Bioorthogonal Noncanonical Amino Acid Tagging Research Articles

Selective analysis of newly synthesized proteins by combining fluorescence correlation spectroscopy with bioorthogonal non-canonical amino acid tagging.

Protein expression is closely related to many biological processes including cell growth, differentiation and signaling. It is a challenge to selectively monitor newly synthesized proteins under both physiological and pathological conditions due to shortage of efficient analytical methods. Here, we proposed a new strategy to selectively monitor newly synthesized proteins in cells by combining fluorescence correlation spectroscopy (FCS) with bioorthogonal noncanonical amino acid tagging (BONCAT) technique. Firstly, homopropargylglycine (HPG), an alkyne surrogate of methionine, was metabolically incorporated into newly synthesized proteins in living cells, and the proteins containing the alkyne functional group were subsequently labeled with chemoselective fluorescence reporters using the Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Then, FCS was used to analyze the newly synthesized proteins based on the difference in the characteristic diffusion times of labeled proteins and free fluorescent dyes. We optimized the conditions of HPG metabolic incorporation and the CuAAC click reaction and applied this new method to study autophagic protein degradation and in situ monitor secreted proteins in cells. Compared to current methods, our method is simple, fast, and without separation, and it may become a promising approach for in situ studying protein expression in living cells.

Translational activity is uncoupled from nucleic acid content in bacterial cells of the human gut microbiota

ABSTRACT Changes in bacterial diversity in the human gut have been associated with many conditions, despite not always reflecting changes in bacterial activity. Methods linking bacterial identity to function are needed for improved understanding of how bacterial communities adapt and respond to their environment, including the gut. Here, we optimized bioorthogonal non-canonical amino acid tagging (BONCAT) for the gut microbiota and combined it with fluorescently activated cell sorting and sequencing (FACS-Seq) to identify the translationally active members of the community. We then used this novel technique to compare with other bulk community measurements of activity and viability: relative nucleic acid content and membrane damage. The translationally active bacteria represent about half of the gut microbiota, and are not distinct from the whole community. The high nucleic acid content bacteria also represent half of the gut microbiota, but are distinct from the whole community and correlate with the damaged subset. Perturbing the community with xenobiotics previously shown to alter bacterial activity but not diversity resulted in stronger changes in the distinct physiological fractions than in the whole community. BONCAT is a suitable method to probe the translationally active members of the gut microbiota, and combined with FACS-Seq, allows for their identification. The high nucleic acid content bacteria are not necessarily the protein-producing bacteria in the community; thus, further work is needed to understand the relationship between nucleic acid content and bacterial metabolism in the human gut. Considering physiologically distinct subsets of the gut microbiota may be more informative than whole-community profiling.

Protein Synthesis in the Developing Neocortex at Near-Atomic Resolution Reveals Ebp1-Mediated Neuronal Proteostasis at the 60S Tunnel Exit

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.

Selective Labeling and Identification of the Tumor Cell Proteome of Pancreatic Cancer In Vivo.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers. Dissecting the tumor cell proteome from that of the non-tumor cells in the PDAC tumor bulk is critical for tumorigenesis studies, biomarker discovery, and development of therapeutics. However, investigating the tumor cell proteome has proven evasive due to the tumor's extremely complex cellular composition. To circumvent this technical barrier, we have combined bioorthogonal noncanonical amino acid tagging (BONCAT) and data-independent acquisition mass spectrometry (DIA-MS) in an orthotopic PDAC model to specifically identify the tumor cell proteome in vivo. Utilizing the tumor cell-specific expression of a mutant tRNA synthetase transgene, this approach provides tumor cells with the exclusive ability to incorporate an azide-bearing methionine analogue into newly synthesized proteins. The azide-tagged tumor cell proteome is subsequently enriched and purified via a bioorthogonal reaction and then identified and quantified using DIA-MS. Applying this workflow to the orthotopic PDAC model, we have identified thousands of proteins expressed by the tumor cells. Furthermore, by comparing the tumor cell and tumor bulk proteomes, we showed that the approach can distinctly differentiate proteins produced by tumor cells from those of non-tumor cells within the tumor microenvironment. Our study, for the first time, reveals the tumor cell proteome of PDAC under physiological conditions, providing broad applications for tumorigenesis, therapeutics, and biomarker studies in various human cancers.

