@article{yamamoto_blackburn_goshe_brown_migoswski_campanhon_moreira_ferreira_soares_2023, title={Tizoxanide Antiviral Activity on Dengue Virus Replication}, volume={15}, ISSN={["1999-4915"]}, DOI={10.3390/v15030696}, abstractNote={Dengue virus is an important circulating arbovirus in Brazil responsible for high morbidity and mortality worldwide, representing a huge economic and social burden, in addition to affecting public health. In this study, the biological activity, toxicity, and antiviral activity against dengue virus type 2 (DENV-2) of tizoxanide (TIZ) was evaluated in Vero cell culture. TIZ has a broad spectrum of action in inhibiting different pathogens, including bacteria, protozoa, and viruses. Cells were infected for 1 h with DENV-2 and then treated for 24 h with different concentrations of the drug. The quantification of viral production indicated the antiviral activity of TIZ. The protein profiles in infected Vero cells treated and not treated with TIZ were analyzed using the label-free quantitative proteomic approach. TIZ was able to inhibit virus replication mainly intracellularly after DENV-2 penetration and before the complete replication of the viral genome. Additionally, the study of the protein profile of infected not-treated and infected-treated Vero cells showed that TIZ interferes with cellular processes such as intracellular trafficking and vesicle-mediated transport and post-translational modifications when added after infection. Our results also point to the activation of immune response genes that would eventually lead to a decrease of DENV-2 production. TIZ is a promising therapeutic molecule for the treatment of DENV-2 infections.}, number={3}, journal={VIRUSES-BASEL}, author={Yamamoto, Kristie A. and Blackburn, Kevin and Goshe, Michael B. and Brown, Dennis T. and Migoswski, Edimilson and Campanhon, Isabele B. and Moreira, Monica F. and Ferreira, Davis F. and Soares, Marcia R.}, year={2023}, month={Mar} }
 @article{ho_blackburn_goshe_williamson_2021, title={Identification of multiple proteins whose interaction with mannitol dehydrogenase is induced by salicylic acid: Implications for unconventional secretion}, ISSN={["1615-9861"]}, DOI={10.1002/pmic.202100091}, abstractNote={Although protein secretion was previously believed to be solely via ER/Golgi pathways, Golgi-independent secretion has now been described in both animals and plants. Secretion of the mannitol catabolic enzyme mannitol dehydrogenase (MTD) in response to the endogenous pathogen response signal salicylic acid (SA) was one of the first reports of unconventional protein secretion in plants. To begin assessing potential secretion-associated MTD protein interactors, we present here high-quality databases describing changes in MTD-interacting proteins following SA treatment of Arabidopsis thaliana cells expressing MTD.}, journal={PROTEOMICS}, author={Ho, Tricia C. and Blackburn, R. Kevin and Goshe, Michael B. and Williamson, John D.}, year={2021}, month={Sep} }
 @article{dinh_paudel_brochu_popowski_gracieux_cores_huang_hensley_harrell_vandergriff_et al._2020, title={Inhalation of lung spheroid cell secretome and exosomes promotes lung repair in pulmonary fibrosis}, volume={11}, ISSN={["2041-1723"]}, url={http://dx.doi.org/10.1038/s41467-020-14344-7}, DOI={10.1038/s41467-020-14344-7}, abstractNote={Abstract Idiopathic pulmonary fibrosis (IPF) is a fatal and incurable form of interstitial lung disease in which persistent injury results in scar tissue formation. As fibrosis thickens, the lung tissue loses the ability to facilitate gas exchange and provide cells with needed oxygen. Currently, IPF has few treatment options and no effective therapies, aside from lung transplant. Here we present a series of studies utilizing lung spheroid cell-secretome (LSC-Sec) and exosomes (LSC-Exo) by inhalation to treat different models of lung injury and fibrosis. Analysis reveals that LSC-Sec and LSC-Exo treatments could attenuate and resolve bleomycin- and silica-induced fibrosis by reestablishing normal alveolar structure and decreasing both collagen accumulation and myofibroblast proliferation. Additionally, LSC-Sec and LSC-Exo exhibit superior therapeutic benefits than their counterparts derived from mesenchymal stem cells in some measures. We showed that an inhalation treatment of secretome and exosome exhibited therapeutic potential for lung regeneration in two experimental models of pulmonary fibrosis.}, number={1}, journal={NATURE COMMUNICATIONS}, publisher={Springer Science and Business Media LLC}, author={Dinh, Phuong-Uyen C. and Paudel, Dipti and Brochu, Hayden and Popowski, Kristen D. and Gracieux, M. Cyndell and Cores, Jhon and Huang, Ke and Hensley, M. Taylor and Harrell, Erin and Vandergriff, Adam C. and et al.}, year={2020}, month={Feb} }
 @article{yamamoto_blackburn_migowski_goshe_brown_ferreira_soares_2020, title={Quantitative proteomic analysis of the tizoxanide effect in vero cells}, volume={10}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-020-71634-2}, abstractNote={Nitazoxanide (NTZ) is effective against helminths and numerous microorganisms, including bacteria and viruses. In vivo, NTZ is metabolized into Tizoxanide (TIZ), which is the active circulating metabolite. With the emergence of SARS-Cov-2 as a Pandemic agent, NTZ became one of the molecules already approved for human use to engage clinical trials, due to results in vitro showing that NTZ was highly effective against the SARS-Cov-2, agent of COVID-19. There are currently several ongoing clinical trials mainly in the USA and Brazil involving NTZ due not only to the in vitro results, but also for its long-known safety. Here, we study the response of Vero cells to TIZ treatment and unveil possible mechanisms for its antimicrobial effect, using a label-free proteomic approach (LC/MS/MS) analysis to compare the proteomic profile between untreated- and TIZ-treated cells. Fifteen differentially expressed proteins were observed related to various biological processes, including translation, intracellular trafficking, RNA processing and modification, and signal transduction. The broad antimicrobial range of TIZ points towards its overall effect in lowering cell metabolism and RNA processing and modification. The decreased levels of FASN, HNRNPH and HNRNPK with the treatment appear to be important for antiviral activity.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Yamamoto, K. A. and Blackburn, K. and Migowski, E. and Goshe, M. B. and Brown, D. T. and Ferreira, D. F. and Soares, M. R.}, year={2020}, month={Sep} }
 @article{schroeter_blackburn_goshe_schweitzer_2019, title={Proteomic method to extract, concentrate, digest and enrich peptides from fossils with coloured (humic) substances for mass spectrometry analyses}, volume={6}, ISSN={["2054-5703"]}, DOI={10.1098/rsos.181433}, abstractNote={Humic substances are breakdown products of decaying organic matter that co-extract with proteins from fossils. These substances are difficult to separate from proteins in solution and interfere with analyses of fossil proteomes. We introduce a method combining multiple recent advances in extraction protocols to both concentrate proteins from fossil specimens with high humic content and remove humics, producing clean samples easily analysed by mass spectrometry (MS). This method includes: (i) a non-demineralizing extraction buffer that eliminates protein loss during the demineralization step in routine methods; (ii) filter-aided sample preparation (FASP) of peptides, which concentrates and digests extracts in one filter, allowing the separation of large humics after digestion; (iii) centrifugal stage tipping, which further clarifies and concentrates samples in a uniform process performed simultaneously on multiple samples. We apply this method to a moa fossil (approx. 800–1000 years) dark with humic content, generating colourless samples and enabling the detection of more proteins with greater sequence coverage than previous MS analyses on this same specimen. This workflow allows analyses of low-abundance proteins in fossils containing humics and thus may widen the range of extinct organisms and regions of their proteomes we can explore with MS.}, number={8}, journal={ROYAL SOCIETY OPEN SCIENCE}, author={Schroeter, Elena R. and Blackburn, Kevin and Goshe, Michael B. and Schweitzer, Mary H.}, year={2019}, month={Aug} }
 @article{mathews_epps_blackburn_goshe_grunden_dunn_2019, title={Public questions spur the discovery of new bacterial species associated with lignin bioconversion of industrial waste}, volume={6}, ISSN={["2054-5703"]}, DOI={10.1098/rsos.180748}, abstractNote={A citizen science project found that the greenhouse camel cricket ( Diestrammena asynamora ) is common in North American homes. Public response was to wonder ‘what good are they anyway?’ and ecology and evolution guided the search for potential benefit. We predicted that camel crickets and similar household species would likely host bacteria with the ability to degrade recalcitrant carbon compounds. Lignocellulose is particularly relevant as it is difficult to degrade yet is an important feedstock for pulp and paper, chemical and biofuel industries. We screened gut bacteria of greenhouse camel crickets and another household insect, the hide beetle ( Dermestes maculatus ) for the ability to grow on and degrade lignocellulose components as well as the lignocellulose-derived industrial waste product black liquor. From three greenhouse camel crickets and three hide beetles, 14 bacterial strains were identified that were capable of growth on lignocellulosic components, including lignin. Cedecea lapagei was selected for further study due to growth on most lignocellulose components. The C. lapagei secretome was identified using LC/MS/MS analysis. This work demonstrates a novel source of lignocellulose-degrading bacteria and introduces an effective workflow to identify bacterial enzymes for transforming industrial waste into value-added products. More generally, our research suggests the value of ecologically guided discovery of novel organisms.}, number={3}, journal={ROYAL SOCIETY OPEN SCIENCE}, author={Mathews, Stephanie L. and Epps, Mary Jane and Blackburn, R. Kevin and Goshe, Michael B. and Grunden, Amy M. and Dunn, Robert R.}, year={2019}, month={Mar} }
