Selected publications citing work performed at Bioproximity


  1. So, Christopher R., Fears, Kenan P., Leary, Dagmar H., Scancella, Jenifer M., Wang, Zheng, Liu, Jinny L., Orihuela, Beatriz, Rittschof, Dan, Spillmann, Christopher M. Wahl, Kathryn J.
    Sequence basis of Barnacle Cement Nanostructure is Defined by Proteins with Silk Homology
    Scientific Reports (2016)
    Journal
    Abstract
    Barnacles adhere by producing a mixture of cement proteins (CPs) that organize into a permanently bonded layer displayed as nanoscale fibers. These cement proteins share no homology with any other marine adhesives, and a common sequence-basis that defines how nanostructures function as adhesives remains undiscovered. Here we demonstrate that a significant unidentified portion of acorn barnacle cement is comprised of low complexity proteins; they are organized into repetitive sequence blocks and found to maintain homology to silk motifs. Proteomic analysis of aggregate bands from PAGE gels reveal an abundance of Gly/Ala/Ser/Thr repeats exemplified by a prominent, previously unidentified, 43 kDa protein in the solubilized adhesive. Low complexity regions found throughout the cement proteome, as well as multiple lysyl oxidases and peroxidases, establish homology with silk-associated materials such as fibroin, silk gum sericin, and pyriform spidroins from spider silk. Distinct primary structures defined by homologous domains shed light on how barnacles use low complexity in nanofibers to enable adhesion, and serves as a starting point for unraveling the molecular architecture of a robust and unique class of adhesive nanostructures.
  1. Ding, Fang, Duan, Yongping, Yuan, Qing, Shao, Jonathan Hartung, John S.
    Serological detection of 'Candidatus Liberibacter asiaticus' in citrus, and identification by GeLC-MS/MS of a chaperone protein responding to cellular pathogens
    Scientific Reports (2016)
    Journal
    Abstract
    We describe experiments with antibodies against ‘Candidatus Liberibacter asiaticus used to detect the pathogen in infected plants. We used scFv selected to bind epitopes exposed on the surface of the bacterium in tissue prints, with secondary monoclonal antibodies directed at a FLAG epitope included at the carboxyl end of the scFv. Unexpectedly, the anti-FLAG secondary antibody produced positive results with CaLas diseased samples when the primary scFv were not used. The anti-FLAG monoclonal antibody (Mab) also identified plants infected with other vascular pathogens. We then identified a paralogous group of secreted chaperone proteins in the HSP-90 family that contained the amino acid sequence DDDDK identical to the carboxy-terminal sequence of the FLAG epitope. A rabbit polyclonal antibody against one of the same epitopes combined with a goat anti-rabbit secondary antibody produced very strong purple color in individual phloem cells, as expected for this pathogen. These results were entirely specific for CaLas-infected citrus. The simplicity, cost and ability to scale the tissue print assay makes this an attractive assay to complement PCR-based assays currently in use. The partial FLAG epitope may itself be useful as a molecular marker for the rapid screening of citrus plants for the presence of vascular pathogens.
  1. Zhang, Mingzhen, Viennois, Emilie, Prasad, Meena, Zhang, Yunchen, Wang, Lixin, Zhang, Zhan, Han, Moon K., Xiao, Bo, Xu, Changlong, Srinivasan, Shanthi Merlin, Didier
    Edible Ginger-Derived Nanoparticles: A Novel Therapeutic Approach for the Prevention and Treatment of Inflammatory Bowel Disease and Colitis-Associated Cancer
    Biomaterials (2016)
    Journal
    Abstract
    There is a clinical need for new, more effective treatments for chronic and debilitating inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis. In this study, we characterized a specific population of nanoparticles derived from edible ginger (GDNPs 2) and demonstrated their efficient colon targeting following oral administration. GDNPs 2 had an average size of ∼230 nm and exhibited a negative zeta potential. These nanoparticles contained high levels of lipids, a few proteins, ∼125 microRNAs (miRNAs), and large amounts of ginger bioactive constituents (6-gingerol and 6-shogaol). We also demonstrated that GDNPs 2 were mainly taken up by intestinal epithelial cells (IECs) and macrophages, and were nontoxic. Using different mouse colitis models, we showed that GDNPs 2 reduced acute colitis, enhanced intestinal repair, and prevented chronic colitis and colitis-associated cancer (CAC). 2D-DIGE/MS analyses further identified molecular target candidates of GDNPs 2 involved in these mouse models. Oral administration of GDNPs 2 increased the survival and proliferation of IECs and reduced the pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β), and increased the anti-inflammatory cytokines (IL-10 and IL-22) in colitis models, suggesting that GDNPs 2 has the potential to attenuate damaging factors while promoting the healing effect. In conclusion, GDNPs 2, nanoparticles derived from edible ginger, represent a novel, natural delivery mechanism for improving IBD prevention and treatment with an added benefit of overcoming limitations such as potential toxicity and limited production scale that are common with synthetic nanoparticles.
  1. Nazemof, Nazila, Couroux, Philippe, Xing, Tim Robert, Laurian S.
    Proteomic Analysis of the Mature Brassica Stigma Reveals Proteins with Diverse Roles in Vegetative and Reproductive Development
    Plant Science (2016)
    Journal
    Abstract
    First characterization of a Brassicaceae mature stigma global proteome. More than 2500 Brassica napus stigma proteins were identified. Stigma proteins had roles in embryo, pistil, root hair and pollen tube development. Brassicaceae and Poaceae stigma proteomes showed conserved and distinct features. The stigma, the specialized apex of the Brassicaceae gynoecium, plays a role in pollen capture, discrimination, hydration, germination, and guidance. Despite this crucial role in reproduction, the global proteome underlying Brassicaceae stigma development and function remains largely unknown. As a contribution towards the characterization of the Brassicaceae dry stigma global proteome, more than 2500 Brassica napus mature stigma proteins were identified using three different gel-based proteomics approaches. Most stigma proteins participated in Metabolic Processes, Responses to Stimulus or Stress, Cellular or Developmental Processes, and Transport. The stigma was found to express a wide variety of proteins with demonstrated roles in cellular and organ development including proteins known to be involved in cellular expansion and morphogenesis, embryo development, as well as gynoecium and stigma development. Comparisons to a corresponding proteome from a very morphologically different Poaceae dry stigma showed a very similar distribution of proteins among different functional categories, but also revealed evident distinctions in protein composition especially in glucosinolate and carotenoid metabolism, photosynthesis, and self-incompatibility. To our knowledge, this study reports the largest Brassicaceae stigma protein dataset described to date.
  1. Luna-Nácar, Milka, Navarrete-Perea, José, Moguel, Bárbara, Bobes, Raúl J., Laclette, Juan P. Carrero, Julio C.
    Proteomic Study of Entamoeba histolytica Trophozoites, Cysts, and Cyst-Like Structures
    PLOS ONE (2016)
    Journal
    Abstract
    The cyst stage of Entamoeba histolytica is a promising therapeutic target against human amoebiasis. Our research team previously reported the production in vitro of Cyst-Like Structures (CLS) sharing structural features with cysts, including rounded shape, size reduction, multinucleation, and the formation of a chitin wall coupled to the overexpression of glucosamine 6-phosphate isomerase, the rate-limiting enzyme of the chitin synthesis pathway. A proteomic study of E. histolytica trophozoites, cysts, and in vitro-produced CLS is reported herein to determine the nature of CLS, widen our knowledge on the cyst stage, and identify possible proteins and pathways involved in the encystment process. Total protein extracts were obtained from E. histolytica trophozoites, CLS, and partially purified cysts recovered from the feces of amoebic human patients; extracts were trypsin-digested and analyzed by LC-MS/MS. In total, 1029 proteins were identified in trophozoites, 550 in CLS, and 411 in cysts, with 539, 299, and 84 proteins unique to each sample, respectively, and only 74 proteins shared by all three stages. About 70% of CLS proteins were shared with trophozoites, even though differences were observed in the relative protein abundance. While trophozoites showed a greater abundance of proteins associated to a metabolically active cell, CLS showed higher expression of proteins related to proteolysis, redox homeostasis, and stress response. In addition, the expression of genes encoding for the cyst wall proteins Jessie and Jacob was detected by RT-PCR and the Jacob protein identified by Western blotting and immunofluorescence in CLS. However, the proteomic profile of cysts as determined by LC-MS/MS was very dissimilar to that of trophozoites and CLS, with almost 40% of hypothetical proteins. Our global results suggest that CLS are more alike to trophozoites than to cysts, and they could be generated as a rapid survival response of trophozoites to a stressful condition, which allows the parasite to survive temporarily inside a chitin-like resistant cover containing Jacob protein. Our findings lead us to suggest that encystment and CLS formation could be distinct stress responses. In addition, we show that cysts express a high number of genes with unknown function, including four new, highly antigenic, possibly membrane-located proteins that could be targets of therapeutic and diagnostic usefulness.
  1. Sparks, J. Alan, Kwon, Taegun, Renna, Luciana, Liao, Fuqi, Brandizzi, Federica Blancaflor, Elison B.
    HLB1 is a Novel Tetratricopeptide Repeat Domain-Containing Protein that Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis
    The Plant Cell (2016)
    Journal
    Abstract
    The endomembrane system plays essential roles in plant development but the proteome responsible for its function and organization still remains largely uncharacterized in plants. Here we report on the identification and characterization of the Hypersensitive to Latrunculin B 1 (HLB1) protein isolated through a forward genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat (TPR) domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi Network (TGN)/early endosome (EE) and tracked along filamentous actin (F-actin) indicating that it could be a novel factor that links post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HopM interactor 7/Brefeldin A-visualized endocytic trafficking defective 1) by co-immunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of a hlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of TGN/EE at the intersection of the exocytic and endocytic pathways in plants.
  1. Aguilo, Francesca, Li, SiDe, Balasubramaniyan, Natarajan, Sancho, Ana, Benko, Sabina, Zhang, Fan, Vashisht, Ajay, Rengasamy, Madhumitha, Andino, Blanca, Chen, Chih-hung, Zhou, Felix, Qian, Chengmin, Zhou, Ming-Ming, Wohlschlegel, James A., Zhang, Weijia, Suchy, Frederick J. Walsh, Martin J.
    Deposition of 5-Methylcytosine on Enhancer RNAs Enables the Coactivator Function of PGC-1α
    Cell Reports (2016)
    Journal
    Abstract
    K779 of PGC-1α is methylated by SET7/9 and demethylated by LSD1 PGC-1α[K779me] recruits SAGA and Mediator at enhancers of target genes PGC-1α[K779me] corresponds with NSUN7 and m5C eRNAs at PGC1α-regulated loci Enrichment of m5C within eRNA coincides with fasting in vivo The Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a transcriptional co-activator that plays a central role in adapted metabolic responses. PGC-1α is dynamically methylated and unmethylated at the residue K779 by the methyltransferase SET7/9 and the Lysine Specific Demethylase 1A (LSD1), respectively. Interactions of methylated PGC-1α[K779me] with the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, the Mediator members MED1 and MED17, and the NOP2/Sun RNA methytransferase 7 (NSUN7) reinforce transcription, and are concomitant with the m5C mark on enhancer RNAs (eRNAs). Consistently, loss of Set7/9 and NSun7 in liver cell model systems resulted in depletion of the PGC-1α target genes Pfkl, Sirt5, Idh3b, and Hmox2, which was accompanied by a decrease in the eRNAs levels associated with these loci. Enrichment of m5C within eRNA species coincides with metabolic stress of fasting in vivo. Collectively, these findings illustrate the complex epigenetic circuitry imposed by PGC-1α at the eRNA level to fine-tune energy metabolism.
  1. Tripathi, Prateek, Rabara, Roel C., Reese, R. Neil, Miller, Marissa A., Rohila, Jai S., Subramanian, Senthil, Shen, Qingxi J., Morandi, Dominique, Bücking, Heike, Shulaev, Vladimir Rushton, Paul J.
    A toolbox of genes, proteins, metabolites and promoters for improving drought tolerance in soybean includes the metabolite coumestrol and stomatal development genes