Ribosomal protein synthesis is decreased in tauopathy as revealed by noncanonical amino acid labelling

AbstractBackgroundTau is a scaffolding protein which serves multiple cellular functions that are perturbed in neurodegenerative diseases, including Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Recently we demonstrated that amyloid‐b, the second hallmark of AD, induces de novo protein synthesis of tau, with this activation being found to be tau‐dependent. This raised the question of whether FTD‐tau by itself affects protein synthesis.MethodIn order to examine protein synthesis, we optimised non‐canonical amino acid (NCAA) labelling to label newly synthesised proteins in vivo in the K369I tau transgenic K3 mouse model of FTD. Combining this technique with either fluorescent non‐canonical amino acid tagging (FUNCAT) or bio‐orthogonal non‐canonical amino acid tagging (BONCAT), we were able to visualise and purify newly synthesised proteins from mouse brain tissue. We also used polysome profiling in primary neurons to further explore the effect of tau on ribosome biogenesis.ResultOur analysis reveals massively decreased protein synthesis in neurons containing pathologically phosphorylated tau, a finding we confirmed in P301L mutant tau transgenic rTg4510 mice. Using quantitative SWATH‐MS proteomics, we identify alterations in the synthesis of 247 proteins in de novo proteome of K3 mice, including the decreased synthesis of the ribosomal subunit proteins RPL23, RPLP0, RPL19 and RPS16, a finding that was validated in both K3 and rTg4510 mice. We also demonstrate that there is a decrease in overall abundance of one of these proteins, RPL23, and that this decrease correlated with the levels of both total and AT8 phosphorylated tau. Lastly, we recapultate these findings in human tissue, with FTD‐brains showing significantly lower levels of RPL23 than healthy controls.ConclusionTogether, our findings present a potential pathomechanism by which pathological tau interferes with cellular functions through the dysregulation of ribosomal protein synthesis.

Temporal Quantitative Profiling of Newly Synthesized Proteins during Aβ Accumulation.

Accumulation of aggregated amyloid beta (Aβ) in the brain is believed to impair multiple cellular pathways and play a central role in Alzheimer's disease pathology. However, how this process is regulated remains unclear. In theory, measuring protein synthesis is the most direct way to evaluate a cell's response to stimuli, but to date, there have been few reliable methods to do this. To identify the protein regulatory network during the development of Aβ deposition in AD, we applied a new proteomic technique to quantitate newly synthesized protein (NSP) changes in the cerebral cortex and hippocampus of 2-, 5-, and 9-month-old APP/PS1 AD transgenic mice. This bio-orthogonal noncanonical amino acid tagging analysis combined PALM (pulse azidohomoalanine labeling in mammals) and HILAQ (heavy isotope labeled AHA quantitation) to reveal a comprehensive dataset of NSPs prior to and post Aβ deposition, including the identification of proteins not previously associated with AD, and demonstrated that the pattern of differentially expressed NSPs is age-dependent. We also found dysregulated vesicle transportation networks including endosomal subunits, coat protein complex I (COPI), and mitochondrial respiratory chain throughout all time points and two brain regions. These results point to a pathological dysregulation of vesicle transportation which occurs prior to Aβ accumulation and the onset of AD symptoms, which may progressively impact the entire protein network and thereby drive neurodegeneration. This study illustrates key pathway regulation responses to the development of AD pathogenesis by directly measuring the changes in protein synthesis and provides unique insights into the mechanisms that underlie AD.

Identification of a herpes simplex virus 1 gene encoding neurovirulence factor by chemical proteomics

Identification of the complete set of translated genes of viruses is important to understand viral replication and pathogenesis as well as for therapeutic approaches to control viral infection. Here, we use chemical proteomics, integrating bio-orthogonal non-canonical amino acid tagging and high-resolution mass spectrometry, to characterize the newly synthesized herpes simplex virus 1 (HSV-1) proteome in infected cells. In these infected cells, host cellular protein synthesis is shut-off, increasing the chance to preferentially detect viral proteomes. We identify nine previously cryptic orphan protein coding sequences whose translated products are expressed in HSV-1-infected cells. Functional characterization of one identified protein, designated piUL49, shows that it is critical for HSV-1 neurovirulence in vivo by regulating the activity of virally encoded dUTPase, a key enzyme that maintains accurate DNA replication. Our results demonstrate that cryptic orphan protein coding genes of HSV-1, and probably other large DNA viruses, remain to be identified.

Activity-based cell sorting reveals responses of uncultured archaea and bacteria to substrate amendment

Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.

Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms.