 @article{lavoie_fazio_blackburn_goshe_carbonell_menegatti_2019, title={Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery}, volume={20}, ISSN={["1422-0067"]}, DOI={10.3390/ijms20071729}, abstractNote={The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of “problematic” HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.}, number={7}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Lavoie, R. Ashton and Fazio, Alice and Blackburn, R. Kevin and Goshe, Michael B. and Carbonell, Ruben G. and Menegatti, Stefano}, year={2019}, month={Apr} }
 @article{shen_bobay_greeley_reyes_rajabu_blackburn_dallas_goshe_ascencio-ibanez_hanley-bowdoin_2018, title={Sucrose Nonfermenting 1-Related Protein Kinase 1 Phosphorylates a Geminivirus Rep Protein to Impair Viral Replication and Infection}, volume={178}, ISSN={["1532-2548"]}, DOI={10.1104/pp.18.00268}, abstractNote={Geminiviruses are single-stranded DNA viruses that infect a wide variety of plants and cause severe crop losses worldwide. The geminivirus replication initiator protein (Rep) binds to the viral replication origin and catalyzes DNA cleavage and ligation to initiate rolling circle replication. In this study, we found that the Tomato golden mosaic virus (TGMV) Rep is phosphorylated at serine-97 by sucrose nonfermenting 1-related protein kinase 1 (SnRK1), a master regulator of plant energy homeostasis and metabolism. Phosphorylation of Rep or the phosphomimic S97D mutation impaired Rep binding to viral DNA. A TGMV DNA-A replicon containing the Rep S97D mutation replicated less efficiently in tobacco (Nicotiana tabacum) protoplasts than in wild-type or Rep phosphorylation-deficient replicons. The TGMV Rep-S97D mutant also was less infectious than the wild-type virus in Nicotiana benthamiana and was unable to infect tomato (Solanum lycopersicum). Nearly all geminivirus Rep proteins have a serine residue at the position equivalent to TGMV Rep serine-97. SnRK1 phosphorylated the equivalent serines in the Rep proteins of Tomato mottle virus and Tomato yellow leaf curl virus and reduced DNA binding, suggesting that SnRK1 plays a key role in combating geminivirus infection. These results established that SnRK1 phosphorylates Rep and interferes with geminivirus replication and infection, underscoring the emerging role for SnRK1 in the host defense response against plant pathogens.}, number={1}, journal={PLANT PHYSIOLOGY}, author={Shen, Wei and Bobay, Benjamin G. and Greeley, Laura A. and Reyes, Maria I. and Rajabu, Cyprian A. and Blackburn, R. Kevin and Dallas, Mary Beth and Goshe, Michael B. and Ascencio-Ibanez, Jose T. and Hanley-Bowdoin, Linda}, year={2018}, month={Sep}, pages={372–389} }
 @article{bender_blackburn_monaghan_derbyshire_menke_zipfel_goshe_zielinski_huber_2017, title={Autophosphorylation-based Calcium (Ca2(+)) Sensitivity Priming and Ca2(+)/Calmodulin Inhibition of Arabidopsis thaliana Ca2(+)-dependent Protein Kinase 28 (CPK28)}, volume={292}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m116.763243}, abstractNote={Plant calcium (Ca2+)-dependent protein kinases (CPKs) represent the primary Ca2+-dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca2+/CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and was inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca2+ activation and might also allow CPK28 to remain active when Ca2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca2+/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca2+ signaling specificity through Ca2+ sensor priming.}, number={10}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Bender, Kyle W. and Blackburn, R. Kevin and Monaghan, Jacqueline and Derbyshire, Paul and Menke, Frank L. H. and Zipfel, Cyril and Goshe, Michael B. and Zielinski, Raymond E. and Huber, Steven C.}, year={2017}, month={Mar}, pages={3988–4002} }
 @article{blackburn_bustamante-marin_yin_goshe_ostrowski_2017, title={Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously Uncharacterized Proteins of High Abundance}, volume={16}, ISSN={["1535-3907"]}, DOI={10.1021/acs.jproteome.6b00972}, abstractNote={Cilia are essential to many diverse cellular processes. Although many major axonemal components have been identified and studied, how they interact to form a functional axoneme is not completely understood. To further our understanding of the protein composition of human airway cilia, we performed a semiquantitative analysis of ciliary axonemes using label-free LC/MSE, which identified over 400 proteins with high confidence. Tubulins were the most abundant proteins identified, with evidence of 20 different isoforms obtained. Twelve different isoforms of axonemal dynein heavy chain were also identified. Absolute quantification of the nontubulin components demonstrated a greater than 75-fold range of protein abundance (RSPH9;1850 fmol vs CCDC103;24 fmol), adding another level of complexity to axonemal structure. Of the identified proteins, ∼70% are known axonemal proteins. In addition, many previously uncharacterized proteins were identified. Unexpectedly, several of these, including ERICH3, C1orf87, and CCDC181, were present at high relative abundance in the cilia. RT-PCR analysis and immunoblotting confirmed cilia-specific expression for eight uncharacterized proteins, and fluorescence microscopy demonstrated unique axonemal localizations. These studies have provided the first quantitative analysis of the ciliary proteome and have identified and characterized several previously unknown proteins as major constituents of human airway cilia.}, number={4}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Blackburn, Kevin and Bustamante-Marin, Ximena and Yin, Weining and Goshe, Michael B. and Ostrowski, Lawrence E.}, year={2017}, month={Apr}, pages={1579–1592} }
 @article{imkampe_halter_huang_schulze_mazzotta_schmidt_manstretta_postel_wierzba_yang_et al._2017, title={The Arabidopsis Leucine-Rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1}, volume={29}, ISSN={["1532-298X"]}, DOI={10.1105/tpc.17.00376}, abstractNote={BAK1 is a coreceptor and positive regulator of multiple ligand binding leucine-rich repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity, and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here, we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in Arabidopsis thaliana BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand binding receptors to prevent BAK1 receptor complex formation.}, number={9}, journal={PLANT CELL}, author={Imkampe, Julia and Halter, Thierry and Huang, Shuhua and Schulze, Sarina and Mazzotta, Sara and Schmidt, Nikola and Manstretta, Raffaele and Postel, Sandra and Wierzba, Micheal and Yang, Yong and et al.}, year={2017}, month={Sep}, pages={2285–2303} }
 @article{saha_paul_herring_dutta_bhattacharya_samaddar_goshe_dasgupta_2016, title={Gatekeeper Tyrosine Phosphorylation of SYMRK Is Essential for Synchronizing the Epidermal and Cortical Responses in Root Nodule Symbiosis}, volume={171}, ISSN={["1532-2548"]}, DOI={10.1104/pp.15.01962}, abstractNote={Symbiosis receptor kinase (SYMRK) is indispensable for activation of root nodule symbiosis (RNS) at both epidermal and cortical levels and is functionally conserved in legumes. Previously, we reported SYMRK to be phosphorylated on "gatekeeper" Tyr both in vitro as well as in planta. Since gatekeeper phosphorylation was not necessary for activity, the significance remained elusive. Herein, we show that substituting gatekeeper with nonphosphorylatable residues like Phe or Ala significantly affected autophosphorylation on selected targets on activation segment/αEF and β3-αC loop of SYMRK. In addition, the same gatekeeper mutants failed to restore proper symbiotic features in a symrk null mutant where rhizobial invasion of the epidermis and nodule organogenesis was unaffected but rhizobia remain restricted to the epidermis in infection threads migrating parallel to the longitudinal axis of the root, resulting in extensive infection patches at the nodule apex. Thus, gatekeeper phosphorylation is critical for synchronizing epidermal/cortical responses in RNS.}, number={1}, journal={PLANT PHYSIOLOGY}, author={Saha, Sudip and Paul, Anindita and Herring, Laura and Dutta, Ayan and Bhattacharya, Avisek and Samaddar, Sandip and Goshe, Michael B. and DasGupta, Maitrayee}, year={2016}, month={May}, pages={71–81} }
 @article{herring_grant_blackburn_haugh_goshe_2015, title={Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks}, volume={988}, ISSN={["1873-376X"]}, DOI={10.1016/j.jchromb.2015.02.017}, abstractNote={Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC/TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites, illustrate the feasibility of this approach in quantitative phosphoproteomic studies.}, journal={JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES}, publisher={Elsevier BV}, author={Herring, Laura E. and Grant, Kyle G. and Blackburn, Kevin and Haugh, Jason M. and Goshe, Michael B.}, year={2015}, month={Apr}, pages={166–174} }
 @article{argo_shi_liu_goshe_2015, title={Performing protein crosslinking using gas-phase cleavable chemical crosslinkers and liquid chromatography-tandem mass spectrometry}, volume={89}, ISSN={["1095-9130"]}, DOI={10.1016/j.ymeth.2015.06.011}, abstractNote={In this article, we describe our methods and protocols using collision-induced dissociative chemical crosslinking-tandem mass spectrometry (CID-CXL-MS/MS) analysis and the practical considerations when implementing these reagents and methodology for protein crosslinking studies. The synthesis of our novel chemical crosslinkers is described as well as their use for effectively labeling protein and protein complexes. Several sample preparation methods for liquid chromatography-tandem mass spectrometry are provided including the enrichment of interpeptide crosslinks. For identification of CID-CXL-MS/MS crosslinks, details regarding MS acquisition parameters and the utilization of various mass spectrometers are addressed along with post-data acquisition analysis to identify interpeptide crosslinks. Once the CID-CXL-MS/MS approach is optimized for a protein target or a set of targets, it can be used as a tool for biological research for studying protein structure when integrated with data obtained using other techniques, such as NMR, X-ray crystallography, and cryo-electron microscopy, or extended to the study of protein-protein interactions in physiological environments.}, journal={METHODS}, author={Argo, Andrew S. and Shi, Chunxiao and Liu, Fan and Goshe, Michael B.}, year={2015}, month={Nov}, pages={64–73} }