    BMC genomics, vol. 17, no. 1 (2016)
    Journal
    Abstract
    The purpose of this project was to identify metabolites, proteins, genes, and promoters associated with water stress responses in soybean. A number of these may serve as new targets for the biotechnological improvement of drought responses in soybean (Glycine max). RESULTS: We identified metabolites, proteins, and genes that are strongly up or down regulated during rapid water stress following removal from a hydroponics system. 163 metabolites showed significant changes during water stress in roots and 93 in leaves. The largest change was a root-specific 160-fold increase in the coumestan coumestrol making it a potential biomarker for drought and a promising target for improving drought responses. Previous reports suggest that coumestrol stimulates mycorrhizal colonization and under certain conditions mycorrhizal plants have improved drought tolerance. This suggests that coumestrol may be part of a call for help to the rhizobiome during stress. About 3,000 genes were strongly up-regulated by drought and we identified regulators such as ERF, MYB, NAC, bHLH, and WRKY transcription factors, receptor-like kinases, and calcium signaling components as potential targets for soybean improvement as well as the jasmonate and abscisic acid biosynthetic genes JMT, LOX1, and ABA1. Drought stressed soybean leaves show reduced mRNA levels of stomatal development genes including FAMA-like, MUTE-like and SPEECHLESS-like bHLH transcription factors and leaves formed after drought stress had a reduction in stomatal density of 22.34 % and stomatal index of 17.56 %. This suggests that reducing stomatal density may improve drought tolerance. MEME analyses suggest that ABRE (CACGT/CG), CRT/DRE (CCGAC) and a novel GTGCnTGC/G element play roles in transcriptional activation and these could form components of synthetic promoters to drive expression of transgenes. Using transformed hairy roots, we validated the increase in promoter activity of GmWRKY17 and GmWRKY67 during dehydration and after 20 μM ABA treatment. CONCLUSIONS: Our toolbox provides new targets and strategies for improving soybean drought tolerance and includes the coumestan coumestrol, transcription factors that regulate stomatal density, water stress-responsive WRKY gene promoters and a novel DNA element that appears to be enriched in water stress responsive promoters.
  1. Taylor, Erin B., Moulana, Mohadetheh, Stuge, Tor B., Quiniou, Sylvie M. A., Bengten, Eva Wilson, Melanie
    A Leukocyte Immune-Type Receptor Subset Is a Marker of Antiviral Cytotoxic Cells in Channel Catfish, Ictalurus punctatus
    The Journal of Immunology, pp. 1502166+ (2016)
    Journal
    Abstract
    Channel catfish, Ictalurus punctatus, leukocyte immune type receptors (LITRs) represent a multigene family that encodes Ig superfamily proteins that mediate activating or inhibitory signaling. In this study, we demonstrate the use of mAb CC41 to monitor viral cytotoxic responses in catfish and determine that CC41 binds to a subset of LITRs on the surface of catfish clonal CTLs. Homozygous gynogenetic catfish were immunized with channel catfish virus (CCV)-infected MHC-matched clonal T cells (G14D-CCV), and PBL were collected at various times after immunization for flow cytometric analyses. The percentage of CC41(+) cells was significantly increased 5 d after primary immunization with G14D-CCV and at 3 d after a booster immunization as compared with control fish only injected with G14D. Moreover, CC41(+) cells magnetically isolated from the PBL specifically killed CCV-infected targets as measured by (51)Cr release assays and expressed messages for CD3γδ, perforin, and at least one of the CD4-like receptors as analyzed by RNA flow cytometry. When MLC effector cells derived from a G14D-CCV-immunized fish were preincubated with CC41 mAb, killing of G14D-CCV targets was reduced by ∼40%, suggesting that at least some LITRs have a role in target cell recognition and/or cytotoxicity. The availability of a LITR-specific mAb has allowed, to our knowledge for the first time, functional characterization of LITRs in an autologous system. In addition, the identification of an LITR subset as a cytotoxic cell marker will allow for more effective monitoring of catfish immune responses to pathogens.
  1. Work, Thierry M., Dagenais, Julie, Breeden, Renee, Schneemann, Anette, Sung, Joyce, Hew, Brian, Balazs, George H. Berestecky, John M.
    Green Turtles (Chelonia mydas) Have Novel Asymmetrical Antibodies
    The Journal of Immunology, pp. 1501332+ (2015)
    PubMed
    Abstract
    Igs in vertebrates comprise equally sized H and L chains, with exceptions such as H chain–only Abs in camels or natural Ag receptors in sharks. In Reptilia, Igs are known as IgYs. Using immunoassays with isotype-specific mAbs, in this study we show that green turtles (Chelonia mydas) have a 5.7S 120-kDa IgY comprising two equally sized H/L chains with truncated Fc and a 7S 200-kDa IgY comprised of two differently sized H chains bound to L chains and apparently often noncovalently associated with an antigenically related 90-kDa moiety. Both the 200- and 90-kDa 7S molecules are made in response to specific Ag, although the 90-kDa molecule appears more prominent after chronic Ag stimulation. Despite no molecular evidence of a hinge, electron microscopy reveals marked flexibility of Fab arms of 7S and 5.7S IgY. Both IgY can be captured with protein G or melon gel, but less so with protein A. Thus, turtle IgY share some characteristics with mammalian IgG. However, the asymmetrical structure of some turtle Ig and the discovery of an Ig class indicative of chronic antigenic stimulation represent striking advances in our understanding of immunology.
  1. Lupsa, Nikolett and Érsek, Barbara and Pócza, Péter and Sarzsinszky, Eszter and Toth, Anett and Bagita, Bence and Bencsik, András and Hegyesi, Hargita and Matolcsy, András and Buzás, Edit Irén and Pós, Zoltán
    CD8+/CD103+ tissue-resident memory T cells display substantial functional plasticity and fine-tune their phenotype to meet local needs..
    Journal of Molecular Cell Biology [pending] (2015)
    Abstract
    In this study we sought a better understanding of the unique phenotypic plasticity displayed by CD8+/CD103+ tissue-resident memory T cells (Trms) in distinct tissue environments. Murine Trm cells were isolated from distinct tissues by automated tissue dissociation and magnetic sorting. Distinctive features of the Trm phenotype in distinct tissues were described by microarray gene expression profiling, Q-PCR, UPLC-MS/MS, and flow cytometry. We found that CD8+ small intestinal Trms frequentlyexhibit an effector-like phenotype, express various chemokines, granzymes A/B, show evidence of degranulation, intense protein synthesis and sustained clonal expansion. In contrast, lung-resident Trms typically express lymphotoxins α/β, but not granzymes or markers of degranulation, and rarely divide due to G2/M arrest. Finally, CD8+ CD103+ T cells in the liver, a tolerogenic environment for CD8+ T cells, are mostly dormant, but often positive for CXCR4, a receptor of the liver-released chemokine SDF-1. We also found, however, that subtle phenotypic changesaffect non-resident CD8+ T effector (Teff) cells infiltrating the same tissues, too. Although far less pronounced, these changes were often analogous to those observed in Trm cells.These data suggest that Trm cells display a unique phenotypic plasticity and adapt to varying environmental conditions by multiple, although not exclusively Trm-restricted mechanisms.
  1. Sancho, Ana, Li, SiDe, Paul, Thankam, Zhang, Fan, Aguilo, Francesca, Vashisht, Ajay, Balasubramaniyan, Natarajan, Leleiko, Neal S., Suchy, Frederick J., Wohlschlegel, James A., Zhang, Weijia Walsh, Martin J.
    CHD6 regulates the topological arrangement of the CFTR locus.
    Human molecular genetics, vol. 24, no. 10, 2724-2732 (2015)
    PubMed
    Abstract
    The control of transcription is regulated through the well-coordinated spatial and temporal interactions between distal genomic regulatory elements required for specialized cell-type and developmental gene expression programs. With recent findings CFTR has served as a model to understand the principles that govern genome-wide and topological organization of distal intra-chromosomal contacts as it relates to transcriptional control. This is due to the extensive characterization of the DNase hypersensitivity sites, modification of chromatin, transcription factor binding sites and the arrangement of these sites in CFTR consistent with the restrictive expression in epithelial cell types. Here, we identified CHD6 from a screen among several chromatin-remodeling proteins as a putative epigenetic modulator of CFTR expression. Moreover, our findings of CTCF interactions with CHD6 are consistent with the role described previously for CTCF in CFTR regulation. Our results now reveal that the CHD6 protein lies within the infrastructure of multiple transcriptional complexes, such as the FACT, PBAF, PAF1C, Mediator, SMC/Cohesion and MLL complexes. This model underlies the fundamental role CHD6 facilitates by tethering cis-acting regulatory elements of CFTR in proximity to these multi-subunit transcriptional protein complexes. Finally, we indicate that CHD6 structurally coordinates a three-dimensional stricture between intragenic elements of CFTR bound by several cell-type specific transcription factors, such as CDX2, SOX18, HNF4α and HNF1α. Therefore, our results reveal new insights into the epigenetic regulation of CFTR expression, whereas the manipulation of CFTR gene topology could be considered for treating specific indications of cystic fibrosis and/or pancreatitis.
  1. Bronisz, Agnieszka, Wang, Yan, Nowicki, Michal O., Peruzzi, Pierpaolo, Ansari, Khairul I., Ogawa, Daisuke, Balaj, Leonora, De Rienzo, Gianluca, Mineo, Marco, Nakano, Ichiro, Ostrowski, Michael C., Hochberg, Fred, Weissleder, Ralph, Lawler, Sean E., Chiocca, E. Antonio Godlewski, Jakub
    Extracellular vesicles modulate the glioblastoma microenvironment via a tumor suppression signaling network directed by miR-1
    Cancer research, vol. 74, no. 3, 738-750 (2014)
    PubMed
    Abstract
    Extracellular vesicles have emerged as important mediators of intercellular communication in cancer, including by conveying tumor-promoting microRNAs between cells, but their regulation is poorly understood. In this study, we report the findings of a comparative microRNA profiling and functional analysis in human glioblastoma that identifies miR-1 as an orchestrator of extracellular vesicle function and glioblastoma growth and invasion. Ectopic expression of miR-1 in glioblastoma cells blocked in vivo growth, neovascularization, and invasiveness. These effects were associated with a role for miR-1 in intercellular communication in the microenvironment mediated by extracellular vesicles released by cancer stem-like glioblastoma cells. An extracellular vesicle-dependent phenotype defined by glioblastoma invasion, neurosphere growth, and endothelial tube formation was mitigated by loading miR-1 into glioblastoma-derived extracellular vesicles. Protein cargo in extracellular vesicles was characterized to learn how miR-1 directed extracellular vesicle function. The mRNA encoding Annexin A2 (ANXA2), one of the most abundant proteins in glioblastoma-derived extracellular vesicles, was found to be a direct target of miR-1 control. In addition, extracellular vesicle-derived miR-1 along with other ANXA2 extracellular vesicle networking partners targeted multiple pro-oncogenic signals in cells within the glioblastoma microenvironment. Together, our results showed how extracellular vesicle signaling promotes the malignant character of glioblastoma and how ectopic expression of miR-1 can mitigate this character, with possible implications for how to develop a unique miRNA-based therapy for glioblastoma management.
  1. Burtnick, Mary N., Brett, Paul J. DeShazer, David
    Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM.
    Infection and immunity, vol. 82, no. 8, 3214-3226 (2014)
    PubMed | Full Text HTML, PDF | Spotlight Article
    Abstract
    Burkholderia pseudomallei, the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provide B. pseudomallei with a growth advantage in vitro and in vivo, but relatively little is known about the full repertoire of exoproducts associated with each system. In this study, we constructed deletion mutations in gspD and gspE, T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles of B. pseudomallei MSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants of B. pseudomallei MSHR668 and B. pseudomallei ΔgspD grown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of ΔgspD strains. Many of these proteins were putative hydrolytic enzymes, including 12 proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities is T2SS dependent. Previous studies had failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of the B. pseudomallei T2SS secretome.
  1. Skeie, Jessica M. Mahajan, Vinit B.
    Proteomic Landscape of the Human Choroid-Retinal Pigment Epithelial Complex
    JAMA Ophthalmology (2014)
    PubMed
    Abstract
    IMPORTANCE:

