Publications by authors named "Emily G Werth"

13 Publications

  • Page 1 of 1

Impact of Systemic versus Intratympanic Dexamethasone Administration on the Perilymph Proteome.

J Proteome Res 2021 08 22;20(8):4001-4009. Epub 2021 Jul 22.

Department of Otolaryngology-Head and Neck Surgery, Columbia University Vagelos College of Physicians and Surgeons, New York, New York 10032, United States.

Glucocorticoids are the first-line treatment for sensorineural hearing loss, but little is known about the mechanism of their protective effect or the impact of route of administration. The recent development of hollow microneedles enables safe and reliable sampling of perilymph for proteomic analysis. Using these microneedles, we investigate the effect of intratympanic (IT) versus intraperitoneal (IP) dexamethasone administration on guinea pig perilymph proteome. Guinea pigs were treated with IT dexamethasone ( = 6), IP dexamethasone ( = 8), or untreated for control ( = 8) 6 h prior to aspiration. The round window membrane (RWM) was accessed via a postauricular approach, and hollow microneedles were used to perforate the RWM and aspirate 1 μL of perilymph. Perilymph samples were analyzed by liquid chromatography-mass spectrometry-based label-free quantitative proteomics. Mass spectrometry raw data files have been deposited in an international public repository (MassIVE proteomics repository at https://massive.ucsd.edu/) under data set # MSV000086887. In the 22 samples of perilymph analyzed, 632 proteins were detected, including the inner ear protein cochlin, a perilymph marker. Of these, 14 proteins were modulated by IP, and three proteins were modulated by IT dexamethasone. In both IP and IT dexamethasone groups, VGF nerve growth factor inducible was significantly upregulated compared to control. The remaining adjusted proteins modulate neurons, inflammation, or protein synthesis. Proteome analysis facilitated by the use of hollow microneedles shows that route of dexamethasone administration impacts changes seen in perilymph proteome. Compared to IT administration, the IP route was associated with greater changes in protein expression, including proteins involved in neuroprotection, inflammatory pathway, and protein synthesis. Our findings show that microneedles can mediate safe and effective intracochlear sampling and hold promise for inner ear diagnostics.
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http://dx.doi.org/10.1021/acs.jproteome.1c00322DOI Listing
August 2021

Formation of Biomolecular Condensates in Bacteria by Tuning Protein Electrostatics.

ACS Cent Sci 2020 Dec 12;6(12):2301-2310. Epub 2020 Nov 12.

Department of Chemical Engineering, Columbia University, New York, New York 10027, United States.

While eukaryotic cells have a myriad of membrane-bound organelles enabling the isolation of different chemical environments, prokaryotic cells lack these defined reaction vessels. Biomolecular condensates-organelles that lack a membrane-provide a strategy for cellular organization without a physical barrier while allowing for the dynamic, responsive organization of the cell. It is well established that intrinsically disordered protein domains drive condensate formation via liquid-liquid phase separation; however, the role of globular protein domains on intracellular phase separation remains poorly understood. We hypothesized that the overall charge of globular proteins would dictate the formation and concentration of condensates and systematically probed this hypothesis with supercharged proteins and nucleic acids in . Within this study, we demonstrated that condensates form via electrostatic interactions between engineered proteins and RNA and that these condensates are dynamic and only enrich specific nucleic acid and protein components. Herein, we propose a simple model for the phase separation based on protein charge that can be used to predict intracellular condensate formation. With these guidelines, we have paved the way to designer functional synthetic membraneless organelles with tunable control over globular protein function.
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http://dx.doi.org/10.1021/acscentsci.0c01146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760465PMC
December 2020

Novel 3D-printed hollow microneedles facilitate safe, reliable, and informative sampling of perilymph from guinea pigs.

Hear Res 2021 02 2;400:108141. Epub 2020 Dec 2.

Department of Otolaryngology - Head and Neck Surgery, Columbia University Vagelos College of Physicians and Surgeons, 180 Fort Washington Avenue, Harkness Pavilion, 8th Floor, New York, NY 10032, United States; Department of Mechanical Engineering, Columbia University, New York, NY, United States. Electronic address:

Background: Inner ear diagnostics is limited by the inability to atraumatically obtain samples of inner ear fluid. The round window membrane (RWM) is an attractive portal for accessing perilymph samples as it has been shown to heal within one week after the introduction of microperforations. A 1 µL volume of perilymph is adequate for proteome analysis, yet the total volume of perilymph within the scala tympani of the guinea pig is limited to less than 5 µL. This study investigates the safety and reliability of a novel hollow microneedle device to aspirate perilymph samples adequate for proteomic analysis.