 @article{ahmed_grant_edwards_rahman_cirit_goshe_haugh_2014, title={Data-driven modeling reconciles kinetics of ERK phosphorylation, localization, and activity states}, volume={10}, ISSN={["1744-4292"]}, DOI={10.1002/msb.134708}, abstractNote={The extracellular signal-regulated kinase (ERK) signaling pathway controls cell proliferation and differentiation in metazoans. Two hallmarks of its dynamics are adaptation of ERK phosphorylation, which has been linked to negative feedback, and nucleocytoplasmic shuttling, which allows active ERK to phosphorylate protein substrates in the nucleus and cytosol. To integrate these complex features, we acquired quantitative biochemical and live-cell microscopy data to reconcile phosphorylation, localization, and activity states of ERK. While maximal growth factor stimulation elicits transient ERK phosphorylation and nuclear translocation responses, ERK activities available to phosphorylate substrates in the cytosol and nuclei show relatively little or no adaptation. Free ERK activity in the nucleus temporally lags the peak in nuclear translocation, indicating a slow process. Additional experiments, guided by kinetic modeling, show that this process is consistent with ERK's modification of and release from nuclear substrate anchors. Thus, adaptation of whole-cell ERK phosphorylation is a by-product of transient protection from phosphatases. Consistent with this interpretation, predictions concerning the dose-dependence of the pathway response and its interruption by inhibition of MEK were experimentally confirmed.}, number={1}, journal={MOLECULAR SYSTEMS BIOLOGY}, publisher={Wiley-Blackwell}, author={Ahmed, Shoeb and Grant, Kyle G. and Edwards, Laura E. and Rahman, Anisur and Cirit, Murat and Goshe, Michael B. and Haugh, Jason M.}, year={2014}, month={Jan} }
 @article{schweitzer_schroeter_goshe_2014, title={Protein Molecular Data from Ancient (>1 million years old) Fossil Material: Pitfalls, Possibilities and Grand Challenges}, volume={86}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/ac500803w}, DOI={10.1021/ac500803w}, abstractNote={Advances in resolution and sensitivity of analytical techniques have provided novel applications, including the analyses of fossil material. However, the recovery of original proteinaceous components from very old fossil samples (defined as >1 million years (1 Ma) from previously named limits in the literature) is far from trivial. Here, we discuss the challenges to recovery of proteinaceous components from fossils, and the need for new sample preparation techniques, analytical methods, and bioinformatics to optimize and fully utilize the great potential of information locked in the fossil record. We present evidence for survival of original components across geological time, and discuss the potential benefits of recovery, analyses, and interpretation of fossil materials older than 1 Ma, both within and outside of the fields of evolutionary biology.}, number={14}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Schweitzer, Mary Higby and Schroeter, Elena R. and Goshe, Michael B.}, year={2014}, month={Jul}, pages={6731–6740} }
 @article{shen_dallas_goshe_hanley-bowdoin_2014, title={SnRK1 Phosphorylation of AL2 Delays Cabbage Leaf Curl Virus Infection in Arabidopsis}, volume={88}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.00761-14}, abstractNote={ABSTRACT Geminivirus AL2/C2 proteins play key roles in establishing infection and causing disease in their plant hosts. They are involved in viral gene expression, counter host defenses by suppressing transcriptional gene silencing, and interfere with the host signaling involved in pathogen resistance. We report here that begomovirus and curtovirus AL2/C2 proteins interact strongly with host geminivirus Rep-interacting kinases (GRIKs), which are upstream activating kinases of the protein kinase SnRK1, a global regulator of energy and nutrient levels in plants. We used an in vitro kinase system to show that GRIK-activated SnRK1 phosphorylates recombinant AL2/C2 proteins from several begomoviruses and to map the SnRK1 phosphorylation site to serine-109 in the AL2 proteins of two New World begomoviruses: Cabbage Leaf Curl Virus (CaLCuV) and Tomato mottle virus . A CaLCuV AL2 S109D phosphomimic mutation did not alter viral DNA levels in protoplast replication assays. In contrast, the phosphomimic mutant was delayed for symptom development and viral DNA accumulation during infection of Arabidopsis thaliana , demonstrating that SnRK1 contributes to host defenses against CaLCuV. Our observation that serine-109 is not conserved in all AL2/C2 proteins that are SnRK1 substrates in vitro suggested that phosphorylation of viral proteins by plant kinases contributes to the evolution of geminivirus-host interactions. IMPORTANCE Geminiviruses are single-stranded DNA viruses that cause serious diseases in many crops. Dicot-infecting geminiviruses carry genes that encode multifunctional AL2/C2 proteins that are essential for infection. However, it is not clear how AL2/C2 proteins are regulated. Here, we show that the host protein kinase SnRK1, a central regulator of energy balance and nutrient metabolism in plants, phosphorylates serine-109 in AL2 proteins of three subgroups of New World begomoviruses, resulting in a delay in viral DNA accumulation and symptom appearance. Our results support SnRK1's antiviral role and reveal a novel mechanism underlying this function. Phylogenetic analysis suggested that AL2 S109 evolved as begomoviruses migrated from the Old World to the New World and may have provided a selective advantage as begomoviruses adapted to a different environment and different plant hosts. This study provides new insights into the interaction of viral pathogens with their plant hosts at the level of viral protein modification by the host.}, number={18}, journal={JOURNAL OF VIROLOGY}, author={Shen, Wei and Dallas, Mary Beth and Goshe, Michael B. and Hanley-Bowdoin, Linda}, year={2014}, month={Sep}, pages={10598–10612} }
 @article{samaddar_dutta_sinharoy_paul_bhattacharya_saha_chien_goshe_dasgupta_2013, title={Autophosphorylation of gatekeeper tyrosine by symbiosis receptor kinase}, volume={587}, ISSN={["0014-5793"]}, DOI={10.1016/j.febslet.2013.07.050}, abstractNote={Plant receptor-like kinases (RLKs) share their evolutionary origin with animal interleukin-1 receptor-associated kinase (IRAK)/Pelle family of soluble kinases and are distinguished by having tyrosine as ‘gatekeeper’. This position is adjacent to the hinge region and is hidden in a hydrophobic pocket of the catalytic cleft of protein kinases and is therefore least probable to be a target for any modification. This communication illustrates the accessibility of the gatekeeper site (Y670) towards both autophosphorylation and dephosphorylation in the recombinant cytoplasmic domain of symbiosis receptor kinase from Arachis hypogaea (AhSYMRK). Autophosphorylation on gatekeeper tyrosine was detected prior to extraction but never under in vitro conditions. We hypothesize gatekeeper phosphorylation to be associated with synthesis/maturation of AhSYMRK and this phenomenon may be prevalent among RLKs.}, number={18}, journal={FEBS LETTERS}, author={Samaddar, Sandip and Dutta, Ayan and Sinharoy, Senjuti and Paul, Anindita and Bhattacharya, Avisek and Saha, Sudip and Chien, Ko-yi and Goshe, Michael B. and DasGupta, Maitrayee}, year={2013}, month={Sep}, pages={2972–2979} }
 @article{olson_liu_tucker_goshe_cavanagh_2013, title={Chemical crosslinking and LC/MS analysis to determine protein domain orientation: Application to AbrB}, volume={431}, ISSN={["1090-2104"]}, DOI={10.1016/j.bbrc.2012.12.124}, abstractNote={To fully understand the modes of action of multi-protein complexes, it is essential to determine their overall global architecture and the specific relationships between domains and subunits. The transcription factor AbrB is a functional homotetramer consisting of two domains per monomer. Obtaining the high-resolution structure of tetrameric AbrB has been extremely challenging due to the independent character of these domains. To facilitate the structure determination process, we solved the NMR structures of both domains independently and utilized gas-phase cleavable chemical crosslinking and LC/MSn analysis to correctly position the domains within the full tetrameric AbrB protein structure.}, number={2}, journal={BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS}, author={Olson, Andrew L. and Liu, Fan and Tucker, Ashley T. and Goshe, Michael B. and Cavanagh, John}, year={2013}, month={Feb}, pages={253–257} }
 @article{bajwa_wang_blackburn_goshe_mitra_williams_bishop_krasnyanski_allen_huber_et al._2013, title={Identification and Functional Analysis of Tomato BRI1 and BAK1 Receptor Kinase Phosphorylation Sites}, volume={163}, ISSN={["1532-2548"]}, DOI={10.1104/pp.113.221465}, abstractNote={Abstract Brassinosteroids (BRs) are plant hormones that are perceived at the cell surface by a membrane-bound receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1). BRI1 interacts with BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) to initiate a signal transduction pathway in which autophosphorylation and transphosphorylation of BRI1 and BAK1, as well as phosphorylation of multiple downstream substrates, play critical roles. Detailed mechanisms of BR signaling have been examined in Arabidopsis (Arabidopsis thaliana), but the role of BRI1 and BAK1 phosphorylation in crop plants is unknown. As a foundation for understanding the mechanism of BR signaling in tomato (Solanum lycopersicum), we used liquid chromatography-tandem mass spectrometry to identify multiple in vitro phosphorylation sites of the tomato BRI1 and BAK1 cytoplasmic domains. Kinase assays showed that both tomato BRI1 and BAK1 are active in autophosphorylation as well as transphosphorylation of each other and specific peptide substrates with a defined sequence motif. Site-directed mutagenesis revealed that the highly conserved kinase domain activation loop residue threonine-1054 was essential for tomato BRI1 autophosphorylation and peptide substrate phosphorylation in vitro. Furthermore, analysis of transgenic lines expressing full-length tomato BRI1-Flag constructs in the weak tomato bri1 allele, curl3-abs1, demonstrated that threonine-1054 is also essential for normal BRI1 signaling and tomato growth in planta. Finally, we cloned the tomato ortholog of TGF-β Receptor Interacting Protein (TRIP1), which was previously shown to be a BRI1-interacting protein and kinase domain substrate in Arabidopsis, and found that tomato TRIP1 is a substrate of both tomato BRI1 and BAK1 kinases in vitro.}, number={1}, journal={PLANT PHYSIOLOGY}, author={Bajwa, Vikramjit S. and Wang, Xiaofeng and Blackburn, R. Kevin and Goshe, Michael B. and Mitra, Srijeet K. and Williams, Elisabeth L. and Bishop, Gerard J. and Krasnyanski, Sergei and Allen, George and Huber, Steven C. and et al.}, year={2013}, month={Sep}, pages={30–42} }