    Differences in geographical protein expression in the human choroid-retinal pigment epithelial (RPE) complex may explain molecular predisposition of regions to ophthalmic diseases such as age-related macular degeneration.

    OBJECTIVE:

    To characterize the proteome of the human choroid-RPE complex and to identify differentially expressed proteins in specific anatomic regions.

    DESIGN, SETTING, AND PARTICIPANTS:

    Experimental study of choroid-RPE tissue from 3 nondiseased eyes. The choroid-RPE complex underwent biopsy from beneath the foveal, macular, and peripheral retina. Protein fractions were isolated and subjected to multidimensional liquid chromatography and tandem mass spectrometry. A bioinformatic pipeline matched peptide spectra to the human proteome, assigned gene ontology classification, and identified protein signaling pathways unique to each of the choroid-RPE regions.

    MAIN OUTCOMES AND MEASURES:

    Mean number of mass spectra, statistically significant differentially expressed proteins, gene ontology classification, and pathway representation.

    RESULTS:

    We identified a mean of 4403 unique proteins in each of the foveal, macular, and peripheral choroid-RPE tissues. Six hundred seventy-one differentially expressed proteins included previously known risk factors for retinal diseases related to oxidative stress, inflammation, and the complement cascade. Gene ontology analysis showed that unique categories in the foveal and macular regions included immune process proteins as well as protein complexes and plasma membrane proteins. The peripheral region contained unique antioxidant activity proteins. Many proteins had the highest expression in the foveal or macular regions, including inflammation-related proteins HLA-A, HLA-B, and HLA-C antigens; intercellular adhesion molecule 1 (ICAM-1); S100; transcription factor ERG; antioxidant superoxide dismutase 1 (SOD1); chloride intracellular channel 6 ion (CLIC6); activators of the complement cascade C1q, C6, and C8; and complement factor H. Proteins with higher expression in the periphery included bestrophin 1 (BEST1), transcription factor RNA binding motif protein 39 (RBM39), inflammatory mediator macrophage migration inhibitory factor, antioxidant SOD3, ion channel voltage-dependent anion-selective channel protein 3 (VDAC3), and complement inhibitor CD55. The complement activation was among the highest represented pathways (P < 7.5e-13).

    CONCLUSIONS AND RELEVANCE:

    This proteomic data set identifies novel molecular signatures in anatomically sensitive regions of the choroid-RPE complex. The findings give mechanistic insight into choroid-RPE function, reveal important choroid-RPE processes, and prioritize new pathways for therapeutic targeting.
  1. Meckes, David G., Gunawardena, Harsha P., Dekroon, Robert M., Heaton, Phillip R., Edwards, Rachel H., Ozgur, Sezgin, Griffith, Jack D., Damania, Blossom Raab-Traub, Nancy
    Modulation of B-cell exosome proteins by gamma herpesvirus infection
    Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 31 (2013)
    PubMed | PMC | Full Text HTML, PDF
    Abstract
    The human gamma herpesviruses, Kaposi sarcoma-associated virus (KSHV) and EBV, are associated with multiple cancers. Recent evidence suggests that EBV and possibly other viruses can manipulate the tumor microenvironment through the secretion of specific viral and cellular components into exosomes, small endocytically derived vesicles that are released from cells. Exosomes produced by EBV-infected nasopharyngeal carcinoma cells contain high levels of the viral oncogene latent membrane protein 1 and viral microRNAs that activate critical signaling pathways in recipient cells. In this study, to determine the effects of EBV and KSHV on exosome content, quantitative proteomics techniques were performed on exosomes purified from 11 B-cell lines that are uninfected, infected with EBV or with KSHV, or infected with both viruses. Using mass spectrometry, 871 proteins were identified, of which ∼360 were unique to the viral exosomes. Analysis by 2D difference gel electrophoresis and spectral counting identified multiple significant changes compared with the uninfected control cells and between viral groups. These data predict that both EBV and KSHV exosomes likely modulate cell death and survival, ribosome function, protein synthesis, and mammalian target of rapamycin signaling. Distinct viral-specific effects on exosomes suggest that KSHV exosomes would affect cellular metabolism, whereas EBV exosomes would activate cellular signaling mediated through integrins, actin, IFN, and NFκB. The changes in exosome content identified in this study suggest ways that these oncogenic viruses modulate the tumor microenvironment and may provide diagnostic markers specific for EBV and KSHV associated malignancies.
  1. Skeie, Jessica M. Mahajan, Vinit B.
    Proteomic interactions in the mouse vitreous-retina complex
    PloS one, vol. 8, no. 11 (2013)
    PubMed | PMC | Full Text HTML, PDF
    Abstract
    PURPOSE:

    Human vitreoretinal diseases are due to presumed abnormal mechanical interactions between the vitreous and retina, and translational models are limited. This study determined whether nonstructural proteins and potential retinal biomarkers were expressed by the normal mouse vitreous and retina.

    METHODS:

    Vitreous and retina samples from mice were collected by evisceration and analyzed by liquid chromatography-tandem mass spectrometry. Identified proteins were further analyzed for differential expression and functional interactions using bioinformatic software.

    RESULTS:

    We identified 1,680 unique proteins in the retina and 675 unique proteins in the vitreous. Unbiased clustering identified protein pathways that distinguish retina from vitreous including oxidative phosphorylation and neurofilament cytoskeletal remodeling, whereas the vitreous expressed oxidative stress and innate immunology pathways. Some intracellular protein pathways were found in both retina and vitreous, such as glycolysis and gluconeogenesis and neuronal signaling, suggesting proteins might be shuttled between the retina and vitreous. We also identified human disease biomarkers represented in the mouse vitreous and retina, including carbonic anhydrase-2 and 3, crystallins, macrophage inhibitory factor, glutathione peroxidase, peroxiredoxins, S100 precursors, and von Willebrand factor.

    CONCLUSIONS:

    Our analysis suggests the vitreous expresses nonstructural proteins that functionally interact with the retina to manage oxidative stress, immune reactions, and intracellular proteins may be exchanged between the retina and vitreous. This novel proteomic dataset can be used for investigating human vitreoretinopathies in mouse models. Validation of vitreoretinal biomarkers for human ocular diseases will provide a critical tool for diagnostics and an avenue for therapeutics.
  1. Tamboli, Irfan Y., Heo, Dongeun Rebeck, G. William
    Extracellular proteolysis of apolipoprotein E (apoE) by secreted serine neuronal protease
    PloS one, vol. 9, no. 3 (2014)
    PubMed | PMC | Full Text HTML, PDF
    Abstract
    Under normal conditions, brain apolipoprotein E (apoE) is secreted and lipidated by astrocytes, then taken up by neurons via receptor mediated endocytosis. Free apoE is either degraded in intraneuronal lysosomal compartments or released. Here we identified a novel way by which apoE undergoes proteolysis in the extracellular space via a secreted neuronal protease. We show that apoE is cleaved in neuronal conditioned media by a secreted serine protease. This apoE cleavage was inhibited by PMSF and О±1-antichymotrypsin, but not neuroserpin-1 or inhibitors of thrombin and cathepsin G, supporting its identity as a chymotrypsin like protease. In addition, apoE incubation with purified chymotrypsin produced a similar pattern of apoE fragments. Analysis of apoE fragments by mass spectrometry showed cleavages occuring at the C-terminal side of apoE tryptophan residues, further supporting our identification of cleavage by chymotrypsin like protease. Hippocampal neurons were more efficient in mediating this apoE cleavage than cortical neurons. Proteolysis of apoE4 generated higher levels of low molecular weight fragments compared to apoE3. Primary glial cultures released an inhibitor of this proteolytic activity. Together, these studies reveal novel mechanism by which apoE can be regulated and therefore could be useful in designing apoE directed AD therapeutic approaches.
  1. Nazemof, Nazila, Couroux, Philippe, Rampitsch, Christof, Xing, Tim Robert, Laurian S.
    Proteomic profiling reveals insights into Triticeae stigma development and function
    Journal of Experimental Botany (2014)
    PubMed | Full Text HTML, PDF
    Abstract
    To our knowledge, this study represents the first high-throughput characterization of a stigma proteome in the Triticeae. A total of 2184 triticale mature stigma proteins were identified using three different gel-based approaches combined with mass spectrometry. The great majority of these proteins are described in a Triticeae stigma for the first time. These results revealed many proteins likely to play important roles in stigma development and pollen-stigma interactions, as well as protection against biotic and abiotic stresses. Quantitative comparison of the triticale stigma transcriptome and proteome showed poor correlation, highlighting the importance of having both types of analysis. This work makes a significant contribution towards the elucidation of the Triticeae stigma proteome and provides novel insights into its role in stigma development and function.
  1. Cocco, Mario, Stephenson, Sophie, Care, Matthew A., Newton, Darren, Barnes, Nicholas A., Davison, Adam, Rawstron, Andy, Westhead, David R., Doody, Gina M. Tooze, Reuben M.
    In vitro generation of long-lived human plasma cells
    Journal of immunology (Baltimore, Md. : 1950), vol. 189, no. 12, 5773-5785 (2012)
    PubMed | Full Text HTML, PDF
    Abstract
    Plasma cells (PCs), the terminal effectors of humoral immunity, are short-lived unless supported by niche environments in which they may persist for years. No model system has linked B cell activation with niche function to allow the in vitro generation of long-lived PCs. Thus, the full trajectory of B cell terminal differentiation has yet to be investigated in vitro. In this article, we describe a robust model for the generation of polyclonal long-lived human PCs from peripheral blood B cells. After a proliferative plasmablast phase, PCs persist in the absence of cell division, with viability limited only by elective culture termination. Conservative predictions for PC life expectancy are 300 d, but with the potential for significantly longer life spans for some cells. These long-lived PCs are preferentially derived from memory B cells, and acquire a CD138(high) phenotype analogous to that of human bone marrow PCs. Analysis of gene expression across the system defines clusters of genes with related dynamics and linked functional characteristics. Importantly, genes in these differentiation clusters demonstrate a similar overall pattern of expression for in vitro and ex vivo PCs. In vitro PCs are fully reprogrammed to a secretory state and are adapted to their secretory load, maintaining IgG secretion of 120 pg/cell/day in the absence of XBP1 mRNA splicing. By establishing a set of conditions sufficient to allow the development and persistence of mature human PCs in vitro, to our knowledge, we provide the first platform with which to sequentially explore and manipulate each stage of human PC differentiation.
  1. Li, Jie, Kil, Catherine, Considine, Kelly, Smarkucki, Bartosz, Stankewich, Michael C., Balgley,2 Brian Vortmeyer, Alexander O.
    Intrinsic indicators for specimen degradation
    Laboratory investigation; a journal of technical methods and pathology, vol. 93, no. 2, 242-253 (2013)
    PubMed | Full Text HTML, PDF
    Abstract
    Variable degrees of molecular degradation occur in human surgical specimens before clinical examination and severely affect analytical results. We therefore initiated an investigation to identify protein markers for tissue degradation assessment. We exposed 4 cell lines and 64 surgical/autopsy specimens to defined periods of time at room temperature before procurement (experimental cold ischemic time (CIT)-dependent tissue degradation model). Using two-dimensional fluorescence difference gel electrophoresis in conjunction with mass spectrometry, we performed comparative proteomic analyses on cells at different CIT exposures and identified proteins with CIT-dependent changes. The results were validated by testing clinical specimens with western blot analysis. We identified 26 proteins that underwent dynamic changes (characterized by continuous quantitative changes, isoelectric changes, and/or proteolytic cleavages) in our degradation model. These changes are strongly associated with the length of CIT. We demonstrate these proteins to represent universal tissue degradation indicators (TDIs) in clinical specimens. We also devised and implemented a unique degradation measure by calculating the quantitative ratio between TDIs’ intact forms and their respective degradation-modified products. For the first time, we have identified protein TDIs for quantitative measurement of specimen degradation. Implementing these indicators may yield a potentially transformative platform dedicated to quality control in clinical specimen analyses.
  1. Cao, Yanyan, Wang, Yichen, Abi Saab, Widian F., Yang, Fajun, Pessin, Jeffrey E. Backer, Jonathan M.
    NRBF2 regulates macroautophagy as a component of Vps34 Complex I
    The Biochemical journal, vol. 461, no. 2, 315-322 (2014)
    PubMed
    Abstract
    Macroautophagy is a physiological cellular response to nutrient stress, which leads to the engulfment of cytosolic contents by a double-walled membrane structure, the phagophore. Phagophores seal to become autophagosomes, which then fuse with lysosomes to deliver their contents for degradation. Macroautophagy is regulated by numerous cellular factors, including the Class III PI3K (phosphoinositide 3-kinase) Vps34 (vacuolar protein sorting 34). The autophagic functions of Vps34 require its recruitment to a complex that includes Vps15, Beclin-1 and Atg14L (autophagy-related 14-like protein) and is known as Vps34 Complex I. We have now identified NRBF2 (nuclear receptor-binding factor 2) as a new member of Vps34 Complex I. NRBF2 binds to complexes that include Vps34, Vps15, Beclin-1 and ATG-14L, but not the Vps34 Complex II component UVRAG (UV radiation resistance-associated gene). NRBF2 directly interacts with Vps15 via the Vps15 WD40 domain as well as other regions of Vps15. The formation of GFP-LC3 (light chain 3) punctae and PE (phosphatidylethanolamine)-conjugated LC3 (LC3-II) in serum-starved cells was inhibited by NRBF2 knockdown in the absence and presence of lysosomal inhibitors, and p62 levels were increased. Thus NRBF2 plays a critical role in the induction of starvation-induced autophagy as a specific member of Vps34 Complex I.
  1. Sohn, Elliott H., Khanna, Aditi, Tucker, Budd A., Abràmoff, Michael D., Stone, Edwin M. Mullins, Robert F.
    Structural and biochemical analyses of choroidal thickness in human donor eyes
    Investigative ophthalmology & visual science, vol. 55, no. 3, 1352-1360 (2014)
    PubMed | Full Text HTML, PDF
    Abstract
    PURPOSE:

    The choroid plays a vital role in the health of the outer retina. While measurements of choroid using optical coherence tomography show altered thickness in aging and macular disease, detailed histopathologic and proteomic analyses are lacking. In this study we sought to evaluate biochemical differences in human donor eyes between very thin and thick choroids.

    METHODS:

    One hundred forty-one eyes from 104 donors (mean age ± standard deviation, 81.5 ± 12.2) were studied. Macular sections were collected, and the distance between Bruch’s membrane and the inner surface of the sclera was measured in control, early/dry age-related macular degeneration (AMD), neovascular AMD, and geographic atrophy eyes. Proteins from the RPE-choroid of eyes with thick and thin choroids were analyzed using two-dimensional electrophoresis and/or mass spectrometry. Two proteins with altered abundance were confirmed using Western blot analysis.

    RESULTS:

    Donor eyes showed a normal distribution of thicknesses. Eyes with geographic atrophy had significantly thinner choroids than age-matched controls or early AMD eyes. Proteomic analysis showed higher levels of the serine protease SERPINA3 in thick choroids and increased levels of tissue inhibitor of metalloproteinases-3 (TIMP3) in thin choroids.

    CONCLUSIONS:

    Consistent with clinical imaging observations, geographic atrophy was associated with choroidal thinning. Biochemical data suggest an alteration in the balance between proteases and protease inhibitors in eyes that lie at the extremes of choroidal thickness. An improved understanding of the basic mechanisms associated with choroidal thinning may guide the development of new therapies for AMD.
  1. Foley, Timothy D., Cantarella, Kristen M., Gillespie, Paul F. Stredny, Edward S.
    Protein Vicinal Thiol Oxidations in the Healthy Brain: Not So Radical Links between Physiological Oxidative Stress and Neural Cell Activities
    Neurochemical research (2014)
    PubMed | DOI
    Abstract
    Reversible oxidations of protein thiols have emerged as alternatives to free radical-mediated oxidative damage with which to consider the impacts of oxidative stress on cellular activities but the scope and pathways of such oxidations in tissues, including the brain, have yet to be fully defined. We report here a characterization of reversible oxidations of glutathione and protein thiols in extracts from rat brains, from two sources, which had been (1) frozen quickly after euthanasia to preserve in vivo redox states and (2) subjected to alkylation upon tissue disruption to trap reduced thiols. Brains were defined, relatively, as Reduced and Moderately Oxidized based on measured ratios of reduced (GSH) to oxidized (GSSG) glutathione. Levels of protein disulfides formed by the cross-linking of closely-spaced (vicinal) protein thiols, but not protein S-glutathionylation, were higher in extracts from the Moderately Oxidized brains compared to the Reduced brains. Moreover, the oxidized vicinal thiol proteome contains proteins that impact cellular energetics, signaling, neurotransmission, and cytoskeletal dynamics among others. These findings argue that kinetically-competent pathways for reversible, two-electron oxidations, of protein vicinal thiols can be activated in healthy brains in response to physiological oxidative stresses. We propose that such oxidations may link oxidative stress to adaptive, but also potentially deleterious, changes in neural cell activities in otherwise healthy brains.
  1. Nakaya, Naoki, Sultana, Afia, Munasinghe, Jeeva, Cheng, Aiwu, Mattson, Mark P. Tomarev, Stanislav I.
    Deletion in the N-terminal half of olfactomedin 1 modifies its interaction with synaptic proteins and causes brain dystrophy and abnormal behavior in mice
    Experimental neurology, vol. 250, 205-218 (2013)
    PubMed
    Abstract
    Olfactomedin 1 (Olfm1) is a secreted glycoprotein that is preferentially expressed in neuronal tissues. Here we show that deletion of exons 4 and 5 from the Olfm1 gene, which encodes a 52 amino acid long region in the N-terminal part of the protein, increased neonatal death and reduced body weight of surviving homozygous mice. Magnetic resonance imaging analyses revealed reduced brain volume and attenuated size of white matter tracts such as the anterior commissure, corpus callosum, and optic nerve. Adult Olfm1 mutant mice demonstrated abnormal behavior in several tests including reduced marble digging, elevated plus maze test, nesting activity and latency on balance beam tests as compared with their wild-type littermates. The olfactory system was both structurally and functionally disturbed by the mutation in the Olfm1 gene as shown by functional magnetic resonance imaging analysis and a smell test. Deficiencies of the olfactory system may contribute to the neonatal death and loss of body weight of Olfm1 mutant. Shotgun proteomics revealed 59 candidate proteins that co-precipitated with wild-type or mutant Olfm1 proteins in postnatal day 1 brain. Olfm1-binding targets included GluR2, Cav2.1, teneurin-4 and Kidins220. Modified interaction of Olfm1 with binding targets led to an increase in intracellular Ca(2+) concentration and activation of ERK1/2, MEK1 and CaMKII in the hippocampus and olfactory bulb of Olfm1 mutant mice compared with their wild-type littermates. Excessive activation of the CaMKII and Ras-ERK pathways in the Olfm1 mutant olfactory bulb and hippocampus by elevated intracellular calcium may contribute to the abnormal behavior and olfactory activity of Olfm1 mutant mice.
  1. Onaivi, E. S., Schanz, N. Lin, Z. C.
    Psychiatric disturbances regulate the innate immune system in CSF of conscious mice
    Translational psychiatry, vol. 4 (2014)
    PubMed | PMC | Full Text HTML, PDF
    Abstract
    Environment may affect brain activity through cerebrospinal fluid (CSF) only if there are regulatory molecules or cascades in CSF that are sensitive to external stimuli. This study was designed to identify regulatory activity present in CSF, better elucidating environmental regulation of brain function. By using cannulation-based sequential CSF sampling coupled with mass spectrometry-based identification and quantification of proteins, we show that the naive mouse CSF harbors, among 22 other pathways, the innate immune system as a main pathway, which was downregulated and upregulated, respectively, by acute stressor (AS) and acute cocaine (AC) administrations. Among novel processes and molecular functions, AS also regulated schizophrenia-associated proteins. Furthermore, AC upregulated exosome-related proteins with a false discovery rate of 1.0 × 10(-)(16). These results suggest that psychiatric disturbances regulate the neuroimmune system and brain disorder-related proteins, presenting a sensitive approach to investigating extracellular mechanisms in conscious and various mouse models of psychiatric
  1. Nayak, Jaladhi, Gastonguay, Adam J., Talipov, Marat R., Vakeel, Padmanabhan, Span, Elise A., Kalous, Kelsey S., Kutty, Raman G., Jensen, Davin R., Pokkuluri, Phani, Sem, Daniel S., Rathore, Rajendra Ramchandran, Ramani
    Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins
    BMC biochemistry, vol. 15, no. 1 (2014)
    PubMed
    Abstract
    The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