Methods: The guinea pig RWM was accessed via a postauricular surgical approach. 3D-printed hollow microneedles with an outer diameter of 100 µm and an inner diameter of 35 µm were used to perforate the RWM and aspirate 1 µL of perilymph. Two perilymph samples were analyzed by liquid chromatography-mass spectrometry-based quantitative proteomics as part of a preliminary study. Hearing was assessed before and after aspiration using compound action potential (CAP) and distortion product otoacoustic emissions (DPOAE). RWMs were harvested 72 h after aspiration and evaluated for healing using confocal microscopy.

Results: There was no permanent damage to hearing at 72 h after perforation as assessed by CAP (n = 7) and DPOAE (n = 8), and all perforations healed completely within 72 h (n = 8). In the two samples of perilymph analyzed, 620 proteins were detected, including the inner ear protein cochlin, widely recognized as a perilymph marker.

Conclusion: Hollow microneedles can facilitate aspiration of perilymph across the RWM at a quality and volume adequate for proteomic analysis without causing permanent anatomic or physiologic dysfunction. Microneedles can mediate safe and effective intracochlear sampling and show great promise for inner ear diagnostics.
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http://dx.doi.org/10.1016/j.heares.2020.108141DOI Listing
February 2021

Label-Free Quantitative Phosphoproteomics for Algae.

Methods Mol Biol 2020 ;2139:197-211

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

The unicellular alga Chlamydomonas reinhardtii is a model photosynthetic organism for the study of microalgal processes. Along with genomic and transcriptomic studies, proteomic analysis of Chlamydomonas has led to an increased understanding of its metabolic signaling as well as a growing interest in the elucidation of its phosphorylation networks. To this end, mass spectrometry-based proteomics has made great strides in large-scale protein quantitation as well as analysis of posttranslational modifications (PTMs) in a high-throughput manner. An accurate quantification of dynamic PTMs, such as phosphorylation, requires high reproducibility and sensitivity due to the substoichiometric levels of modified peptides, which can make depth of coverage challenging. Here we present a method using TiO-based phosphopeptide enrichment paired with label-free LC-MS/MS for phosphoproteome quantification. Three technical replicate samples in Chlamydomonas were processed and analyzed using this approach, quantifying a total of 1775 phosphoproteins with a total of 3595 phosphosites. With a median CV of 21% across quantified phosphopeptides, implementation of this method for differential studies provides highly reproducible analysis of phosphorylation events. While the culturing and extraction methods used are specific to facilitate coverage in algal species, this approach is widely applicable and can easily extend beyond algae to other photosynthetic organisms with minor modifications.
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http://dx.doi.org/10.1007/978-1-0716-0528-8_15DOI Listing
March 2021

Time Course of Changes in Sorafenib-Treated Hepatocellular Carcinoma Cells Suggests Involvement of Phospho-Regulated Signaling in Ferroptosis Induction.

Proteomics 2020 05 18;20(10):e2000006. Epub 2020 May 18.

Department of Biological Sciences, Columbia University, New York, NY, 10027, USA.

Ferroptosis is a form of regulated, non-apoptotic cell death characterized by excessive lipid peroxidation that can be triggered by inhibition of the cystine-glutamate antiporter, system X . Sorafenib, an FDA-approved multi-kinase inhibitor drug that is used for treatment of hepatocellular carcinoma (HCC), has been shown to induce ferroptosis. Protein phosphorylation changes upon sorafenib treatment have been previously reported in patient studies and in cell culture. However, early phosphorylation changes during induction of ferroptosis are not reported. This work highlights these changes through a time course from 7 to 60 min of sorafenib treatment in human (SKHep1) HCC cells. A total of 6170 unique phosphosites from 2381 phosphoproteins are quantified, and phosphorylation changes occur after as little as 30 min of sorafenib treatment. By 60 min, notable changes included phosphosites significantly changing on p53 (P04637), CAD protein (P27708), and proteins important for iron homeostasis, such as heavy chain ferritin (FTH1; P02794), heme oxygenase 1 (HMOX1; P09601), and PCBP1 (Q15365). Additional sites on proteins in key regulatory pathways are identified, including sites in ferroptosis-related proteins, indicating the likely involvement of phospho-regulated signaling during ferroptosis induction.
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http://dx.doi.org/10.1002/pmic.202000006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305944PMC
May 2020

Receptor-Like Kinase Phosphorylation of Arabidopsis Heterotrimeric G-Protein Gα -Subunit AtGPA1.