 @article{liu_wu_sweedler_goshe_2012, title={An enhanced protein crosslink identification strategy using CID-cleavable chemical crosslinkers and LC/MSn analysis}, volume={12}, ISSN={["1615-9861"]}, DOI={10.1002/pmic.201100352}, abstractNote={We describe a novel two-step LC/MS(n) strategy to effectively and confidently identify numerous crosslinked peptides from complex mixtures. This method incorporates the use of our gas-phase cleavable crosslinking reagent, disuccinimidyl-succinamyl-aspartyl-proline (SuDP), and a new data-processing algorithm CXLinkS (Cleavable Crosslink Selection), which enables unequivocal crosslink peptide selection and identification on the basis of mass measurement accuracy, high resolving power, and the unique fragmentation pattern of each crosslinked peptide. We demonstrate our approach with well-characterized monomeric and multimeric protein systems with and without database searching restrictions where inter-peptide crosslink identification is increased 8-fold over our previously published data-dependent LC/MS³ method and discuss its applicability to other CID-cleavable crosslinkers and more complex protein systems.}, number={3}, journal={PROTEOMICS}, author={Liu, Fan and Wu, Cong and Sweedler, Jonathan V. and Goshe, Michael B.}, year={2012}, month={Feb}, pages={401–405} }
 @article{chien_blackburn_liu_goshe_2012, title={Proteomic and Phosphoproteomic Analysis of Chicken Embryo Fibroblasts Infected with Cell Culture-Attenuated and Vaccine Strains of Marek's Disease Virus}, volume={11}, ISSN={["1535-3907"]}, DOI={10.1021/pr300471y}, abstractNote={Vaccination is an effective strategy to reduce the loss of chickens in the poultry industry caused by Marek's Disease (MD), an avian lymphoproliferative disease. The vaccines currently used are from attenuated serotype 1 Marek's disease virus (MDV) or naturally nononcogenic MDV strains. To prepare for future immunity breaks, functional genomic and proteomic studies have been used to better understand the underlying mechanisms of MDV pathogenicity and the effects induced by the vaccine viruses. In this study, a combined approach of quantitative GeLC-MSE and qualitative ERLIC/IMAC/LC-MS/MS analysis were used to identify abundance changes of proteins and the variations of phosphorylation status resulting from the perturbations due to infection with an attenuated oncogenic virus strain (Md11/75C) and several nononcogenic virus strains (CVI988, FC126 and 301B) in vitro. Using this combined approach, several signal transduction pathways mapped by the identified proteins were found to be altered at both the level of protein abundance and phosphorylation. On the basis of this study, a kinase-dependent pathway to regulate phosphorylation of 4E-BP1 to modulate assembly of the protein translation initiation complex was revealed. The differences of 4E-BP1 phosphorylation patterns as well as the measured abundance changes among several other proteins that regulate host transcriptional and translational activities across the virus strains used in this study provide new insight for future functional and biochemical characterization of specific proteins involved in MDV pathogenesis.}, number={12}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Chien, Ko-yi and Blackburn, Kevin and Liu, Hsiao-Ching and Goshe, Michael B.}, year={2012}, month={Dec}, pages={5663–5677} }
 @misc{kota_goshe_2011, title={Advances in qualitative and quantitative plant membrane proteomics}, volume={72}, ISSN={["0031-9422"]}, DOI={10.1016/j.phytochem.2011.01.027}, abstractNote={The membrane proteome consists of integral and membrane-associated proteins that are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are only expressed during certain developmental stages or in response to environmental stress. These proteins can undergo post-translational modifications in response to these different conditions, allowing them to transiently associate with the membrane or other membrane proteins. Along with their increased size, hydrophobicity, and the additional organelle and cellular features of plant cells relative to mammalian systems, the characterization of the plant membrane proteome presents unique challenges for effective qualitative and quantitative analysis using mass spectrometry (MS) analysis. Here, we present the latest advancements developed for the isolation and fractionation of plant organelles and their membrane components amenable to MS analysis. Separations of membrane proteins from these enriched preparations that have proven effective are discussed for both gel- and liquid chromatography-based MS analysis. In this context, quantitative membrane proteomic analyses using both isotope-coded and label-free approaches are presented and reveal the potential to establish a wider-biological interpretation of the function of plant membrane proteins that will ultimately lead to a more comprehensive understanding of plant physiology and their response mechanisms.}, number={10}, journal={PHYTOCHEMISTRY}, author={Kota, Uma and Goshe, Michael B.}, year={2011}, month={Jul}, pages={1040–1060} }
 @article{chien_liu_goshe_2011, title={Development and Application of a Phosphoproteomic Method Using Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC), IMAC, and LC-MS/MS Analysis to Study Marek's Disease Virus Infection}, volume={10}, ISSN={["1535-3893"]}, DOI={10.1021/pr2002403}, abstractNote={Marek's Disease (MD) is an avian neoplastic disease caused by Marek's Disease Virus (MDV). The mechanism of virus transition between the lytic and latent cycle is still being investigated; however, post-translational modifications, especially phosphorylation, have been thought to play an important role. Previously, our group has used strong cation exchange chromatography in conjunction with reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the changes in global proteomic expression upon MDV infection (Ramaroson , M. F.; Ruby, J.; Goshe, M. B.; Liu , H.-C. S. J. Proteome Res. 2008, 7, 4346-4358). Here, we extend our study by developing an effective separation and enrichment approach to investigate the changes occurring in the phosphoproteome using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) to fractionate peptides from chicken embryo fibroblast (CEF) digests and incorporating a subsequent IMAC enrichment step to selectively target phosphorylated peptides for LC-MS/MS analysis. To monitor the multidimensional separation between mock- and MDV-infected CEF samples, a casein phosphopeptide mixture was used as an internal standard. With LC-MS/MS analysis alone, no CEF phosphopeptides were detected, while with ERLIC fractionation only 1.2% of all identified peptides were phosphorylated. However, the incorporation of IMAC enrichment with ERLIC fractionation provided a 50-fold increase in the percentage of identified phosphopeptides. Overall, a total of 581 unique phosphopeptides were identified (p < 0.05) with those of the MDV-infected CEF sample containing nearly twice as many as the mock-infected control of which 11% were unique to MDV proteins. The changes in the phosphoproteome are discussed including the role that microtubule-associated proteins may play in MDV infection mechanisms.}, number={9}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Chien, Ko-yi and Liu, Hsiao-Ching and Goshe, Michael B.}, year={2011}, month={Sep}, pages={4041–4053} }
 @article{mbeunkui_goshe_2011, title={Investigation of solubilization and digestion methods for microsomal membrane proteome analysis using data-independent LC-MSE}, volume={11}, ISSN={["1615-9861"]}, DOI={10.1002/pmic.200900698}, abstractNote={To evaluate the implementation of various denaturants and their efficacy in bottom-up membrane proteomic methods using LC-MS analysis, microsomes isolated from tomato roots were treated with MS-compatible surfactants (RapiGest SF Surfactant from Waters and PPS Silent Surfactant from Protein Discovery), a chaotropic reagent (guanidine hydrochloride), and an organic solvent (methanol). Peptides were analyzed in triplicate sample and technical replicates by data-independent LC-MS(E) analysis. Overall, 2333 unique peptides matching to 662 unique proteins were detected with the order of denaturant method efficacy being RapiGest SF Surfactant, PPS Silent Surfactant, guanidine hydrochloride, and methanol. Using bioinformatic analysis, 103 proteins were determined to be integral membrane proteins. When normalizing the data as a percentage of the overall number of peptides and proteins identified for each method, the order for integral membrane protein identification efficacy was methanol, guanidine hydrochloride, RapiGest SF Surfactant, and PPS Silent Surfactant. Interestingly, only 8% of the proteins were identified in all four methods with the silent surfactants having the greatest overlap at 17%. GRAVY analysis at the protein and peptide level indicated that methanol and guanidine hydrochloride promoted detection of hydrophobic proteins and peptides, respectively; however, trypsin activity in the presence of each denaturant was determined as a major factor contributing to peptide identification by LC-MS(E) . These results reveal the complementary nature of each denaturant method, which can be used in an integrated approach to provide a more effective bottom-up analysis of membrane proteomes than can be achieved using only a single denaturant.}, number={5}, journal={PROTEOMICS}, author={Mbeunkui, Flaubert and Goshe, Michael B.}, year={2011}, month={Mar}, pages={898–911} }