Selected publications by our scientists


  1. Wu LQ, Embree HD, Balgley BM, Smith PJ, Payne GF.
    Utilizing renewable resources to create functional polymers: chitosan-based associative thickener
    Environmental science & technology. 2002 Aug;36(15):3446-3454.
    DOI
    Abstract
    There is a growing interest in utilizing renewable resources and exploiting biological reactions for environmentally friendly products and processes. We report the use of the enzyme tyrosinase to graft the natural phenol, catechin, onto the biopolymer chitosan. Chemical evidence for grafting was obtained by UV/visible spectrophotometry and electrospray mass spectrometry. Rheological measurements demonstrate that the catechin-modified chitosan behaves as an associative thickener. Specifically, the viscosity increases dramatically with concentration of this modified chitosan. Furthermore, when the catechin-modified chitosan is dissolved at low concentrations (0.6% w/w), steady shear measurements show shear thinning behavior, while oscillatory measurements show weak gel behavior. These results demonstrate the potential for utilizing renewable resources and biochemical processing to functionalize biopolymers to offer technically useful properties. To suggest the relative environmental impacts of chitosan derivatives with existing water-soluble polymers, we used the framework of a life cycle assessment.
  1. Chen J, Balgley BM, DeVoe DL, Lee CS.
    Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis.
    Analytical chemistry. 2003 Jul;75(13):3145-3152
    DOI
    Abstract
    An integrated proteome concentration/separation approach involving on-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures. Upon completion of analyte focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of ∼240. In addition to analyte focusing, CIEF as the first separation dimension resolves proteins/peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identification of low-abundance proteins. The CIEF-based multidimensional separation/concentration platform enables the identification of a greater number of yeast soluble proteins than methods presented in the literature, yet requires a protein loading of only 9.6 μg. This protein loading is 2−3 orders of magnitude lower than those employed by the reported non-gel-based proteome techniques. The distribution of a codon adaptation index value for identified yeast proteins approximates to that predicted for the entire yeast proteome and supports the capability of CIEF-based proteome separation technology for achieving comprehensive proteome analysis. By reducing the inner diameter of chromatography columns from 180 μm to 100 μm, the required protein loading is further decreased from 9.6 μg to 960 ng, illustrating the potential usage of this proteome technology for the analysis of protein profiles within small cell populations or limited tissue samples
  1. Wang Y, Balgley BM, Rudnick PA, Evans EL, DeVoe DL, Lee CS.
    Integrated capillary isoelectric focusing/nano-reversed phase liquid chromatography coupled with ESI-MS for characterization of intact yeast proteins.
    Journal of proteome research. 2005;4(1):36-42
    DOI
    Abstract
    By obtaining a peak capacity of 1440 from combined capillary isoelectric focusing/nano-reversed phase liquid chromatography and acquiring 3-5 electrospray ionization protein envelopes over the elution of any chromatography peak using Q-TOF-MS, an overall system capacity of 4320-7200 is achieved for the analysis of intact proteins obtained from the soluble fraction of S. cerevisiae. A total of 534 distinct protein masses are identified over a wide mass range of 5-70 kDa, requiring a protein loading of less than 10 μg.
  1. Wang Y, Balgley BM, Rudnick PA, Lee CS.
    Effects of chromatography conditions on intact protein separations for top-down proteomics.
    Journal of chromatography A. 2005 May;1073(1-2):35-41
    DOI
    Abstract
    For top-down proteomics, nano-reversed phase liquid chromatography (RPLC) plays a major role in both single and multidimensional protein separations in an effort to increase the overall peak capacity for the resolution of complex protein mixtures prior to mass spectrometry analysis. Effects of various chromatography conditions, including alkyl chain length in the stationary phase, capillary column temperature, and ion-pairing agent, on the resolution of intact proteins are studied using nano-RPLC-electrospray ionization-mass spectrometry. Optimal chromatography conditions include the use of C18 column heated at 60 °C and the addition of trifluoroacetic acid instead of heptafluorobutyric acid as the ion-paring agent in the mobile phase. Under optimized chromatography conditions, there are no significant differences in the separation performance of yeast cell lysates present in the native versus denatured states. Denatured yeast proteins resolved and eluted from nano-RPLC can be subjected to proteolytic digestion in an on- or off-line approach to provide improved protein sequence coverage toward protein identification in a combined top-down/bottom-up proteome platform.
  1. Rudnick PA, Wang Y, Evans E, Lee CS, Balgley BM.
    Large scale analysis of MASCOT results using a Mass Accuracy-based THreshold (MATH) effectively improves data interpretation.
    Journal of proteome research. 2005;4(4):1353-1360
    DOI
    Abstract
    In this work, we describe the effects of mass tolerance settings and database size on the performance of MASCOT and interpretations based on the Identity Threshold. Database size and mass tolerance settings, in large excess over the actual mass error, negatively affect the performance of the algorithm and interpretive power of MASCOT. False negative identifications can be significantly reduced by calculating a mass accuracy-based threshold by searching a reversed database.
  1. Wang YX, Zhou Y, Balgley BM, Cooper JW, Lee CS, DeVoe DL.
    Electrospray interfacing of polymer microfluidics to MALDI-MS.
    Electrophoresis. 2005 Oct;26(19):3631-3640.
    DOI
    Abstract
    The off-line coupling of polymer microfluidics to MALDI-MS is presented using electrospray deposition. Using polycarbonate microfluidic chips with integrated hydrophobic membrane electrospray tips, peptides and proteins are deposited onto a stainless steel target followed by MALDI-MS analysis. Microchip electrospray deposition is found to yield excellent spatial control and homogeneity of deposited peptide spots, and significantly improved MALDI-MS spectral reproducibility compared to traditional target preparation methods. A detection limit of 3.5 fmol is demonstrated for angiotensin. Furthermore, multiple electrospray tips on a single chip provide the ability to simultaneously elute parallel sample streams onto a MALDI target for high-throughput multiplexed analysis. Using a three-element electrospray tip array with 150 µm spacing, the simultaneous deposition of bradykinin, fibrinopeptide, and angiotensin is achieved with no cross talk between deposited samples. In addition, in-line proteolytic digestion of intact proteins is successfully achieved during the electrospray process by binding trypsin within the electrospray membrane, eliminating the need for on-probe digestion prior to MALDI-MS. The technology offers promise for a range of microfluidic platforms designed for high-throughput multiplexed proteomic analyses in which simultaneous on-chip separations require an effective interface to MS.
  1. Wang Y, Balgley BM, Lee CS.
    Tissue proteomics using capillary isoelectric focusing-based multidimensional separations.
    Expert review of proteomics. 2005 Oct;2(5):659-667.
    DOI
    Abstract
    The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.
  1. Wang Y, Rudnick PA, Evans EL, Li J, Zhuang Z, Devoe DL, et al.
    Proteome analysis of microdissected tumor tissue using a capillary isoelectric focusing-based multidimensional separation platform coupled with ESI-tandem MS.
    Analytical chemistry. 2005 Oct;77(20):6549-6556.
    DOI
    Abstract
    This study demonstrates the ability to perform sensitive proteome analysis on the limited protein quantities available through tissue microdissection. Capillary isoelectric focusing combined with nano-reversed-phase liquid chromatography in an automated and integrated platform not only provides systematic resolution of complex peptide mixtures based on their differences in isoelectric point and hydrophobicity but also eliminates peptide loss and analyte dilution. In comparison with strong cation exchange chromatography, the significant advantages of electrokinetic focusing-based separations include high resolving power, high concentration and narrow analyte bands, and effective usage of electrospray ionization-tandem MS toward peptide identifications. Through the use of capillary isoelectric focusing-based multidimensional peptide separations, a total of 6866 fully tryptic peptides were detected, leading to the identification of 1820 distinct proteins. Each distinct protein was identified by at least one distinct peptide sequence. These high mass accuracy and high-confidence identifications were generated from three proteome runs of a single glioblastoma multiforme tissue sample, each run consuming only 10 μg of total protein, an amount corresponding to 20 000 selectively isolated cells. Instead of performing multiple runs of multidimensional separations, the overall peak capacity can be greatly enhanced for mining deeper into tissue proteomics by increasing the number of CIEF fractions without an accompanying increase in sample consumption.
  1. Guo T, Rudnick PA, Wang W, Lee CS, Devoe DL, Balgley BM.
    Characterization of the human salivary proteome by capillary isoelectric focusing/nanoreversed-phase liquid chromatography coupled with ESI-tandem MS.
    Journal of proteome research. 2006 Jun;5(6):1469-1478.
    DOI
    Abstract
    Saliva is a readily available body fluid with great diagnostic potential. The foundation for saliva-based diagnostics, however, is the development of a complete catalog of secreted and “leaked” proteins detectable in saliva. By employing a capillary isoelectric focusing-based multidimensional separation platform coupled with electrospray ionization tandem mass spectrometry, a total of 5338 distinct peptides were sequenced, leading to the identification of 1381 distinct proteins.
  1. Shi SR, Liu C, Balgley BM, Lee C, Taylor CR.
    Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.
    J Histochem Cytochem. 2006 Jun;54(6):739-743.
    DOI
    Abstract
    A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.
  1. An Y, Cooper JW, Balgley BM, Lee CS.
    Selective enrichment and ultrasensitive identification of trace peptides in proteome analysis using transient capillary isotachophoresis/zone electrophoresis coupled with nano-ESI-MS.
    Electrophoresis. 2006 Sep;27(18):3599-3608.
    DOI
    Abstract
    Besides the complexity in protein samples of biological origin, probably the greatest challenge presently facing comprehensive proteome analysis is related to the large variation of protein relative abundances (>6 orders of magnitude), having potential biological significance in mammalian systems. As demonstrated in this work, transient capillary ITP/zone electrophoresis (CITP/CZE) provides selective analyte enrichment through electrokinetic stacking and extremely high resolving power toward protein and peptide mixtures. The result of the CITP process is that major components may be diluted, but trace compounds are concentrated. The on-column transition of CITP to CZE minimizes additional band broadening while providing superior analyte resolution. Online coupling of transient CITP/CZE with nano-ESI-MS allows ultrasensitive detection of trace peptides at levels of subnanomolar concentration or subfemtomole mass in complex peptide mixtures. More importantly, selective enrichment of trace peptides enables the identification and sequence analysis of low-abundance peptides co-migrated with highly abundant species at a concentration ratio of 1:500 000. The combined CITP/CZE-nano-ESI-MS system is demonstrated to be at least one to two orders of magnitude more sensitive than that attained in conventional electrophoretic and chromatographic-based proteome technologies over a wide dynamic concentration range, potentially allowing comprehensive analysis of protein profiles within a small cell population and limited tissue samples using conventional mass spectrometers. Furthermore, the speed of CITP/CZE separation and the lack of column equilibration in CITP/CZE not only improve the throughput of proteome analysis, but also facilitate its seamless integration with other separation technologies in a multidimensional protein identification platform.
  1. Guo T, Lee CS, Wang W, DeVoe DL, Balgley BM.
    Capillary separations enabling tissue proteomics-based biomarker discovery.
    Electrophoresis. 2006 Sep;27(18):3523-3532.
    DOI
    Abstract
