employ it as an internal standard for histone PTM quantitation.

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation. In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required. Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry.

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

A Nature-Inspired Metal-Organic Framework Discriminator for Differential Diagnosis of Cancer Cell Subtypes.

Metabolic glycan labeling (MGL) followed by bioorthogonal chemistry provides a powerful tool for tumor imaging and therapy. However, selectively metabolic labeling of cells or tissues of interest remains a challenge. Particularly, owing to tumor heterogeneity including tumor subtypes and interpatient heterogeneity, it is far more difficult to realize tumor-cell-selective metabolic labeling for precise diagnosis. Inspired by nature, we designed azidosugar-functionalized metal-organic frameworks camouflaged with cancer cell membranes to accomplish cancer-cell-selective MGL in vivo. With abundant receptors, this biomimetic platform not only selectively targets homotypic cells but also realizes different breast cancer subtype-selective MGL. Moreover, the endo/lysosomal-escaped ZIF-8 can make azidosugar escape from lysosomes and accelerate its metabolic incorporation. This strategy also takes advantage of cancer-tissue-derived cell membranes, which may have huge potential for personalized diagnosis and therapy.

A Nature‐Inspired Metal–Organic Framework Discriminator for Differential Diagnosis of Cancer Cell Subtypes

AbstractMetabolic glycan labeling (MGL) followed by bioorthogonal chemistry provides a powerful tool for tumor imaging and therapy. However, selectively metabolic labeling of cells or tissues of interest remains a challenge. Particularly, owing to tumor heterogeneity including tumor subtypes and interpatient heterogeneity, it is far more difficult to realize tumor‐cell‐selective metabolic labeling for precise diagnosis. Inspired by nature, we designed azidosugar‐functionalized metal–organic frameworks camouflaged with cancer cell membranes to accomplish cancer‐cell‐selective MGL in vivo. With abundant receptors, this biomimetic platform not only selectively targets homotypic cells but also realizes different breast cancer subtype‐selective MGL. Moreover, the endo/lysosomal‐escaped ZIF‐8 can make azidosugar escape from lysosomes and accelerate its metabolic incorporation. This strategy also takes advantage of cancer‐tissue‐derived cell membranes, which may have huge potential for personalized diagnosis and therapy.

Examining Myc-Dependent Translation Changes in Cellular Homeostasis and Cancer.

A central component of Myc's role as a master coordinator of energy metabolism and biomass accumulation is its ability to increase the rate of protein synthesis, driving cell cycle progression, and proliferation. Importantly, Myc-induced alterations in both global and specific mRNA translation is a key determinant of Myc's oncogenic function. Herein, we provide five assays to enable researchers to measure global protein synthesis changes, to identify the translatome uniquely regulated by Myc and to investigate the mechanisms generating the tailored Myc translation network. Metabolic labeling of cells with 35S-containing methionine and cysteine in culture and O-propargyl-puromycin (OP-Puro) incorporation in vivo are presented as methods to measure the overall rate of global protein synthesis. Isolation of polysome-associated mRNAs followed by quantitative real-time PCR (qRT-PCR) and the toeprint assay enable the detection of altered translation of specific mRNAs and isoforms, and visualization of differential ribosomal engagement at start codons uniquely mediated by Myc activation, respectively. Finally, the translation initiation reporter assay is utilized to uncover the molecular mechanism mediating altered translation initiation of a specific mRNA. Together, the protocols detailed in this chapter can be used to illuminate how and to what degree Myc-dependent regulation of translation influences homeostatic cellular functions as well as tumorigenesis.

Aptamer-T Cell Targeted Therapy for Tumor Treatment Using Sugar Metabolism and Click Chemistry.

The development of a tumor-targeted immunotherapy is highly required. The most advanced application is the use of CD19 chimeric antigen receptor (CAR)T (CAR-T) cells to B cell malignancies, but there are still side effects including potential carcinogenicity of lentiviral or retroviral insertion into the host cell genome. Here, we developed a nonviral aptamer-T cell targeted strategy for tumor therapy. Tumor cells surface-specific ssDNA aptamers were conjugated to CD3+T cells (aptamer-T cells) using N-azidomannosamine (ManNAz) sugar metabolic cell labeling and click chemistry. We found that the aptamer-T cells could specifically target and bind to tumor cells (such as SGC-7901 gastric cancer cell and CT26 colon carcinoma cell) in vitro and in mice after adoptively transfer in. Aptamer-T cells led to significant regression in tumor volume due to being enriched at tumor microenvironment and producing strong cytotoxicity activities of CD3+T cells with enhanced perforin, granzyme B, CD107a, CD69, and FasL expression. Moreover, aptamer-T displayed even stronger antitumor effects than an anti-PD1 immune-checkpoint monoclonal antibody (mAb) treatment in mice and combination with anti-PD1 yielded synergic antitumor effects. This study uncovers the strong potential of the adoptive nonviral aptamer-T cell strategy as a feasible and efficacious approach for tumor-targeted immunotherapy application.