 @article{wang_lockney_goshe_franzen_2011, title={Mass Spectrometric Detection of Targeting Peptide Bioconjugation to Red clover necrotic mosaic virus}, volume={22}, ISSN={["1520-4812"]}, DOI={10.1021/bc2001769}, abstractNote={Plant virus nanoparticle (PVN) formulations constructed from Red clover necrotic mosaic virus by drug infusion and targeting peptide conjugation can be employed as drug delivery tools. In this investigation, we studied the cross-linked structures formed by application of sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sSMCC) and succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester (SMPEG) as heterobifunctional linkers in the bioconjugation process. The plant virus formulations using several targeting peptides cross-linked to the plant virus capsid were characterized by LC/MSE analysis, which produced at least 69% sequence coverage using trypsin and chymotrypsin digestion. The results showed evidence for several types of modification located in three domains of the capsid protein. Extensive linker modifications on lysines or cysteines were detected in all the domains, including both intended peptide–capsid cross-links and unintended intracapsid cross-links. Surprisingly, the most extensive peptide modification was observed in the R domain, which is thought to be quite inaccessible to peptides and cross-linking reagents in solution, since it is on the interior of the virus. These results show that heterobifunctional linkers may not be the most efficient method for attachment of peptides to plant virus capsids. As an alternative conjugation strategy, maleimide peptides were used to conjugate with the virus in a one-step reaction. Analysis by LC/MSE showed that these one-step maleimide coupling reactions were more specific, such as modifications of C154 and to a lesser extent C267, and provide a means for achieving more effective PVN formulations.}, number={10}, journal={BIOCONJUGATE CHEMISTRY}, author={Wang, Ruqi and Lockney, Dustin M. and Goshe, Michael B. and Franzen, Stefan}, year={2011}, month={Oct}, pages={1970–1982} }
 @article{liu_goshe_2010, title={Combinatorial Electrostatic Collision-Induced Dissociative Chemical Cross-linking Reagents for Probing Protein Surface Topology}, volume={82}, ISSN={["1520-6882"]}, DOI={10.1021/ac101030w}, abstractNote={To ascertain more information on protein domain orientation and complex structure associations using chemical cross-linking, we have developed a combination of electrostatic collision-induced dissociative cross-linking reagents that differentially react with protein surfaces which are effectively analyzed by liquid chromatography-tandem mass spectrometry using ion trap multistage collision-induced dissociation. Implementing our original design and methodology based on disuccinimidyl-succinamyl-aspartyl-proline (SuDP) (Soderblom, E. J.; Goshe, M. B. Anal. Chem 2006, 78, 8059-8068. Soderblom, E. J.; Bobay, B. G.; Cavanagh, J.; Goshe, M. B. Rapid Commun Mass Spectrom 2007, 21, 3395-3408.), disuccinimidyl-succinamyl-valyl-proline (SuVP) was synthesized. The SuDP and SuVP reagents are the same except for the valyl and aspartyl groups which provide a distinctive chemical feature to each reagent. When performing labeling reactions using various protein-to-cross-linker ratios at pH 7.5, the negatively charged SuDP and neutral SuVP were used to label bovine serum albumin and hemoglobin. After protein digestion, the resulting peptides were analyzed using four different ion trap LC/MS(3) acquisition methods incorporating multistage CID. The more polar BSA surface resulted in a number of unique interpeptide and intrapeptide cross-links for each reagent whereas the less polarized surface of hemoglobin produced similar results for both reagents. Based on the identification of dead-end products (i.e., a cross-link modification containing a hydrolyzed end) for each protein, the aminolysis reactivity of each modified lysyl side chain revealed a preference for reacting with each reagent according to its local electrostatic surface environment. Overall, combinatorial application of SuDP and SuVP chemical labeling produces a set of unique interpeptide, intrapeptide, and dead-end cross-linked products that provides protein structural information according to its electrostatic surface topology which has the potential to be used to more comprehensively probe protein structure and dynamics.}, number={14}, journal={ANALYTICAL CHEMISTRY}, author={Liu, Fan and Goshe, Michael B.}, year={2010}, month={Jul}, pages={6215–6223} }
 @article{blackburn_cheng_williamson_goshe_2010, title={Data-independent liquid chromatography/mass spectrometry (LC/MSE) detection and quantification of the secreted Apium graveolens pathogen defense protein mannitol dehydrogenase}, volume={24}, DOI={10.1002/rcm.4476}, abstractNote={Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, mannitol dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted mannitol. In the current study, LC/MS(E) was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MS(E) can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis.}, number={7}, journal={Rapid Communications in Mass Spectrometry}, author={Blackburn, K. and Cheng, F. Y. and Williamson, J. D. and Goshe, M. B.}, year={2010}, pages={1009–1016} }
 @article{blackburn_mbeunkui_mitra_mentzel_goshe_2010, title={Improving Protein and Proteome Coverage through Data-Independent Multiplexed Peptide Fragmentation}, volume={9}, ISSN={["1535-3907"]}, DOI={10.1021/pr100144z}, abstractNote={Performance differences in protein and proteome characterization achieved by data-independent acquisition (DIA) LC/MS(E) and data-dependent acquisition (DDA) LC/MS/MS approaches were investigated. LC/MS(E) is a novel mode of generating product ion data for all coeluting precursors in parallel as opposed to LC/MS/MS where coeluting precursors must be serially fragmented one at a time. During LC/MS(E) analysis, alternating MS scans of "normal" and "elevated" collision energy are collected at regular intervals, providing nearly a 100% duty cycle for precursor detection and fragmentation because all precursors are fragmented across their full chromatographic elution profile. This is in contrast to DDA-based MS/MS where serial selection of precursor ions is biased toward interrogation and detection of the highest abundance sample components by virtue of the intensity-driven interrogation scheme employed. Both modes of acquisition were applied to a simple four-protein standard mixture with a 16-fold dynamic range in concentration, an in-gel digest of the Arabidopsis thaliana protein FLS2 purified by immunoprecipitation, and a solution-digested tomato leaf proteome sample. Dramatic improvement for individual protein sequence coverage was obtained for all three samples analyzed by the DIA approach, particularly for the lowest abundance sample components. In many instances, precursors readily detected and identified during DIA were either interrogated by MS/MS during DDA at inopportune points in their chromatographic elution profiles resulting in poor quality product ion spectra or not interrogated at all. Detailed evaluation of both DDA and DIA raw data and timing of the MS-to-MS/MS switching events clearly revealed the fundamental limitations of serial MS/MS interrogation and the advantages of parallel fragmentation by DIA for more comprehensive protein identification and characterization which holds promise for enhanced isoform and post-translational modification analysis.}, number={7}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Blackburn, Kevin and Mbeunkui, Flaubert and Mitra, Srijeet K. and Mentzel, Tobias and Goshe, Michael B.}, year={2010}, month={Jul}, pages={3621–3637} }
 @article{mbeunkui_scholl_opperman_goshe_bird_2010, title={Proteomic and Bioinformatic Analysis of the Root-Knot Nematode Meloidogyne hapla: The Basis for Plant Parasitism}, volume={9}, ISSN={1535-3893 1535-3907}, url={http://dx.doi.org/10.1021/pr1006069}, DOI={10.1021/pr1006069}, abstractNote={On the basis of the complete genome sequence of the root-knot nematode Melodogyne hapla, we have deduced and annotated the entire proteome of this plant-parasite to create a database of 14 420 proteins. We have made this database, termed HapPep3, available from the Superfamily repository of model organism proteomes (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY). To experimentally confirm the HapPep3 assignments using proteomics, we applied a data-independent LC/MSE analysis to M. hapla protein extracts fractionated by SDS-PAGE. A total of 516 nonredundant proteins were identified with an average of 9 unique peptides detected per protein. Some proteins, including examples with complex gene organization, were defined by more than 20 unique peptide matches, thus, providing experimental confirmation of computational predictions of intron/exon structures. On the basis of comparisons of the broad physicochemical properties of the experimental and computational proteomes, we conclude that the identified proteins reflect a true and unbiased sampling of HapPep3. Conversely, HapPep3 appears to broadly cover the protein space able to be experimentally sampled. To estimate the false discovery rate, we queried human, plant, and bacterial databases for matches to the LC/MSE-derived peptides, revealing fewer than 1% of matches, most of which were to highly conserved proteins. To provide a functional comparison of the acquired and deduced proteomes, each was subjected to higher order annotation, including comparisons of Gene Ontology, protein domains, signaling, and localization predictions, further indicating concordance, although those proteins that did deviate seem to be highly significant. Approximately 20% of the experimentally sampled proteome was predicted to be secreted, and thus potentially play a role at the host−parasite interface. We examined reference pathways to determine the extent of proteome similarity of M. hapla to that of the free-living nematode, Caenorhabditis elegans, revealing significant similarities and differences. Collectively, the analyzed protein set provides an initial foundation to experimentally dissect the basis of plant parasitism by M. hapla.}, number={10}, journal={Journal of Proteome Research}, publisher={American Chemical Society (ACS)}, author={Mbeunkui, Flaubert and Scholl, Elizabeth H. and Opperman, Charles H. and Goshe, Michael B. and Bird, David McK.}, year={2010}, month={Oct}, pages={5370–5381} }