    Development of the capability to enable large-scale proteome studies, analogous to comprehensive gene expression analysis, will clearly have far-reaching impacts on protein biomarker investigations of human diseases such as cancer through interrogation of the archived fresh frozen and formalin-fixed and paraffin-embedded tissue collections. This review therefore focuses on the most recent advances in microdissection techniques and proteome platforms for procuring homogeneous subpopulations of tumor cells or structures and performing comprehensive analysis of protein profiles within tissue specimens, respectively. Developments in capillary separations capable of providing extremely high resolving power and selective analyte enrichment are particularly highlighted for their roles within the broader context of a state-of-the-art integrated tissue proteome effort. The capabilities of CIEF-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. These proteome technological advances combined with recently developed tissue microdissection techniques provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.
  1. Wang W, Guo T, Rudnick PA, Song T, Li J, Zhuang Z, et al.
    Membrane proteome analysis of microdissected ovarian tumor tissues using capillary isoelectric focusing/reversed-phase liquid chromatography-tandem MS.
    Analytical chemistry. 2007 Feb;79(3):1002-1009.
    DOI
    Abstract
    This work expands our tissue proteome capabilities from the analysis of soluble proteins in previous studies to the examination of membrane proteins within the pellets of enriched and selectively isolated tumor cells procured from microdissected tissue specimens. The pellets of targeted ovarian tumor cells are treated by two different membrane protein extraction methods, including the use of detergent and organic solvent. The detergent-based membrane protein preparation protocol not only extracts proteins effectively from cell pellets but also is compatible with subsequent proteome analysis using combined capillary isoelctric focusing/nano reversed-phase liquid chromatography separations coupled with nano electrospray ionization mass spectrometry. Among proteins identified from an amount of pellet equivalent to 20 000 cells, 773 proteins are predicted to contain one or more transmembrane domains, corresponding to 22% membrane proteome coverage within the SwissProt Human protein sequence entries.
  1. Wang W, Guo T, Song T, Lee CS, Balgley BM.
    Comprehensive yeast proteome analysis using a capillary isoelectric focusing-based multidimensional separation platform coupled with ESI-MS/MS.
    PROTEOMICS. 2007;7(8):1178-1187.
    DOI
    Abstract
    As demonstrated in this study, a CIEF-based multidimensional separation platform not only is compatible with the detergent-based membrane protein preparation protocol, but also achieves both the largest yeast membrane proteome coverage and the most comprehensive analysis of the yeast proteome to date. By using a 1% false discovery rate for total peptide identifications, a total of 2513 distinct yeast proteins are identified from the SDS-solubilized fraction with an average of 5.4 peptides leading to each protein identification. Among proteins identified from the SDS-solubilized fraction, 407 proteins are predicted to contain at least two or more transmembrane domains using TMHMM (www.cbs.dtu.dk/services/TMHMM-2.0/), corresponding to 46% yeast membrane proteome coverage. Only four additional membrane proteins are identified in the soluble and urea-solubilized fractions, affirming the utility of SDS extraction for enriching the membrane proteome. By combining proteome results obtained from the soluble, urea-solubilized, and SDS-solubilized fractions, a single yeast proteome analysis yields the identification of 3632 distinct yeast proteins, corresponding to 55% theoretical yeast proteome coverage or 70% of proteins predicted to be expressed during log-phase growth in rich media.
  1. Martin DN, Balgley B, Dutta S, Chen J, Rudnick P, Cranford J, et al.
    Proteomic analysis of steroid-triggered autophagic programmed cell death during Drosophila development.
    Cell Death and Differentiation;aop(current).
    DOI
    Abstract
    Two morphological forms of programmed cell death, apoptosis and autophagic cell death, remove unneeded or damaged cells during animal development. Although the mechanisms that regulate apoptosis are well studied, little is known about autophagic cell death. A shotgun proteome analysis of purified dying larval salivary glands in Drosophila was used to identify proteins that are expressed during autophagic programmed cell death. A total of 5661 proteins were identified from stages before and after the onset of cell death. Analyses of these data enabled us to identify proteins from a number of interesting categories including regulators of transcription, the apoptosis, autophagy, lysosomal, and ubiquitin proteasome degradation pathways, and proteins involved in growth control. Several of the identified proteins, including the serine/threonine kinase warts (Wts), were not detected using whole-genome DNA microarrays, providing support for the importance of such high-throughput proteomic technology. Wts regulates cell-cycle arrest and apoptosis, and significantly, mutations in wts prevent destruction of salivary glands.
  1. Guo T, Wang W, Rudnick PA, Song T, Li J, Zhuang Z, et al.
    Proteome analysis of microdissected formalin-fixed and Paraffin-embedded tissue specimens.
    J Histochem Cytochem. 2007 Jul;55(7):763-772.
    DOI
    Abstract
    Targeted proteomics research, based on the enrichment of disease-relevant proteins from isolated cell populations selected from high-quality tissue specimens, offers great potential for the identification of diagnostic, prognostic, and predictive biological markers for use in the clinical setting and during preclinical testing and clinical trials, as well as for the discovery and validation of new protein drug targets. Formalin-fixed and paraffin-embedded (FFPE) tissue collections, with attached clinical and outcome information, are invaluable resources for conducting retrospective protein biomarker investigations and performing translational studies of cancer and other diseases. Combined capillary isoelectric focusing/nano-reversed-phase liquid chromatography separations equipped with nano-electrospray ionization-tandem mass spectrometry are employed for the studies of proteins extracted from microdissected FFPE glioblastoma tissues using a heat-induced antigen retrieval (AR) technique. A total of 14,478 distinct peptides are identified, leading to the identification of 2733 non-redundant SwissProt protein entries. Eighty-three percent of identified FFPE tissue proteins overlap with those obtained from the pellet fraction of fresh-frozen tissue of the same patient. This large degree of protein overlapping is attributed to the application of detergent-based protein extraction in both the cell pellet preparation protocol and the AR technique.
  1. Fang X, Yang L, Wang W, Song T, Lee CS, DeVoe DL, et al.
    Comparison of electrokinetics-based multidimensional separations coupled with electrospray ionization-tandem mass spectrometry for characterization of human salivary proteins.
    Analytical chemistry. 2007 Aug;79(15):5785-5792.
    DOI
    Abstract
    The foundation for saliva-based diagnostics is the development of a complete catalog of secreted proteins detectable in saliva. Besides protein complexity, the greatest bioanalytical challenge facing comprehensive analysis of saliva samples is related to the large variation of protein relative abundances including the presence of high-abundance proteins such as amylases, mucins, proline-rich proteins (PRPs), and secretory IgA complex. Among a number of electrokinetic separation techniques, transient capillary isotachophoresis/capillary zone electrophoresis (CITP/CZE) specifically targets trace amounts of proteins and thus reduces the range of relative protein abundances for providing unparallel advantages toward the identification of low-abundance proteins. By employing a CITP/CZE-based multidimensional separation platform coupled with electrospray ionization-tandem mass spectrometry (ESI-tandem MS), a total of 6112 fully tryptic peptides are sequenced at a 1% false discovery rate (FDR), leading to the identification of 1479 distinct human SwissProt protein entries. By comparing with capillary isoelectric focusing (CIEF) as another electrokinetics-based stacking approach, CITP/CZE not only offers a broad field of application but also is less prone to protein/peptide precipitation during the analysis. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. Furthermore, when evaluating the protein sequence coverage by the number of distinct peptides mapping to each protein identification, the CITP-based proteome technology similarly achieves the superior performance with 674 proteins (46%) having three or more distinct peptides, 288 (19%) having two distinct peptides, and 517 (35%) having a single distinct peptide.
  1. Balgley BM, Laudeman T, Yang L, Song T, Lee CS.
    Comparative Evaluation of Tandem MS Search Algorithms Using a Target-Decoy Search Strategy.
    Mol Cell Proteomics. 2007 Sep;6(9):1599-1608.
    DOI
    Abstract
    Peptide identification of tandem mass spectra by a variety of available search algorithms forms the foundation for much of modern day mass spectrometry-based proteomics. Despite the critical importance of proper evaluation and interpretation of the results generated by these algorithms there is still little consistency in their application or understanding of their similarities and differences. A survey was conducted of four tandem mass spectrometry peptide identification search algorithms, including Mascot, Open Mass Spectrometry Search Algorithm, Sequest, and X! Tandem. The same input data, search parameters, and sequence library were used for the searches. Comparisons were based on commonly used scoring methodologies for each algorithm and on the results of a target-decoy approach to sequence library searching. The results indicated that there is little difference in the output of the algorithms so long as consistent scoring procedures are applied. The results showed that some commonly used scoring procedures may lead to excessive false discovery rates. Finally an alternative method for the determination of an optimal cutoff threshold is proposed.
  1. Li J, Yin C, Okamoto H, Mushlin H, Balgley BM, Lee CS, et al.
    Identification of a novel proliferation-related protein, WHSC1 4a, in human gliomas.
    Neuro-oncology. 2008 Feb;10(1):45-51.
    DOI
    Abstract
    Dynamic changes in the expression of multiple genes appear to be common features that distinguish transformed cells from their normal counterparts. We compared the proteomic profiles of four glioblastoma multiforme (GBM) tissue samples and four normal brain cortex samples to examine the molecular basis of gliomagenesis. Trypsin-digested protein samples were separated by capillary isoelectric focusing with nano-reversed-phase liquid chromatography and were profiled by mass spectrometric sequencing. Wolf-Hirschhorn syndrome candidate 1 (WHSC1), along with 103 other proteins, was found only in the GBM proteomes. Western blot and immunohistochemistry verified our proteomic findings and demonstrated that 30-kDa WHSC1 expression increases with ascending tumor proliferation activity. RNA interference could suppress glioma cell growth by blocking WHSC1 expression. Our novel findings encourage the application of proteomic techniques in cancer research.
  1. Mathivanan S, Ahmed M, Ahn NG, Alexandre H, Amanchy R, Andrews PC, et al.
    Human Proteinpedia enables sharing of human protein data.
    Nature Biotechnology;26(2):164-167.
    DOI
    Abstract
    Proteomic technologies, such as yeast two-hybrid, mass spectrometry (MS), protein/peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, it is not fruitful for the biomedical community to contribute these types of data to centralized repositories.
  1. Xu H, Yang L, Wang W, Shi SR, Liu C, Liu Y, et al.
    Antigen retrieval for proteomic characterization of formalin-fixed and paraffin-embedded tissues.
    Journal of proteome research. 2008 Mar;7(3):1098-1108.
    DOI
    Abstract
    Formalin-fixed and paraffin-embedded tissues represent the vast majority of archived tissue. Access to such tissue specimens via shotgun-based proteomic analyses may open new avenues for both prospective and retrospective translational research. We evaluate the effects of fixation time on antigen retrieval for the purposes of shotgun proteomics. For the first time, we demonstrate the capability of a capillary isotachophoresis-based platform for the shotgun proteomic analysis of proteins recovered from FFPE tissues.
  1. Fang X, Wang W, Yang L, Chandrasekaran K, Kristian T, Balgley BM, et al.
    Application of capillary isotachophoresis-based multidimensional separations coupled with electrospray ionization-tandem mass spectrometry for characterization of mouse brain mitochondrial proteome.