Proteomics 2019 12 10;19(24):e1900265. Epub 2019 Dec 10.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.

As molecular on-off switches, heterotrimeric G protein complexes, comprised of a Gα subunit and an obligate Gβγ dimer, transmit extracellular signals received by G protein-coupled receptors (GPCRs) to cytoplasmic targets that respond to biotic and abiotic stimuli. Signal transduction is modulated by phosphorylation of GPCRs and G protein complexes. In Arabidopsis thaliana, the Gα subunit AtGPA1 is phosphorylated by the receptor-like kinase (RLK) BRI1-associated Kinase 1 (BAK1), but the extent that other RLKs phosphorylates AtGPA1 is unknown. Twenty-two trans-phosphorylation sites on AtGPA1 are mapped by 12 RLKs hypothesized to act in the Arabidopsis G protein signaling pathway. Cis-phosphorylation sites are also identified on these RLKs, some newly shown to be dual specific kinases. Multiple sites are present in the core AtGPA1 functional units, including pSer52 and/or pThr53 of the conserved P-loop that directly binds nucleotide/phosphate, pThr164, and pSer175 from αE helix in the intramolecular domain interface for nucleotide exchange and GTP hydrolysis, and pThr193 and/or pThr194 in Switch I (SwI) that coordinates nucleotide exchange and protein partner binding. Several AtGPA1 S/T phosphorylation sites are potentially nucleotide-dependent phosphorylation patterns, such as Ser52/Thr53 in the P-loop and Thr193 and/or Thr194 in SwI.
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http://dx.doi.org/10.1002/pmic.201900265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014827PMC
December 2019

Investigating the effect of target of rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets.

New Phytol 2019 01 24;221(1):247-260. Epub 2018 Jul 24.

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.

Target of rapamycin (TOR) kinase is a conserved regulator of cell growth whose activity is modulated in response to nutrients, energy and stress. Key proteins involved in the pathway are conserved in the model photosynthetic microalga Chlamydomonas reinhardtii, but the substrates of TOR kinase and downstream signaling network have not been elucidated. Our study provides a new resource for investigating the phosphorylation networks governed by the TOR kinase pathway in Chlamydomonas. We used quantitative phosphoproteomics to investigate the effects of inhibiting Chlamydomonas TOR kinase on dynamic protein phosphorylation. Wild-type and AZD-insensitive Chlamydomonas strains were treated with TOR-specific chemical inhibitors (rapamycin, AZD8055 and Torin1), after which differentially affected phosphosites were identified. Our quantitative phosphoproteomic dataset comprised 2547 unique phosphosites from 1432 different proteins. Inhibition of TOR kinase caused significant quantitative changes in phosphorylation at 258 phosphosites, from 219 unique phosphopeptides. Our results include Chlamydomonas homologs of TOR signaling-related proteins, including a site on RPS6 with a decrease in phosphorylation. Additionally, phosphosites on proteins involved in translation and carotenoid biosynthesis were identified. Follow-up experiments guided by these phosphoproteomic findings in lycopene beta/epsilon cyclase showed that carotenoid levels are affected by TORC1 inhibition and carotenoid production is under TOR control in algae.
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http://dx.doi.org/10.1111/nph.15339DOI Listing
January 2019

The phosphorylated redox proteome of Chlamydomonas reinhardtii: Revealing novel means for regulation of protein structure and function.

Redox Biol 2018 07 4;17:35-46. Epub 2018 Apr 4.