In this study, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM) analysis, and we demonstrate the applicability of the method for marine prokaryotes. Enumeration of active marine bacteria was performed by combining the DNA stain SYBR Green with detection of protein production with BONCAT. After optimization of incubation condition and substrate concentration on monoculture of Escherichia coli, we applied and modified the method to natural marine samples. We found that between 10 and 30% of prokaryotes in natural communities were active. The method is replicable, fast, and allow high sample throughput when using FCM. We conclude that the combination of BONCAT and FCM is an alternative to current methods for quantifying active populations in aquatic environments.

Dynamics of protein synthesis in the initial steps of strobilation in the model cestode parasite Mesocestoides corti (syn. vogae)

Mesocestoides corti (syn. vogae) is a useful model for developmental studies of platyhelminth parasites of the Cestoda class, such as Taenia spp. or Echinococcus spp. It has been used in studies to characterize cestode strobilation, i.e. the development of larvae into adult worms. So far, little is known about the initial molecular events involved in cestode strobilation and, therefore, we carried out a study to characterize newly synthesized (NS) proteins upon strobilation induction. An approach based on bioorthogonal noncanonical amino acid tagging and mass spectrometry was used to label, isolate, identify, and quantify NS proteins in the initial steps of M. corti strobilation. Overall, 121 NS proteins were detected exclusively after induction of strobilation, including proteins related to development pathways, such as insulin and notch signaling. Metabolic changes that take place in the transition from the larval stage to adult worm were noted in special NS protein subsets related to developmental processes, such as focal adhesion, cell leading edge, and maintenance of location. The data shed light on mechanisms underlying early steps of cestode strobilation and enabled identification of possible developmental markers. We also consider the use of developmental responsive proteins as potential drug targets for developing novel anthelmintics. Biological significanceLarval cestodiases are life-threatening parasitic diseases that affect both man and domestic animals worldwide. Cestode parasites present complex life cycles, in which they undergo major morphological and physiological changes in the transition from one life-stage to the next. One of these transitions occurs during cestode strobilation, when the mostly undifferentiated and non-segmented larval or pre-adult form develops into a fully segmented and sexually differentiated (strobilated) adult worm. Although the proteomes of bona fide larvae and strobialted adults have been previously characterized for a few cestode species, little is still known about the dynamic of protein synthesis during the early steps of cestode strobilation. Now, the assessment of newly synthesized (NS) proteins within the first 48 h of strobilation the model cestode M. corti allowed to shed light on molecular mechanisms that are triggered by strobilation induction. The functional analyses of this repertoire of over a hundred NS proteins pointed out to changes in metabolism and activation of classical developmental signaling pathways in early strobilation. Many of the identified NS proteins may become valuable cestode developmental markers and their involvement in vital processes make them also good candidate targets for novel anthelmintic drugs.

Neuronal subclass-selective proteomic analysis in Caenorhabditis elegans

Neurons are categorised into many subclasses, and each subclass displays different morphology, expression patterns, connectivity and function. Changes in protein synthesis are critical for neuronal function. Therefore, analysing protein expression patterns in individual neuronal subclass will elucidate molecular mechanisms for memory and other functions. In this study, we used neuronal subclass-selective proteomic analysis with cell-selective bio-orthogonal non-canonical amino acid tagging. We selected Caenorhabditis elegans as a model organism because it shows diverse neuronal functions and simple neural circuitry. We performed proteomic analysis of all neurons or AFD subclass neurons that regulate thermotaxis in C. elegans. Mutant phenylalanyl tRNA synthetase (MuPheRS) was selectively expressed in all neurons or AFD subclass neurons, and azido-phenylalanine was incorporated into proteins in cells of interest. Azide-labelled proteins were enriched and proteomic analysis was performed. We identified 4,412 and 1,834 proteins from strains producing MuPheRS in all neurons and AFD subclass neurons, respectively. F23B2.10 (RING-type domain-containing protein) was identified only in neuronal cell-enriched proteomic analysis. We expressed GFP under the control of the 5′ regulatory region of F23B2.10 and found GFP expression in neurons. We expect that more single-neuron specific proteomic data will clarify how protein composition and abundance affect characteristics of neuronal subclasses.

Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition.