    Electrophoresis. 2008 May;29(10):2215-2223.
    DOI
    Abstract
    By employing a capillary ITP (CITP)/CZE-based proteomic technology, a total of 1795 distinct mouse Swiss-Prot protein entries (or 1705 nonredundant proteins) are identified from synaptic mitochondria isolated from mouse brain. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. The degree of peptide overlapping among CITP fractions is even lower than that achieved using combined CIEF/nano-RP LC separations for the analysis of the same mitochondrial sample. When evaluating the protein sequence coverage by the number of distinct peptides mapping to each mitochondrial protein identification, CITP/CZE similarly achieves superior performance with 1041 proteins (58%) having 3 or more distinct peptides, 233 (13%) having 2 distinct peptides, and 521 (29%) having a single distinct peptide. The reproducibility of protein identifications is found to be around 86% by comparing proteins identified from repeated runs of the same mitochondrial sample. The analysis of the mouse mitochondrial proteome by two CITP/CZE runs results in the detection of 2095 distinct mouse Swiss-Prot protein entries (or 1992 nonredundant proteins), corresponding to 59% coverage of the updated Maestro mitochondrial reference set. The collective analysis from combined CITP/CZE and CIEF-based proteomic studies yields the identification of 2191 distinct mitochondrial protein entries (or 2082 nonredundant proteins), corresponding to 76% coverage of the MitoP2-database reference set.
  1. Balgley BM, Wang W, Song T, Fang X, Yang L, Lee CS.
    Evaluation of confidence and reproducibility in quantitative proteomics performed by a capillary isoelectric focusing-based proteomic platform coupled with a spectral counting approach.
    ELECTROPHORESIS. 2008;29(14):3047-3054.
    DOI
    Abstract
    Multidimensional separations of the peptides resulting from enzymatic digestions of complex protein mixtures prior to MS/MS, namely shotgun proteomics, is increasingly utilized for large-scale identification and quantitation of proteins. Inherent to the performance of proteomic measurements is the resolving power of each of the separations both separately and in combination. By simply raising the number of CIEF fractions, the resulting enhancement in the overall peak capacity of combined CIEF/nano-RPLC separations greatly reduces the complexity of eluted peptides prior to MS detection and sequencing and increases the proteome coverage. The capabilities of the CIEF-based proteome platform coupled with the spectral counting approach to confidently and reproducibly quantify proteins and changes in protein expression levels among samples are evaluated. Analytical reproducibility of relative protein abundance is determined to exhibit a Pearson R2 value greater than 0.99 and a CV of 14.1%. The platform is demonstrated to be capable of measuring changes in protein expression as low as 1.5-fold, with confidence following multiple testing adjustment.
  1. Eisenhofer G, Huynh TT, Elkahloun A, Morris JC, Bratslavsky G, Linehan WM, et al.
    Differential expression of the regulated catecholamine secretory pathway in different hereditary forms of pheochromocytoma.
    American journal of physiology Endocrinology and metabolism. 2008 Nov;295(5).
    DOI
    Abstract
    Pheochromocytomas in patients with von Hippel-Lindau (VHL) syndrome and multiple endocrine neoplasia type 2 (MEN 2) differ in the types and amounts of catecholamines produced and the resulting signs and symptoms. We hypothesized the presence of different processes of catecholamine release reflecting differential expression of components of the regulated secretory pathway among the two types of hereditary tumors. Differences in catecholamine secretion from tumors in patients with VHL syndrome (n = 47) and MEN 2 (n = 32) were examined using measurements of catecholamines in tumor tissue, urine, and plasma, the last of which was under baseline conditions in all subjects and in a subgroup of patients who received intravenous glucagon to provoke catecholamine release. Microarray and proteomics analyses, quantitative PCR, and Western blotting were used to assess expression of tumor tissue secretory pathway components. The rate constant for baseline catecholamine secretion was 20-fold higher in VHL than in MEN 2 tumors (0.359 ± 0.094 vs. 0.018 ± 0.009 day−1), but catecholamine release was responsive only to glucagon in MEN 2 tumors. Compared with tumors from MEN 2 patients, those from VHL patients were characterized by reduced expression of numerous components of the regulated secretory pathway (e.g., SNAP25, syntaxin, rabphilin 3A, annexin A7, calcium-dependent secretion activator). The mutation-dependent differences in expression of secretory pathway components indicate a more mature regulated secretory pathway in MEN 2 than VHL tumors. These data provide a unique mechanistic link to explain how variations in the molecular machinery governing exocytosis may contribute to clinical differences in the secretion of neurotransmitters or hormones and the subsequent presentation of a disease.
  1. Yan W, Apweiler R, Balgley BM, Boontheung P, Bundy JL, Cargile BJ, et al.
    Systematic comparison of the human saliva and plasma proteomes.
    PROTEOMICS – CLINICAL APPLICATIONS. 2009 Jan;3(1):116-134.
    DOI
    Abstract
    The proteome of human salivary fluid has the potential to open new doors for disease biomarker discovery. A recent study to comprehensively identify and catalog the human ductal salivary proteome led to the compilation of 1166 proteins. The protein complexity of both saliva and plasma is large, suggesting that a comparison of these two proteomes will provide valuable insight into their physiological significance and an understanding of the unique and overlapping disease diagnostic potential that each fluid provides. To create a more comprehensive catalog of human salivary proteins, we have first compiled an extensive list of proteins from whole saliva (WS) identified through MS experiments. The WS list is thereafter combined with the proteins identified from the ductal parotid, and submandibular and sublingual (parotid/SMSL) salivas. In parallel, a core dataset of the human plasma proteome with 3020 protein identifications was recently released. A total of 1939 nonredundant salivary proteins were compiled from a total of 19 474 unique peptide sequences identified from whole and ductal salivas; 740 out of the total 1939 salivary proteins were identified in both whole and ductal saliva. A total of 597 of the salivary proteins have been observed in plasma. Gene ontology (GO) analysis showed similarities in the distributions of the saliva and plasma proteomes with regard to cellular localization, biological processes, and molecular function, but revealed differences which may be related to the different physiological functions of saliva and plasma. The comprehensive catalog of the salivary proteome and its comparison to the plasma proteome provides insights useful for future study, such as exploration of potential biomarkers for disease diagnostics.
  1. Lee CS, Balgley BM.
    Capillary isoelectric focusing/reversed phase liquid chromatography/mass spectrometry.
    Methods in molecular biology (Clifton, NJ). 2009;492:233-240.
    DOI
    Abstract
    The vast number of proteins present in the proteome of a typical organism requires that separations be performed on the mixture prior to introduction into a mass spectrometer for protein identification and quantification. An integrated protein separation platform, combining capillary isoelectric focusing (CIEF) with reversed phase liquid chromatography (RPLC), is described to provide high resolving power for the analysis of complex protein mixtures. Thus, the proteins are systematically resolved according to their differences in isoelectric point and hydrophobicity using combined CIEF/RPLC separations. A key feature of the CIEF-based multidimensional separation platform is the elimination of protein loss and dilution in an integrated platform while achieving comprehensive and ultrasensitive analysis of protein profiles within small cell populations or limited tissue samples.
  1. Balgley BM, Guo T, Zhao K, Fang X, Tavassoli FA, Lee CS.
    Evaluation of archival time on shotgun proteomics of formalin-fixed and paraffin-embedded tissues.
    Journal of proteome research. 2009 Feb;8(2):917-925.
    DOI
    Abstract
    Evaluation of effects of archival time on FFPE tissue represents one of the critical parameters which need to be addressed for robust application of archival specimens and to ensure confident high impact inferences can be made. Optimized protein extraction/digestion procedures for handling FFPE tissues are coupled with the capillary isotachophoresis-based proteome technology to investigate the effects of archival time on protein expression profiles within uterine mesenchymal tumor tissue blocks.
  1. Fang X, Balgley BM, Wang W, Park DM, Lee CS.
    Comparison of multidimensional shotgun technologies targeting tissue proteomics.
    Electrophoresis. 2009 Dec;30(23):4063-4070.
    DOI
    Abstract
    A compelling need exists for the development of technologies that facilitate and accelerate the discovery of novel protein biomarkers with therapeutic and diagnostic potential. Comparisons among shotgun proteome technologies, including capillary isotachophoresis (CITP)-based multidimensional separations and multidimensional LC system, are therefore performed in this study regarding their abilities to address the challenges of protein complexity and relative abundance inherent in glioblastoma multiforme-derived cancer stem cells. Comparisons are conducted using a single processed protein digest with equal sample loading, identical second-dimension separation (RPLC) and MS conditions, and consistent search parameters and cutoff established by the target-decoy determined false-discovery rate. Besides achieving superior overall proteome performance in total peptide, distinct peptide, and distinct protein identifications; analytical reproducibility of the CITP proteome platform coupled with the spectral counting approach are determined by a Pearson R2 value of 0.98 and a CV of 15% across all proteins quantified. In contrast, extensive fraction overlapping in strong cation exchange greatly limits the ability of multidimensional LC separations for mining deeper into the tissue proteome as evidenced by the poor coverage in various protein functional categories and key protein pathways. The CITP proteomic technology, equipped with selective analyte enrichment and ultrahigh resolving power, is expected to serve as a critical component in the overall toolset required for biomarker discovery via shotgun proteomic analysis of tissue specimens.
  1. Fang X, Balgley BM, Lee CS.
    Recent advances in capillary electrophoresis-based proteomic techniques for biomarker discovery.
    Electrophoresis. 2009 Dec;30(23):3998-4007.
    DOI
    Abstract
    A compelling need exists for the development of technologies that facilitate and accelerate the discovery of novel protein biomarkers with therapeutic and diagnostic potential. The inherent disadvantage of biomarker dilution in complex biological fluids such as serum/plasma, urine, and saliva necessitates highly sensitive analytical approaches, often exceeding the dynamic range of currently available proteomic platforms. Thus, investigative studies directed at tissues obtained from the primary site of pathology probably afford the best opportunity for the discovery of disease biomarkers. This review therefore focuses on the most recent advances in capillary electrophoresis-based single and multidimensional separations coupled with ESI-MS for performing comprehensive and comparative analysis of protein expression profiles within clinical specimens. Advanced sample preparation techniques, including tissue microdissection, detergent-based membrane protein extraction, and heat-induced protein retrieval, further enable targeted protein profiling of both fresh-frozen, formalin-fixed, and paraffin-embedded tissues. Comparative proteomics involving measurements in changes of biological pathways or functional processes are expected to provide relevant disease-associated markers and networks, molecular relationships among different stages of disease, and molecular mechanisms that drive the progression of disease. From a practical perspective, the evaluation of comparative proteomic dataset within a biological context is essential for high-throughput data validation, prioritization of follow-on biomarker selection, and validation experiments.
  1. Jinawath N, Vasoontara C, Jinawath A, Fang X, Zhao K, Yap KLL, et al.
    Oncoproteomic analysis reveals co-upregulation of RELA and STAT5 in carboplatin resistant ovarian carcinoma.
    PloS one. 2010 Jun;5(6):e11198+.
    DOI
    Abstract
    Background