 @article{gagnon_ju_goshe_maxwell_franzen_2009, title={A role for hydrophobicity in a DielsAlder reaction catalyzed by pyridyl-modified RNA}, volume={37}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkp177}, abstractNote={New classes of RNA enzymes or ribozymes have been obtained by in vitro evolution and selection of RNA molecules. Incorporation of modified nucleotides into the RNA sequence has been proposed to enhance function. DA22 is a modified RNA containing 5-(4-pyridylmethyl) carboxamide uridines, which has been selected for its ability to promote a Diels-Alder cycloaddition reaction. Here, we show that DA_TR96, the most active member of the DA22 RNA sequence family, which was selected with pyridyl-modified nucleotides, accelerates a cycloaddition reaction between anthracene and maleimide derivatives with high turnover. These widely used reactants were not used in the original selection for DA22 and yet here they provide the first demonstration of DA_TR96 as a true multiple-turnover catalyst. In addition, the absence of a structural or essential kinetic role for Cu(2+), as initially postulated, and nonsequence-specific hydrophobic interactions with the anthracene substrate have led to a reevaluation of the pyridine modification's role. These findings broaden the catalytic repertoire of the DA22 family of pyridyl-modified RNAs and suggest a key role for the hydrophobic effect in the catalytic mechanism.}, number={9}, journal={NUCLEIC ACIDS RESEARCH}, author={Gagnon, Keith T. and Ju, Show-Yi and Goshe, Michael B. and Maxwell, E. Stuart and Franzen, Stefan}, year={2009}, month={May}, pages={3074–3082} }
 @article{cheng_blackburn_lin_goshe_williamson_2009, title={Absolute Protein Quantification by LC/MSE for Global Analysis of Salicylic Acid-induced Plant Protein Secretion Responses}, volume={8}, ISSN={["1535-3907"]}, DOI={10.1021/pr800649s}, abstractNote={The plant cell wall is a dynamic cellular compartment consisting of a complex matrix of components that can change dramatically in response to environmental stresses. During pathogen attack, for instance, a wide spectrum of proteins that participate in various sequential processes involved in plant defense is secreted into the cell wall. In this study, a mass spectrometry, data-independent acquisition approach known as LC/MS (E) was used to assess temporal changes in the cell wall proteome in response to different levels of an endogenous inducer of plant disease defense responses, salicylic acid (SA). LC/MS (E) was used as a label-free method that enabled simultaneous protein identification and absolute femtomole quantification of each protein secreted into the extracellular matrix. A total of 74 secreted proteins were identified, 63 of which showed increased specific secretion in response to SA. A majority of this induced secretion occurred within 2 h of treatment, indicating that many proteins are involved in the early stages of plant defenses. We also identified a number of apparently nonclassically secreted proteins, suggesting that, as in many nonplant systems, Golgi/ER-independent mechanisms exist for plant protein secretion. These results provide new insight into plant apoplastic defense mechanisms and demonstrate that LC/MS (E) is a powerful tool for obtaining both relative and absolute proteome-scale quantification that can be applied to complex, time- and dose-dependent experimental designs.}, number={1}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Cheng, Fang-yi and Blackburn, Kevin and Lin, Yu-min and Goshe, Michael B. and Williamson, John D.}, year={2009}, month={Jan}, pages={82–93} }
 @misc{chien_goshe_2009, title={Advances in Quantitative Mass Spectrometry Analysis: Weighing in on Isotope Coding and Label-Free Approaches for Expression and Functional Proteomics}, volume={5}, ISSN={["1875-6727"]}, DOI={10.2174/157341109787846126}, number={2}, journal={CURRENT ANALYTICAL CHEMISTRY}, author={Chien, Ko-yi and Goshe, Michael B.}, year={2009}, month={Apr}, pages={166–185} }
 @article{mitra_walters_clouse_goshe_2009, title={An Efficient Organic Solvent Based Extraction Method for the Proteomic Analysis of Arabidopsis Plasma Membranes}, volume={8}, ISSN={["1535-3907"]}, DOI={10.1021/pr801044y}, abstractNote={Membrane proteins are involved in diverse cellular processes and are an integral component of many signaling cascades, but due to their highly hydrophobic nature and the complexities associated with studying these proteins in planta, alternative methods are being developed to better characterize these proteins on a proteome-wide scale. In our previous work (Mitra, S. K.et al. J. Proteome Res. 2007, 6, (5), 1933−50), methanol-assisted solubilization was determined to facilitate the identification of both hydrophobic and hydrophilic membrane proteins compared to Brij-58 solubilization and was particularly effective for leucine-rich repeat receptor-like kinases (LRR RLKs). To improve peptide identification and to overcome sample losses after tryptic digestion, we have developed an effective chloroform extraction method to promote plasma membrane protein identification. The use of chloroform extraction over traditional solid-phase extraction (SPE) prior to off-line strong cation exchange liquid chromatography (SCXC) and reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis facilitated the removal of chlorophylls, major contaminants of plant tissue preparations that can affect downstream analysis, in addition to the effective removal of trypsin used in the digestion. On the basis of a statistically derived 5% false discovery rate, the chloroform extraction procedure increased the identification of unique peptides for plasma membrane proteins over SPE by 70% which produced nearly a 2-fold increase in detection of membrane transporters and LRR RLKs without increased identification of contaminating Rubisco and ribosomal peptides. Overall, the combined use of methanol and chloroform provides an effective method to study membrane proteins and can be readily applied to other tissues and cells types for proteomic analysis.}, number={6}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Mitra, Srijeet K. and Walters, Benjamin T. and Clouse, Steven D. and Goshe, Michael B.}, year={2009}, month={Jun}, pages={2752–2767} }
 @article{blackburn_goshe_2009, title={Mass Spectrometry Bioinformatics: Tools for Navigating the Proteomics Landscape}, volume={5}, ISSN={["1875-6727"]}, DOI={10.2174/157341109787846135}, abstractNote={Central to the successful implementation of a proteomics pipeline are appropriate bioinformatics tools to provide a level of automation and standardization to the qualitative identification of proteins, quantitation of protein changes, data capture and storage, and integration with other data platforms. Many of these efforts are in various stages of maturity; however, the identification, or recognition, of peptides/proteins from mass spectrometry data, arguably the most developed area, continues to remain challenging due to the ever increasing size of proteomic datasets. Confident peptide and protein identification, including assignment of any post-translational modifications, is a necessary prerequisite for any proteomic study aimed at elucidating biological and physiological responses. This review includes discussions of bioinformatic approaches for the qualitative identification of peptides/proteins from mass spectrometry data as well as the software tools and the analytical considerations required for analysis. Issues related to placing a proteomics dataset in a larger biological context which includes splice variants, variations in databases, and neglected proteomes are also discussed. Keywords: Mass spectrometry, Peptide identification, Product ion spectrum, Database searching, Proteomics, Bioinformatics}, number={2}, journal={CURRENT ANALYTICAL CHEMISTRY}, author={Blackburn, Kevin and Goshe, Michael B.}, year={2009}, month={Apr}, pages={131–143} }
 @article{oh_wang_kota_goshe_clouse_huber_2009, title={Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis}, volume={106}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0810249106}, abstractNote={Brassinosteroids (BRs) are essential growth-promoting hormones that regulate many aspects of plant growth and development. Two leucine-rich repeat receptor-like kinases (LRR-RLKs) are involved in BR perception and signal transduction: brassinosteroid insensitive 1 (BRI1), which is the BR receptor, and its coreceptor BRI1-associated kinase 1 (BAK1). Both proteins are classified as serine/threonine protein kinases, but here we report that recombinant cytoplasmic domains of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases. With BRI1, Tyr-831 and Tyr-956 are identified as autophosphorylation sites in vitro and in vivo. Interestingly, Tyr-956 in kinase subdomain V is essential for activity, because the Y956F mutant is catalytically inactive and thus this site cannot be simply manipulated by mutagenesis. In contrast, Tyr-831 in the juxtamembrane domain is not essential for kinase activity but plays an important role in BR signaling in vivo, because expression of BRI1(Y831F)-Flag in transgenic bri1–5 plants results in plants with larger leaves (but altered leaf shape) and early flowering relative to plants expressing wild-type BRI1-Flag. Acidic substitutions of Tyr-831 restored normal leaf size (but not shape) and normal flowering time. This is an example where a specific tyrosine residue has been shown to play an important role in vivo in plant receptor kinase function. Interestingly, 6 additional LRR-RLKs (of the 23 tested) were also found to autophosphorylate on tyrosine in addition to serine and threonine, suggesting that tyrosine signaling should be considered with other plant receptor kinases as well.}, number={2}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Oh, Man-Ho and Wang, Xiaofeng and Kota, Uma and Goshe, Michael B. and Clouse, Steven D. and Huber, Steven C.}, year={2009}, month={Jan}, pages={658–663} }
 @article{schaff_mbeunkui_blackburn_bird_goshe_2008, title={A sequence-anchored genetic linkage map for the moss, Physcomitrella patens}, volume={56}, number={5}, journal={Plant Journal}, author={Schaff, J. E. and Mbeunkui, F. and Blackburn, K. and Bird, D. M. and Goshe, M. B.}, year={2008}, pages={840–854} }
 @article{ramaroson_ruby_goshe_liu_2008, title={Changes in the Gallus gallus proteorne induced by Marek's disease virus}, volume={7}, ISSN={["1535-3893"]}, DOI={10.1021/pr800268h}, abstractNote={Marek's disease virus (MDV) is a highly oncogenic avian herpesvirus. We have used a modified MudPIT analysis to examine the effect of MDV infection on the chicken proteome. We identified 3561 unique nonphosphorylated peptides, representing 1460 chicken proteins, in a mock-infected sample versus 4240 unique nonphosphorylated peptides, representing 1676 proteins, in an MDV-infected sample. Of these unique peptides, 59.1% from the mock- and 49.6% from the MDV-infected samples were detected in both samples, and for the represented proteins, 69.1% from the mock- and 60.2% from the MDV-infected samples were common to both samples. In terms of phosphorylation, 357 and 506 phosphopeptides, representing 342 and 483 proteins, were detected in the mock- and MDV-infected samples, respectively. At the phosphopeptide level, 10.1% from the mock- and 7.1% from the MDV-infected samples overlapped, and for the represented phosphoproteins, 12.0% from the mock- and 8.5% from the MDV-infected samples were common to both samples. There were no significant differences in the hydropathicity values and number of transmembrane domains of the identified protein sets. Subtle differences were observed for subcellular localizations of the identified proteins. These results suggest that MDV infection may alter host cell biochemistry by perturbing the host's proteomic composition.}, number={10}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Ramaroson, Mialy F. and Ruby, James and Goshe, Michael B. and Liu, Hsiao-Ching}, year={2008}, month={Oct}, pages={4346–4358} }