Targeting infected host cells in vivo via responsive azido-sugar mediated metabolic cell labeling followed by click reaction

The early in vivo diagnosis of infectious disease foci is largely hindered by invasion and concealment of pathogens in host cells, making it difficult for conventional probes to detect and analyze intracellular pathogens. Taking advantage of the excessively produced reactive oxygen species (ROS) within host cells, herein we report the design of thiol-hemiketal blocked N-azidoacetyl galactosamine (Ac3GalNAzSP), an azido unnatural sugar bearing an unprecedent designed ROS-responsive moiety for targeted labelling of infected host cells. Ac3GalNAzSP showed great stability under physiological conditions, specifically released active unnatural sugar in host cells overproducing ROS, metabolically labeled infected host cells with azido groups, and enabled targeting in vivo infection sites by subsequent Click Chemistry reactions, substantiating an unprecedented approach for targeting infected host cells. This technique could be a powerful tool for early in vivo diagnosis and targeted treatment of infectious disease.

In vivo cancer targeting via glycopolyester nanoparticle mediated metabolic cell labeling followed by click reaction

We developed glycopolyesters (GPs) via azido-sugar initiated ring-opening polymerization of O-carboxyanhydrides (OCAs) and achieved efficient in vivo cancer targeting via GP-nanoparticle (GP-NP) mediated metabolic cell labeling followed by Click reaction. GP-NP shows controlled release of azido-sugars and can efficiently label LS174T colon cancer cells with azido groups in tumor-bearing mice. The exogenously introduced azido groups render excellent in vivo cancer targeting and retention of dibenzocyclooctyne-Cy5 (DBCO-Cy5) with an increasing tumor retention enhancement over time (68% at 6 h, 105% at 24 h, and 191% at 48 h) compared to control mice without azido labeling. The tumor accumulation of DBCO-doxorubicin is also significantly enhanced in GP-NP pretreated mice, resulting in improved in vivo anticancer efficacy. This study, for the first time, proposes the use of azido-sugar initiated polymerization of OCAs to form sugar delivery vehicles with high stability and controlled release, and demonstrates the increasing tumor targeting effect of DBCO-cargo over time by azido-modified tumor cells.

Identification and Characterization of Canine Ligament Progenitor Cells and Their Extracellular Matrix Niche.

Ligaments are prone to injury and degeneration in humans and animals, however the healing potential of ligament is poor and current treatment options ineffective. Stem cell-based therapies hold potential for treatment of ligament injuries. This study aimed to characterize a ligament progenitor cell (LPC) population and to identify specific niche components which could promote the survival and function of LPCs. LPCs were isolated from canine cranial cruciate ligament and characterized for clonogenicity, multipotency and marker expression. The extracellular matrix (ECM) composition was characterized by the novel application of a metabolic labeling and mass spectrometry technique. LPCs demonstrated clonogenicity, multipotency, and stem cell marker expression. A number of different collagens, glycoproteins, and proteoglycans were identified in the LPC niche using proteomics. Metabolic labeling of cells demonstrated unique turnover profiles for distinct ECM protein groups, indicating the importance of certain niche components for LPC survival and function. The newly synthesized niche components identified in this study could be exploited to aid identification of LPCs and to promote their survival and function for potential ligament repair strategies.

A Methodology for Comprehensive Analysis of Toll-Like Receptor Signaling in Macrophages.

A combination of high-throughput, multiplexed, quantitative methods with computational modeling and statistical approaches is required to obtain system-level understanding of biological function. Mass spectrometry (MS)-based proteomics has emerged as a preferred tool for the analysis of changes in protein abundance and their post-translational modification (PTM) levels at a global scale, comparable with genomic experiments and generating data suitable for use in mathematical modeling of signaling pathways. Here we describe a set of parallel bottom-up proteomic approaches to detect and quantify the global protein changes in total intracellular proteins, their phosphorylation, and the proteins released by active and passive secretion or shedding mechanisms (referred to as the secretome as reviewed in Makridakis and Vlahou, J Proteome 73:2291-2305, 2010) in response to the stimulation of Toll-like receptors (TLRs) with specific ligands in cultured macrophages. The method includes protocols for metabolic labeling of cells (SILAC: stable isotope labeling by amino acids in cell culture; Ong et al., Mol Cell Proteomics 1:376-386, 2002), ligand stimulation, cell lysis and media collection, in-gel and in-solution modification and digestion of proteins, phosphopeptide enrichment for phosphoproteomics, and LC-MS/MS analysis. With these methods, we can not only reliably quantify the relative changes in the TLR signaling components (Sjoelund et al., J Proteome Res 13:5185-5197, 2014) but also use the data as constraints for mathematical modeling.

ADAM12-L is a direct target of the miR-29 and miR-200 families in breast cancer.