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States. Electronic address:

Post-translational modifications (PTMs) are covalent modifications to protein residues which may alter both conformation and activity, thereby modulating signaling and metabolic processes. While PTMs have been largely investigated independently, examination into how different modification interact, or crosstalk, will reveal a more complete understanding of the reciprocity of signaling cascades across numerous pathways. Combinatorial reversible thiol oxidation and phosphorylation in eukaryotes is largely recognized, but rigorous approaches for experimental discovery are underdeveloped. To begin meaningful interrogation of PTM crosstalk in systems biology research, knowledge of targeted proteins must be advanced. Herein, we demonstrate protein-level enrichment of reversibly oxidized proteoforms in Chlamydomonas reinhardtii with subsequent phosphopeptide analysis to determine the extent of phosphorylation in the redox thiol proteome. Label-free quantification was used to quantify 3353 oxidized Cys-sites on 1457 enriched proteins, where sequential phosphopeptide enrichment measured 1094 sites of phosphorylation on 720 proteins with 23% (172 proteins) also identified as reversibly oxidized. Proteins identified with both reversible oxidation and phosphorylation were involved in signaling transduction, ribosome and translation-related machinery, and metabolic pathways. Several redox-modified Calvin-Benson cycle proteins were found phosphorylated and many kinases/phosphatases involved in phosphorylation-dependent photosynthetic state transition and stress-response pathways had sites of reversible oxidation. Identification of redox proteins serves as a crucial element in understanding stress response in photosynthetic organisms and beyond, whereby knowing the ensemble of modifications co-occurring with oxidation highlights novel mechanisms for cellular control.
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http://dx.doi.org/10.1016/j.redox.2018.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006682PMC
July 2018

Tyrosine phosphorylation switching of a G protein.

J Biol Chem 2018 03 30;293(13):4752-4766. Epub 2018 Jan 30.

Departments of Biology, Chapel Hill, North Carolina 27599; Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599. Electronic address:

Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein-coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that posttranslational modification of the Gα subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr in the Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr-dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound Gα substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism "substrate phosphoswitching."
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http://dx.doi.org/10.1074/jbc.RA117.000163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880138PMC
March 2018

Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics.

Plant J 2017 01 17;89(2):416-426. Epub 2017 Jan 17.

Department of Chemistry, University of North Carolina at Chapel Hill, 125 South Road, CB#3290, Chapel Hill, NC, 2759934, USA.

The identification of dynamic protein phosphorylation events is critical for understanding kinase/phosphatase-regulated signaling pathways. To date, protein phosphorylation and kinase expression have been examined independently in photosynthetic organisms. Here we present a method to study the global kinome and phosphoproteome in tandem in a model photosynthetic organism, the alga Chlamydomonas reinhardtii (Chlamydomonas), using mass spectrometry-based label-free proteomics. A dual enrichment strategy targets intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment. To increase depth of coverage, both data-dependent and data-independent (via SWATH, Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) mass spectrometric acquisitions were performed to obtain a more than 50% increase in coverage of the enriched Chlamydomonas kinome over coverage found with no enrichment. The quantitative phosphoproteomic dataset yielded 2250 phosphopeptides and 1314 localized phosphosites with excellent reproducibility across biological replicates (90% of quantified sites with coefficient of variation below 11%). This approach enables simultaneous investigation of kinases and phosphorylation events at the global level to facilitate understanding of kinase networks and their influence in cell signaling events.
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http://dx.doi.org/10.1111/tpj.13384DOI Listing
January 2017

Label-free quantitative proteomic analysis of pre-flowering PMeV-infected Carica papaya L.

J Proteomics 2017 01 23;151:275-283. Epub 2016 Jun 23.

Núcleo de Biotecnologia, Universidade Federal do Espírito Santo, Av. Marechal Campos, 1498, Vitória, ES 29040-090, Brazil.

Papaya meleira virus (PMeV) infects papaya (Carica papaya L.) and leads to Papaya Sticky Disease (PSD) or "Meleira", characterized by a spontaneous exudation of latex from fruits and leaves only in the post-flowering developmental stage. The latex oxidizes in contact with air and accumulates as a sticky substance on the plant organs, impairing papaya fruit's marketing and exportation. To understand pre-flowering C. papaya resistance to PMeV, an LC-MS/MS-based label-free proteomics approach was used to assess the differential proteome of PMeV-infected pre-flowering C. papaya vs. uninfected (control) plants. In this study, 1333 proteins were identified, of which 111 proteins showed a significant abundance change (57 increased and 54 decreased) and supports the hypothesis of increased photosynthesis and reduction of 26S-proteassoma activity and cell-wall remodeling. All of these results suggest that increased photosynthetic activity has a positive effect on the induction of plant immunity, whereas the reduction of caspase-like activity and the observed changes in the cell-wall associated proteins impairs the full activation of defense response based on hypersensitive response and viral movement obstruction in pre-flowering C. papaya plants.