 @article{galvao_kota_soderblom_goshe_boss_2008, title={Characterization of a new family of protein kinases from Arabidopsis containing phosphoinositide 3/4-kinase and ubiquitin-like domains}, volume={409}, ISSN={["1470-8728"]}, DOI={10.1042/bj20070959}, abstractNote={At least two of the genes predicted to encode type II PI4K (phosphoinositide 4-kinase) in Arabidopsis thaliana (thale cress), namely AtPI4Kγ4 and AtPI4Kγ7, encode enzymes with catalytic properties similar to those of members of the PIKK (phosphoinositide kinase-related kinase) family. AtPI4Kγ4 and AtPI4Kγ7 undergo autophosphorylation and phosphorylate serine/threonine residues of protein substrates, but have no detectable lipid kinase activity. AtPI4Kγ4 and AtPI4Kγ7 are members of a subset of five putative AtPI4Ks that contain N-terminal UBL (ubiquitin-like) domains. In vitro analysis of AtPI4Kγ4 indicates that it interacts directly with, and phosphorylates, two proteins involved in the ubiquitin–proteasome system, namely UFD1 (ubiquitin fusion degradation 1) and RPN10 (regulatory particle non-ATPase 10). On the basis of the present results, we propose that AtPI4Kγ4 and AtPI4Kγ7 should be designated UbDKγ4 and UbDKγ7 (ubiquitin-like domain kinases γ4 and γ7). These UBL-domain-containing AtPI4Ks correspond to a new PIKK subfamily of protein kinases. Furthermore, UFD1 and RPN10 phosphorylation represents an additional mechanism by which their function can be regulated.}, journal={BIOCHEMICAL JOURNAL}, author={Galvao, Rafaelo M. and Kota, Uma and Soderblom, Erik J. and Goshe, Michael B. and Boss, Wendy F.}, year={2008}, month={Jan}, pages={117–127} }
 @article{wang_kota_he_blackburn_li_goshe_huber_clouse_2008, title={Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling}, volume={15}, ISSN={["1534-5807"]}, DOI={10.1016/j.devcel.2008.06.011}, abstractNote={Brassinosteroids (BRs) regulate plant development through a signal transduction pathway involving the BRI1 and BAK1 transmembrane receptor kinases. The detailed molecular mechanisms of phosphorylation, kinase activation, and oligomerization of the BRI1/BAK1 complex in response to BRs are uncertain. We demonstrate that BR-dependent activation of BRI1 precedes association with BAK1 in planta, and that BRI1 positively regulates BAK1 phosphorylation levels in vivo. BRI1 transphosphorylates BAK1 in vitro on specific kinase-domain residues critical for BAK1 function. BAK1 also transphosphorylates BRI1, thereby quantitatively increasing BRI1 kinase activity toward a specific substrate. We propose a sequential transphosphorylation model in which BRI1 controls signaling specificity by direct BR binding followed by substrate phosphorylation. The coreceptor BAK1 is then activated by BRI1-dependent transphosphorylation and subsequently enhances signaling output through reciprocal BRI1 transphosphorylation. This model suggests both conservation and distinct differences between the molecular mechanisms regulating phosphorylation-dependent kinase activation in plant and animal receptor kinases.}, number={2}, journal={DEVELOPMENTAL CELL}, author={Wang, Xiaofeng and Kota, Uma and He, Kai and Blackburn, Kevin and Li, Jia and Goshe, Michael B. and Huber, Steven C. and Clouse, Steven D.}, year={2008}, month={Aug}, pages={220–235} }
 @article{whitehurst_soderblom_west_hernandez_goshe_brown_2007, title={Location and role of free cysteinyl residues in the Sindbis virus E1 and E2 glycoproteins}, volume={81}, ISSN={["0022-538X"]}, DOI={10.1128/JVI.02859-06}, abstractNote={ABSTRACT Sindbis virus is a single-stranded positive-sense RNA virus. It is composed of 240 copies of three structural proteins: E1, E2, and capsid. These proteins form a mature virus particle composed of two nested T=4 icosahedral shells. A complex network of disulfide bonds in the E1 and E2 glycoproteins is developed through a series of structural intermediates as virus maturation occurs (M. Mulvey and D. T. Brown, J. Virol. 68:805-812, 1994; M. Carleton et al., J. Virol. 71:1558-1566, 1997). To better understand the nature of this disulfide network, E1 and E2 cysteinyl residues were labeled with iodoacetamide in the native virus particle and analyzed by liquid chromatography-tandem mass spectrometry. This analysis identified cysteinyl residues of E1 and E2, which were found to be label accessible in the native virus particle, as well as those that were either label inaccessible or blocked by their involvement in disulfide bonds. Native virus particles alkylated with iodoacetamide demonstrated a 4-log decrease in viral infectivity. This suggests that the modification of free cysteinyl residues results in the loss of infectivity by destabilizing the virus particle or that a rearrangement of disulfide bonds, which is required for infectivity, is blocked by the modification. Although modification of these residues prevented infectivity, it did not alter the ability of virus to fuse cells after exposure to acidic pH; thus, modification of free cysteinyl residues biochemically separated the process of infection from the process of membrane fusion.}, number={12}, journal={JOURNAL OF VIROLOGY}, author={Whitehurst, Christopher B. and Soderblom, Erik J. and West, Michelle L. and Hernandez, Raquel and Goshe, Michael B. and Brown, Dennis T.}, year={2007}, month={Jun}, pages={6231–6240} }
 @article{mitra_gantt_ruby_clouse_goshe_2007, title={Membrane proteomic analysis of Arabidopsis thaliana using alternative solubilization techniques}, volume={6}, ISSN={["1535-3907"]}, DOI={10.1021/pr060525b}, abstractNote={This study presents a comparative proteomic analysis of the membrane subproteome of whole Arabidopsis seedlings using 2% Brij-58 or 60% methanol to enrich and solubilize membrane proteins for strong cation exchange fractionation and reversed-phase liquid chromatography-tandem mass spectrometry (LC−MS/MS). A total of 441 proteins were identified by our Brij-58 method, and 300 proteins were detected by our methanol-based solubilization approach. Although the total number of proteins obtained using the nonionic detergent was higher than the total obtained by organic solvent, the ratio of predicted membrane proteins to total proteins identified indicates up to an 18.6% greater enrichment efficiency using methanol. Using two different bioinformatics approaches, between 31.0 and 40.0% of the total proteins identified by the methanol-based method were classified as containing at least one putative transmembrane domain as compared to 22.0−23.4% for Brij-58. In terms of protein hydrophobicity as determined by the GRAVY index, it was revealed that methanol was more effective than Brij-58 for solubilizing membrane proteins ranging from −0.4 (hydrophilic) to +0.4 (hydrophobic). Methanol was also approximately 3-fold more effective than Brij-58 in identifying leucine-rich repeat receptor-like kinases. The ability of methanol to effectively solubilize and denature both hydrophobic and hydrophilic proteins was demonstrated using bacteriorhodopsin and cytochrome c, respectively, where both proteins were identified with at least 82% sequence coverage from a single reversed-phase LC−MS/MS analysis. Overall, our data show that methanol is a better alternative for identifying a wider range of membrane proteins than the nonionic detergent Brij-58. Keywords: Arabidopsis • proteomics • liquid chromatography • mass spectrometry • membrane proteins • phosphorylation • leucine-rich repeat receptor-like kinases}, number={5}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Mitra, Srijeet K. and Gantt, John A. and Ruby, James F. and Clouse, Steven D. and Goshe, Michael B.}, year={2007}, pages={1933–1950} }
 @article{soderblom_bobay_cavanagh_goshe_2007, title={Tandem mass spectrometry acquisition approaches to enhance identification of protein-protein interactions using low-energy collision-induced dissociative chemical crosslinking reagents}, volume={21}, ISSN={["0951-4198"]}, DOI={10.1002/rcm.3213}, abstractNote={Chemical crosslinking combined with mass spectrometry is a useful tool for studying the topological organization of multiprotein interactions, but it is technically challenging to identify peptides involved in a crosslink using tandem mass spectrometry (MS/MS) due to the presence of product ions originating from both peptides within the same crosslink. We have previously developed a novel set of collision-induced dissociative chemical crosslinking reagents (CID-CXL reagents) that incorporate a labile bond within the linker which readily dissociates at a single site under low-energy collision-induced dissociation (CID) to enable independent isolation and sequencing of the crosslinked peptides by traditional MS/MS and database searching. Alternative low-energy CID events were developed within the in-source region by increasing the multipole DC offset voltage (ISCID) or within the ion trap by increasing the collisional excitation (ITCID). Both dissociation events, each having their unique advantages, occur without significant backbone fragmentation to the peptides, thus permitting subsequent CID to be applied to these distinct peptide ions for generation of suitable product ion spectra for database searching. Each approach was developed and applied to a chemical crosslinking study involving the N-terminal DNA-binding domain of AbrB (AbrBN), a transition-state regulator in Bacillus subtilis. A total of thirteen unique crosslinks were identified using the ITCID approach which represented a significant improvement over the eight unique crosslinks identified using the ISCID approach. The ability to segregate intrapeptide and interpeptide crosslinks using ITCID represents the first step towards high-throughput analysis of protein-protein crosslinks using our CID-CXL reagents. Copyright © 2007 John Wiley & Sons, Ltd.}, number={21}, journal={RAPID COMMUNICATIONS IN MASS SPECTROMETRY}, author={Soderblom, Erik J. and Bobay, Benjamin G. and Cavanagh, John and Goshe, Michael B.}, year={2007}, pages={3395–3408} }