BackgroundADAM12-L and ADAM12-S represent two major splice variants of human metalloproteinase-disintegrin 12 mRNA, which differ in their 3′-untranslated regions (3′UTRs). ADAM12-L, but not ADAM12-S, has prognostic and chemopredictive values in breast cancer. Expression levels of the two ADAM12 splice variants in clinical samples are highly discordant, suggesting post-transcriptional regulation of the ADAM12 gene. The miR-29, miR-30, and miR-200 families have potential target sites in the ADAM12-L 3′UTR and they may negatively regulate ADAM12-L expression.MethodsmiR-29b/c, miR-30b/d, miR-200b/c, or control miRNA mimics were transfected into SUM159PT, BT549, SUM1315MO2, or Hs578T breast cancer cells. ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR, and ADAM12-L protein was detected by Western blotting. Direct targeting of the ADAM12-L 3′UTR by miRNAs was tested using an ADAM12-L 3′UTR luciferase reporter. The rate of ADAM12-L translation was evaluated by metabolic labeling of cells with 35S cysteine/methionine. The roles of endogenous miR-29b and miR-200c were tested by transfecting cells with miRNA hairpin inhibitors.ResultsTransfection of miR-29b/c mimics strongly decreased ADAM12-L mRNA levels in SUM159PT and BT549 cells, whereas ADAM12-S levels were not changed. ADAM12-L, but not ADAM12-S, levels were also significantly diminished by miR-200b/c in SUM1315MO2 cells. In Hs578T cells, miR-200b/c mimics impeded translation of ADAM12-L mRNA. Importantly, both miR-29b/c and miR-200b/c strongly decreased steady state levels of ADAM12-L protein in all breast cancer cell lines tested. miR-29b/c and miR-200b/c also significantly decreased the activity of an ADAM12-L 3′UTR reporter, and this effect was abolished when miR-29b/c and miR-200b/c target sequences were mutated. In contrast, miR-30b/d did not elicit consistent and significant effects on ADAM12-L expression. Analysis of a publicly available gene expression dataset for 100 breast tumors revealed a statistically significant negative correlation between ADAM12-L and both miR-29b and miR-200c. Inhibition of endogenous miR-29b and miR-200c in SUM149PT and SUM102PT cells led to increased ADAM12-L expression.ConclusionsThe ADAM12-L 3′UTR is a direct target of miR-29 and miR-200 family members. Since the miR-29 and miR-200 families play important roles in breast cancer progression, these results may help explain the different prognostic and chemopredictive values of ADAM12-L and ADAM12-S in breast cancer.

Characterization of mammalian selenoprotein o: a redox-active mitochondrial protein.

Selenoproteins exhibit diverse biological functions, most of which are associated with redox control. However, the functions of approximately half of mammalian selenoproteins are not known. One such protein is Selenoprotein O (SelO), the largest mammalian selenoprotein with orthologs found in a wide range of organisms, including bacteria and yeast. Here, we report characterization of mammalian SelO. Expression of this protein could be verified in HEK 293T cells by metabolic labeling of cells with 75Se, and it was abolished when selenocysteine was replaced with serine. A CxxU motif was identified in the C-terminal region of SelO. This protein was reversibly oxidized in a time- and concentration-dependent manner in HEK 293T cells when cells were treated with hydrogen peroxide. This treatment led to the formation of a transient 88 kDa SelO-containing complex. The formation of this complex was enhanced by replacing the CxxU motif with SxxC, but abolished when it was replaced with SxxS, suggesting a redox interaction of SelO with another protein through its Sec residue. SelO was localized to mitochondria and expressed across mouse tissues. Its expression was little affected by selenium deficiency, suggesting it has a high priority for selenium supply. Taken together, these results show that SelO is a redox-active mitochondrial selenoprotein.

Tracking the Subcellular Fate of 20(S)-Hydroxycholesterol with Click Chemistry Reveals a Transport Pathway to the Golgi

Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. Although the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl derivative of 20(S)-hydroxycholesterol has allowed us to directly visualize this oxysterol by attaching an azide fluorophore through cyclo-addition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. Although previous models have proposed nonvesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.

Neutron encoded labeling for peptide identification.

Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, "Amino Acid Counter", which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis.

Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry

The post-translational modification of proteins with N-acetylglucosamine (O-GlcNAc) is involved in the regulation of a wide variety of cellular processes and associated with a number of chronic diseases. Despite its emerging biological significance, the systematic identification of O-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved O-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copper-mediated Click chemistry for purification of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC-MS/MS afforded the identification of around 1500 O-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound O-GlcNAc peptides using selective β-elimination enabled the identification of 185 O-GlcNAc modification sites on 80 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the O-GlcNAcase inhibitor GlcNAcstatin G on the level of O-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signaling pathway showed significantly increased O-GlcNAcylation levels in response to the drug, which further strengthens the link of O-GlcNAc protein modification to cellular nutrient sensing and response.

Chemical Reporters for Monitoring RNA Synthesis and Poly(A) Tail Dynamics

A versatile "clickable" nucleoside: Metabolic labeling of cells is useful in studying the dynamics of biological molecules. N(6) pA can be utilized by all three mammalian RNA polymerases, as well as poly(A) polymerase. Because of its alkyne modification, RNA labeled with N(6) pA can be visualized and purified by using click chemistry.