Aberrant protein synthesis and protein expression are a hallmark of many conditions ranging from cancer to Alzheimer's. Blood-based biomarkers indicative of changes in proteomes have long been held to be potentially useful with respect to disease prognosis and treatment. However, most biomarker efforts have focused on unlabeled plasma proteomics that include nonmyeloid origin proteins with no attempt to dynamically tag acute changes in proteomes. Herein we report a method for evaluating de novo protein synthesis in whole blood liquid biopsies. Using a modification of the "bioorthogonal noncanonical amino acid tagging" (BONCAT) protocol, rodent whole blood samples were incubated with l-azidohomoalanine (AHA) to allow incorporation of this selectively reactive non-natural amino acid within nascent polypeptides. Notably, failure to incubate the blood samples with EDTA prior to implementation of azide-alkyne "click" reactions resulted in the inability to detect probe incorporation. This live-labeling assay was sensitive to inhibition with anisomycin and nascent, tagged polypeptides were localized to a variety of blood cells using FUNCAT. Using labeled rodent blood, these tagged peptides could be consistently identified through standard LC/MS-MS detection of known blood proteins across a variety of experimental conditions. Furthermore, this assay could be expanded to measure de novo protein synthesis in human blood samples. Overall, we present a rapid and convenient de novo protein synthesis assay that can be used with whole blood biopsies that can quantify translational change as well as identify differentially expressed proteins that may be useful for clinical applications.

Abstract 523: Analysis of Mesenchymal Stem Cell Proteomes In Situ in the Ischemic Heart

Cell therapy for myocardial infarction is promising but largely unsuccessful partially due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects. To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to engineer a mutant methionyl-tRNA synthetase (MetRS L274G ) in MSC, which charges the methionine analogue azidonorleucine (ANL) to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction. Murine MSCs were transduced with lentivirus MetRS L274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRS L274G -transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricle by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and their GO terms are enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 35, 131 and 84 proteins were significantly up-regulated and 12, 38 and 27 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular microvesicles, inflammatory response and apoptosis and in the KEGG pathways of complements and coagulation cascades, lysosomes, and regulators of actin cytoskeleton. MetRS L274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

Expression of Annexin A2 Promotes Cancer Progression in Estrogen Receptor Negative Breast Cancers.

When breast cancer progresses to a metastatic stage, survival rates decline rapidly and it is considered incurable. Thus, deciphering the critical mechanisms of metastasis is of vital importance to develop new treatment options. We hypothesize that studying the proteins that are newly synthesized during the metastatic processes of migration and invasion will greatly enhance our understanding of breast cancer progression. We conducted a mass spectrometry screen following bioorthogonal noncanonical amino acid tagging to elucidate changes in the nascent proteome that occur during epidermal growth factor stimulation in migrating and invading cells. Annexin A2 was identified in this screen and subsequent examination of breast cancer cell lines revealed that Annexin A2 is specifically upregulated in estrogen receptor negative (ER-) cell lines. Furthermore, siRNA knockdown showed that Annexin A2 expression promotes the proliferation, wound healing and directional migration of breast cancer cells. In patients, Annexin A2 expression is increased in ER- breast cancer subtypes. Additionally, high Annexin A2 expression confers a higher probability of distant metastasis specifically for ER- patients. This work establishes a pivotal role of Annexin A2 in breast cancer progression and identifies Annexin A2 as a potential therapeutic target for the more aggressive and harder to treat ER- subtype.

Bioorthogonal non-canonical amino acid tagging reveals translationally active subpopulations of the cystic fibrosis lung microbiota

Culture-independent studies of cystic fibrosis lung microbiota have provided few mechanistic insights into the polymicrobial basis of disease. Deciphering the specific contributions of individual taxa to CF pathogenesis requires comprehensive understanding of their ecophysiology at the site of infection. We hypothesize that only a subset of CF microbiota are translationally active and that these activities vary between subjects. Here, we apply bioorthogonal non-canonical amino acid tagging (BONCAT) to visualize and quantify bacterial translational activity in expectorated sputum. We report that the percentage of BONCAT-labeled (i.e. active) bacterial cells varies substantially between subjects (6-56%). We use fluorescence-activated cell sorting (FACS) and genomic sequencing to assign taxonomy to BONCAT-labeled cells. While many abundant taxa are indeed active, most bacterial species detected by conventional molecular profiling show a mixed population of both BONCAT-labeled and unlabeled cells, suggesting heterogeneous growth rates in sputum. Differentiating translationally active subpopulations adds to our evolving understanding of CF lung disease and may help guide antibiotic therapies targeting bacteria most likely to be susceptible.

Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis.

BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG); L-methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools.

Cell-specific non-canonical amino acid labelling identifies changes in the de novo proteome during memory formation.