Biological Significance: The papaya (Carica papaya L.) fruit's production is severely limited by the occurrence of Papaya meleira virus (PMeV) infection, which causes Papaya Sticky Disease (PSD). Despite the efforts to understand key features involved with the plant×virus interaction, PSD management is still largely based on the observation of the first disease symptoms in the field, followed by the elimination of the diseased plants. However, C. papaya develops PSD only after flowering, i.e. about six-months after planting, and the virus inoculum sources are kept in field. The development of PMeV resistant genotypes is impaired by the limited knowledge about C. papaya resistance against viruses. The occurrence of a resistance/tolerance mechanism to PSD symptoms development prior to C. papaya flowering is considered in this study. Thus, field-grown and PMeV-infected C. papaya leaf samples were analyzed using proteomics, which revealed the modulation of photosynthesis-, 26S proteasome- and cell-wall remodeling-associated proteins. The data implicate a role for those systems in C. papaya resistance to viruses and support the idea of a partial resistance induction in the plants at pre-flowering stage. The specific proteins presented in the manuscript represent a starting point to the selection of key genes to be used in C. papaya improvement to PMeV infection resistance. The presented data also contribute to the understanding of virus-induced disease symptoms development in plants, of interest to the plant-virus interaction field.
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http://dx.doi.org/10.1016/j.jprot.2016.06.025DOI Listing
January 2017

Quantifying reversible oxidation of protein thiols in photosynthetic organisms.

J Am Soc Mass Spectrom 2015 Apr 20;26(4):631-40. Epub 2015 Feb 20.

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.

Photosynthetic organisms use dynamic post-translational modifications to survive and adapt, which include reversible oxidative modifications of protein thiols that regulate protein structure, function, and activity. Efforts to quantify thiol modifications on a global scale have relied upon peptide derivatization, typically using isobaric tags such as TMT, ICAT, or iTRAQ that are more expensive, less accurate, and provide less proteome coverage than label-free approaches--suggesting the need for improved experimental designs for studies requiring maximal coverage and precision. Herein, we present the coverage and precision of resin-assisted thiol enrichment coupled to label-free quantitation for the characterization of reversible oxidative modifications on protein thiols. Using C. reinhardtii and Arabidopsis as model systems for algae and plants, we quantified 3662 and 1641 unique cysteinyl peptides, respectively, with median coefficient of variation (CV) of 13% and 16%. Further, our method is extendable for the detection of protein abundance changes and stoichiometries of cysteine oxidation. Finally, we demonstrate proof-of-principle for our method, and reveal that exogenous hydrogen peroxide treatment regulates the C. reinhardtii redox proteome by increasing or decreasing the level of oxidation of 501 or 67 peptides, respectively. As protein activity and function is controlled by oxidative modifications on protein thiols, resin-assisted thiol enrichment coupled to label-free quantitation can reveal how intracellular and environmental stimuli affect plant survival and fitness through oxidative stress.
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http://dx.doi.org/10.1007/s13361-014-1073-yDOI Listing
April 2015

Phosphoproteomics in photosynthetic organisms.

Electrophoresis 2014 Dec 10;35(24):3441-51. Epub 2014 Jul 10.

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

As primarily sessile organisms, photosynthetic species survive in dynamic environments by using elegant signaling pathways to manifest molecular responses to extracellular cues. These pathways exploit phosphorylation of specific amino acids (e.g. serine, threonine, tyrosine), which impact protein structure, function, and localization. Despite substantial progress in implementation of phosphoproteomics to understand photosynthetic organisms, researchers still struggle to translate a biological question into an experimental strategy and vice versa. This review evaluates the current status of phosphoproteomics in photosynthetic organisms and concludes with recommendations based on current knowledge.
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http://dx.doi.org/10.1002/elps.201400154DOI Listing
December 2014
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