 @article{liu_soderblom_goshe_2006, title={A mass spectrometry-based proteomic approach to study Marek's Disease Virus gene expression}, volume={135}, ISSN={0166-0934}, url={http://dx.doi.org/10.1016/j.jviromet.2006.02.001}, DOI={10.1016/j.jviromet.2006.02.001}, abstractNote={Marek's Disease Virus (MDV) is an avian herpesvirus that causes a lymphoproliferative disorder in chickens. MDV transitions between a lytic phase in which new viruses are produced and a latent phase in which the virus lays dormant. The mechanism controlling this lytic-to-latent switch remains unclear. To better understand the lytic phase of MDV infection, a mass spectrometry-based strategy was developed to identify viral proteins and to qualitatively examine their abundance in lytically infected chicken embryo fibroblast (CEF) cells. A combination of strong cation exchange chromatography (SCXC) and microcapillary reversed-phase liquid chromatography-tandem mass spectrometry (murpLC/MS/MS) was used to resolve peptides from tryptic digests of MDV-infected CEF cell lysates. Peptides were identified by searching the tandem mass spectra against a protein database containing both MDV proteins and all currently available Gallus gallus proteins using the SEQUEST algorithm. A total of 427 MDV peptides, corresponding to 82 unique proteins, were identified, with 56 of them detected with at least two unique peptides. Overall, nearly 80% of all putative MDV proteins expressed in infected CEF cells were identified. We anticipate that this approach will be a viable method for determining how viral and host proteome changes occurring in Marek's Disease pathogenesis regulate the switch between the lytic and latent phases of the MDV life cycle.}, number={1}, journal={Journal of Virological Methods}, publisher={Elsevier BV}, author={Liu, Hsiao-Ching S. and Soderblom, Erik J. and Goshe, Michael B.}, year={2006}, month={Jul}, pages={66–75} }
 @article{soderblom_goshe_2006, title={Collision-induced dissociative chemical cross-linking reagents and methodology: Applications to protein structural characterization using tandem mass spectrometry analysis}, volume={78}, ISSN={["0003-2700"]}, DOI={10.1021/ac0613840}, abstractNote={Chemical cross-linking combined with mass spectrometry is a viable approach to study the low-resolution structure of protein and protein complexes. However, unambiguous identification of the residues involved in a cross-link remains analytically challenging. To enable a more effective analysis across various MS platforms, we have developed a novel set of collision-induced dissociative cross-linking reagents and methodology for chemical cross-linking experiments using tandem mass spectrometry (CID-CXL-MS/MS). These reagents incorporate a single gas-phase cleavable bond within their linker region that can be selectively fragmented within the in-source region of the mass spectrometer, enabling independent MS/MS analysis for each peptide. Initial design concepts were characterized using a synthesized cross-linked peptide complex. Following verification and subsequent optimization of cross-linked peptide complex dissociation, our reagents were applied to homodimeric glutathione S-transferase and monomeric bovine serum albumin. Cross-linked residues identified by our CID-CXL-MS/MS method were in agreement with published crystal structures and previous cross-linking studies using conventional approaches. Common LC/MS/MS acquisition approaches such as data-dependent acquisition experiments using ion trap mass spectrometers and product ion spectral analysis using SEQUEST were shown to be compatible with our CID-CXL-MS/MS reagents, obviating the requirement for high resolution and high mass accuracy measurements to identify both intra- and interpeptide cross-links.}, number={23}, journal={ANALYTICAL CHEMISTRY}, author={Soderblom, Erik J. and Goshe, Michael B.}, year={2006}, month={Dec}, pages={8059–8068} }
 @article{feeney_soderblom_goshe_clark_2006, title={Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker}, volume={47}, ISSN={1046-5928}, url={http://dx.doi.org/10.1016/j.pep.2005.10.005}, DOI={10.1016/j.pep.2005.10.005}, abstractNote={Coupled with over-expression in host organisms, fusion protein systems afford economical methods to obtain large quantities of target proteins in a fast and efficient manner. Some proteases used for these purposes cleave C-terminal to their recognition sequences and do not leave extra amino acids on the target. However, they are often inefficient and are frequently promiscuous, resulting in non-specific cleavages of the target protein. To address these issues, we created a fusion protein system that utilizes a highly efficient enzyme and leaves no residual amino acids on the target protein after removal of the affinity tag. We designed a glutathione S -transferase (GST)-fusion protein vector with a caspase-3 consensus cleavage sequence located between the N-terminal GST tag and a target protein. We show that the enzyme efficiently cleaves the fusion protein without leaving excess amino acids on the target protein. In addition, we used an engineered caspase-3 enzyme that is highly stable, has increased activity relative to the wild-type enzyme, and contains a poly-histidine tag that allows for efficient removal of the enzyme after cleavage of the fusion protein. Although we have developed this system using a GST tag, the system is amenable to any commercially available affinity tag.}, number={1}, journal={Protein Expression and Purification}, publisher={Elsevier BV}, author={Feeney, Brett and Soderblom, Erik J. and Goshe, Michael B. and Clark, A. Clay}, year={2006}, month={May}, pages={311–318} }
 @article{wang_goshe_soderblom_phinney_kuchar_li_asami_yoshida_huber_clouse_2005, title={Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase}, volume={17}, ISSN={["1532-298X"]}, DOI={10.1105/tpc.105.031393}, abstractNote={Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.}, number={6}, journal={PLANT CELL}, author={Wang, XF and Goshe, MB and Soderblom, EJ and Phinney, BS and Kuchar, JA and Li, J and Asami, T and Yoshida, S and Huber, SC and Clouse, SD}, year={2005}, month={Jun}, pages={1685–1703} }
 @article{bobay_benson_naylor_feeney_clark_goshe_strauch_thompson_cavanagh_2004, title={Evaluation of the DNA Binding Tendencies of the Transition State Regulator AbrB†}, volume={43}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi048399h}, DOI={10.1021/bi048399h}, abstractNote={Global transition state regulator proteins represent one of the most diverse classes of prokaryotic transcription factors. One such transition state regulator, AbrB from Bacillus subtilis, is known to bind more than 60 gene targets yet displays specificity within this target set by binding each promoter with a different affinity. Microelectrospray ionization mass spectrometry (microESI-MS), circular dichroism, fluorescence, UV spectroscopy, and molecular modeling were used to elucidate differences among AbrB, DNA, and AbrB-DNA complexes. MicroESI-MS analysis of AbrB confirmed its stable macromolecular state as being tetrameric and verified the same stoichiometric state in complex with DNA targets. MicroESI-MS, circular dichroism, and fluorescence provided relative binding affinities for AbrB-DNA interactions in a qualitative manner. UV spectroscopy was used in a quantitative manner to determine solution phase dissociation constants for AbrB-DNA complexes. General DNA structural parameters for all known natural AbrB binding sequences were also studied and significant similarities in topological constraints (stretch, opening, and propeller twist) were observed. It is likely that these parameters contribute to the differential binding proclivities of AbrB. In addition to providing an improved understanding of transition state regulator-DNA binding properties and structural tendencies of target promoters, this comprehensive and corroborative spectroscopic study endorses the use of microESI-MS for rapidly ascertaining qualitative binding trends in noncovalent systems in a high-throughput manner.}, number={51}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Bobay, Benjamin G. and Benson, Linda and Naylor, Stephen and Feeney, Brett and Clark, A. Clay and Goshe, Michael B. and Strauch, Mark A. and Thompson, Richele and Cavanagh, John}, year={2004}, month={Dec}, pages={16106–16118} }
 @article{blonder_goshe_xiao_camp_wingerd_davis_smith_2004, title={Global Analysis of the Membrane Subproteome of Pseudomonas aeruginosaUsing Liquid Chromatography-Tandem Mass Spectrometry}, volume={3}, ISSN={1535-3893 1535-3907}, url={http://dx.doi.org/10.1021/pr034074w}, DOI={10.1021/pr034074w}, abstractNote={Pseudomonas aeruginosa is one of the most significant opportunistic bacterial pathogens in humans causing infections and premature death in patients with cystic fibrosis, AIDS, severe burns, organ transplants, or cancer. Liquid chromatography coupled online with tandem mass spectrometry was used for the large-scale proteomic analysis of the P. aeruginosa membrane subproteome. Concomitantly, an affinity labeling technique, using iodoacetyl-PEO biotin to tag cysteinyl-containing proteins, permitted the enrichment and detection of lower abundance membrane proteins. The application of these approaches resulted in the identification of 786 proteins. A total of 333 proteins (42%) had a minimum of one transmembrane domain (ranging from 1 to14) and 195 proteins were classified as hydrophobic based on their positive GRAVY values (ranging from 0.01 to 1.32). Key integral inner and outer membrane proteins involved in adaptation and antibiotic resistance were conclusively identified, including the detection of 53% of all predicted opr-type porins (outer integral membrane proteins) and all the components of the mexA-mexB-oprM transmembrane protein complex. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of P. aeruginosa and for prokaryotes in general. Keywords: proteome • membrane proteins • low abundance • LC−MS/MS • affinity labeling}, number={3}, journal={Journal of Proteome Research}, publisher={American Chemical Society (ACS)}, author={Blonder, Josip and Goshe, Michael B. and Xiao, Wenzhong and Camp, David G. and Wingerd, Mark and Davis, Ronald W. and Smith, Richard D.}, year={2004}, month={Jun}, pages={434–444} }