The formation of spatial long-term memory (LTM) requires the de novo synthesis of distinct sets of proteins; however, a non-biased examination of the de novo proteome in this process is lacking. Here, we generated a novel mouse strain, which enables cell-type-specific labelling of newly synthesised proteins with non-canonical amino acids (NCAAs) by genetically restricting the expression of the mutant tRNA synthetase, NLL-MetRS, to hippocampal neurons. By combining this labelling technique with an accelerated version of the active place avoidance task and bio-orthogonal non-canonical amino acid tagging (BONCAT) followed by SWATH quantitative mass spectrometry, we identified 156 proteins that were altered in synthesis in hippocampal neurons during spatial memory formation. In addition to observing increased synthesis of known proteins important in memory-related processes, such as glutamate receptor recycling, we also identified altered synthesis of proteins associated with mRNA splicing as a potential mechanism involved in spatial LTM formation.

Probing the active fraction of soil microbiomes using BONCAT-FACS

The ability to link soil microbial diversity to soil processes requires technologies that differentiate active microbes from extracellular DNA and dormant cells. Here, we use BONCAT (bioorthogonal non-canonical amino acid tagging) to measure translationally active cells in soils. We compare the active population of two soil depths from Oak Ridge (Tennessee, USA) and find that a maximum of 25–70% of the extractable cells are active. Analysis of 16S rRNA sequences from BONCAT-positive cells recovered by fluorescence-activated cell sorting (FACS) reveals that the phylogenetic composition of the active fraction is distinct from the total population of extractable cells. Some members of the community are found to be active at both depths independently of their abundance rank, suggesting that the incubation conditions favor the activity of similar organisms. We conclude that BONCAT-FACS is effective for interrogating the active fraction of soil microbiomes in situ and provides a new approach for uncovering the links between soil processes and specific microbial groups.

1961-P: Identification of a Novel Brown/Beige Fat-Derived Adipokine

Fat cells or adipocytes play a central role in obesity, diabetes, and metabolic disease. White adipocytes store excess energy in the form of triglycerol, and brown adipocytes dissipate chemical energy into heat. In addition to these bioenergetics functions, adipocytes also have important endocrine functions. While several adipocyte-derived secreted factors, or adipokines, have been shown to have important physiological roles, bioinformatic analyses suggest that adipocytes may secrete more than 1,000 unique polypeptides. The vast majority of these gene products remain uncharacterized, representing a major opportunity for discovering new biology with translational potential. To that end, we have employed chemical and genetic tools to label and enrich circulating proteins in a cell-selective manner. Using bio-orthogonal non-canonical amino acid tagging (BONCAT) along with mass spectrometry we have characterized the secretome of white, brown, and beige adipocytes. We have identified more than 800 unique proteins, with approximately 300 showing fat cell type specific enrichment. Our studies have focused on a brown and beige fat specific protein encoded by a previously uncharacterized open reading frame (BAT-ORF). The mRNA encoding this protein is expressed in UCP1+thermogenic adipocytes, induced by cold and repressed by warm temperature, and upregulated following thiazolidinedione treatment. We have found that the BAT-ORF gene encodes two isoforms, only one of which is secreted. We have generated mice with genetic deletion of each isoform using CRISPR-Cas9. Preliminary data suggests these mice have decreased energy expenditure. Complete characterization of these mice is underway and data will be presented. We will also present a method using genetically engineered mouse models that allows us to specifically label and enrich secreted proteins in the blood in a cell-type specific fashion. Disclosure C.J. Choi: None. A.A. Crane: None. P. Cohen: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-17-ACE-17 to P.C.)

Comparative analysis of differentially secreted proteins in serum-free and serum-containing media by using BONCAT and pulsed SILAC

Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the majority of mass spectrometric cell secretome studies have been performed using serum-free medium (SFM). On the other hand, serum-containing medium (SCM) is not recommended very much because the secretome obtained with SCM is easily contaminated with fetal bovine serum (FBS) proteins. In this study, through the combination of bioorthogonal non-canonical amino acid tagging (BONCAT) and pulsed-SILAC (pSILAC), we analyzed differentially secreted proteins between SFM and SCM in a cancer-derived human cell, U87MG, and a mesenchymal stem cell derived from human Wharton’s jelly (hWJ-MSCs). In most cases, the bioinformatic tools predicted a protein to be truly secretory when the secretion level of the protein was more in SCM than in SFM. In the case of hWJ-MSCs, the amount of proteins secreted in SCM for 24 hours was larger than that of SFM (log2 fold change = 0.96), even considering different cell proliferation rates. hWJ-MSCs proteins secreted more in SCM included several positive markers of MSC paracrine factors implicated in angiogenesis, neurogenesis and osteogenesis, and upstream regulators of cell proliferation. Our study suggests the analysis of the secretome should be processed in SCM that promotes cell proliferation and secretion.