    Ovarian cancer is one of the most lethal types of female malignancy. Although most patients are initially responsive to platinum-based chemotherapy, almost all develop recurrent chemoresistant tumors and succumb to their diseases. Elucidating the pathogenesis underlying drug resistance is fundamental to the development of new therapeutics, leading to improved clinical outcomes in these patients.

    Methods and Findings

    We compared the proteomes of paired primary and recurrent post-chemotherapy ovarian high-grade serous carcinomas from nine ovarian cancer patients using CIEF/Nano-RPLC coupled with ESI-Tandem MS. As compared to their primary tumors, more than half of the recurrent tumors expressed higher levels of several proteins including CP, FN1, SYK, CD97, AIF1, WNK1, SERPINA3, APOD, URP2, STAT5B and RELA (NF-κB p65), which were also validated by quantitative RT-PCR. Based on shRNA screening for the upregulated genes in in vitro carboplatin-resistant cells, we found that simultaneous knockdown of RELA and STAT5B was most effective in sensitizing tumor cells for carboplatin treatment. Similarly, the NF-κB inhibitor, BMS-345541, and the STAT5 inhibitor, Dasatinib, significantly enhanced cell sensitivity to carboplatin. Moreover, both RELA and STAT5 are known to bind to the promoter region of Bcl-X, regulating its promoter activity. In this regard, augmented Bcl-xL expression was detected in carboplatin-resistant cells. Combined ectopic expression of RELA and STAT5B enhanced Bcl-xL promoter activity while treatment with BMS-345541 and Dasatinib decreased it. Chromatin immunoprecipitation of the Bcl-X promoter region using a STAT5 antibody showed induction of RELA and STAT5 DNA-binding segments both in naïve cells treated with a high concentration of carboplatin as well as in carboplatin-resistant cells.

    Conclusions

    Proteomic analysis identified RELA and STAT5 as two major proteins associated with carboplatin resistance in ovarian tumors. Our results further showed that NF-κB and STAT5 inhibitor could sensitize carboplatin-resistant cells and suggest that such inhibitors can be used to benefit patients with carboplatin-resistant recurrent ovarian cancer.
  1. Balgley BM.
    Application of Shotgun Proteomics to Formalin-Fixed and Paraffin-Embedded Tissues.
    John Wiley & Sons, Inc.; 2010.
    DOI
    Abstract

    This chapter contains sections titled:

    • The Promise and Challenge of Shotgun Proteomics in Archival, Formalin-Fixed, Paraffin-Embedded Tissues (FFPE)
    • Development of Shotgun Proteomics in FFPE Tissues
    • Evaluation of Laser Capture Microdissection of FFPE Tissues
    • Shotgun Proteome Analysis of Microdissected Formalin-Fixed Brain Tumor Tissue
    • Evaluation of Confidence and Reproducibility of Quantitative Shotgun Proteomic Analyses
    • Evaluation of Confidence and Reproducibility of Quantitative Shotgun Proteomic Analyses of FFPE Tissues
    • Shotgun Proteomics for the Analysis of Archival FFPE Tissues
    • Future Directions for Shotgun Proteomics Applied to FFPE Tissues
  1. Shi SR, Balgley BM, Taylor CR.
    Symbiosis of Immunohistochemistry and Proteomics: Marching to a New ERA.
    John Wiley & Sons, Inc.; 2010.
    DOI
    Abstract
      Symbiosis of immunohistochemistry and proteomics – marching to a new era;
        protocol for Matrix-assisted laser desorption-ionization (MALDI) imaging mass spectrometry (IMS);
          IHC method, increasing demands for standardization – and true quantification of protein analytes by weight.
  1. Chien Y, Scuoppo C, Wang X, Fang X, Balgley B, Bolden JE, et al.
    Control of the senescence-associated secretory phenotype by NF-kappaB promotes senescence and enhances chemosensitivity.
    Genes & development. 2011 Oct
    DOI
    Abstract
    Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells. We found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-κB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-κB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. Our results demonstrate that NF-κB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-κB that contributes to the outcome of cancer therapy.
  1. Fang X, Wang C, Balgley BM, Zhao K, Wang W, He F, et al.
    Targeted Tissue Proteomic Analysis of Human Astrocytomas.
    J Proteome Res. 2012 Jul
    DOI
    Abstract
    Complicating proteomic analysis of whole tissues is the obvious problem of cell heterogeneity in tissues, which often results in misleading or confusing molecular findings. Thus, the coupling of tissue microdissection for tumor cell enrichment with capillary isotachophoresis-based selective analyte concentration not only serves as a synergistic strategy to characterize low abundance proteins, but it can also be employed to conduct comparative proteomic studies of human astrocytomas. A set of fresh frozen brain biopsies were selectively microdissected to provide an enriched, high quality, and reproducible sample of tumor cells. Despite sharing many common proteins, there are significant differences in the protein expression level among different grades of astrocytomas. A large number of proteins, such as plasma membrane proteins EGFR and Erbb2, are up-regulated in glioblastoma. Besides facilitating the prioritization of follow-on biomarker selection and validation, comparative proteomics involving measurements in changes of pathways are expected to reveal the molecular relationships among different pathological grades of gliomas and potential molecular mechanisms that drive gliomagenesis.
  1. McPhee CK, Balgley BM, Nelson C, Hill JH, Batlevi Y, Fang X, et al.
    Identification of factors that function in Drosophila salivary gland cell death during development using proteomics.
    Cell death and differentiation. 2013 Feb;20(2):218-225.
    DOI
    Abstract
    Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.
  1. Lu J, Ksendzovsky A, Yang C, Mehta GU, Yong RL, Weil RJ, et al.
    CNTF receptor subunit α as a marker for glioma tumor-initiating cells and tumor grade: laboratory investigation.
    Journal of neurosurgery. 2012 Dec;117(6):1022-1031.
    DOI
    Abstract
    OBJECT

    Tumor-initiating cells are uniquely resilient to current treatment modalities and play an important role in tumor resistance and recurrence. The lack of specific tumor-initiating cell markers to identify and target these cells presents a major obstacle to effective directed therapy.

    METHODS

    To identify tumor-initiating cell markers in primary brain tumors, the authors compared the proteomes of glioma tumor-initiating cells to their differentiated progeny using a novel, nongel/shotgun-based, multidimensional liquid-chromatography protein separation technique. An in vivo xenograft model was used to demonstrate the tumorigenic and stem cell properties of these cells. Western blot and immunofluorescence analyses were used to confirm findings of upregulated ciliary neurotrophic factor receptor subunit–α (CNTFRα) in undifferentiated tumor-initiating cells and gliomas of increasing tumor grade. Sequencing of the CNTFRα coding regions was performed for mutation analysis. Finally, antibody-dependent cell-mediated cytotoxicity was used to establish the role of CNTFRα as a potential immunotherapeutic target.

    RESULTS

    Ciliary neurotrophic factor receptor subunit–α expression was increased in tumor-initiating cells and was decreased in the cells’ differentiated progeny, and expression levels increased with glioma grade. Mutations of CNTFRα are not common in gliomas. Functional studies using CNTF treatment in glioma tumor-initiating cells showed induction of differentiation through the CNTFRα pathway. Treatment with anti-CNTFRα antibody resulted in increased antibody-dependent cell-mediated cytotoxicity in CNTFRα expressing DAOY cells but not in cell lines that lack CNTFRα.

    CONCLUSIONS

    These data indicate that CNTFRα plays a role in the formation or maintenance of tumor-initiating cells in gliomas, is a marker that correlates with histological grade, may underlie treatment resistance in some cases, and is a potential therapeutic target.
  1. Li J, Kil C, Considine K, Smarkucki B, Stankewich MC, Balgley B, et al.
    Intrinsic indicators for specimen degradation.
    Laboratory investigation; a journal of technical methods and pathology. 2013 Feb;93(2):242-253.
    DOI
    Abstract
    Variable degrees of molecular degradation occur in human surgical specimens before clinical examination and severely affect analytical results. We therefore initiated an investigation to identify protein markers for tissue degradation assessment. We exposed 4 cell lines and 64 surgical/autopsy specimens to defined periods of time at room temperature before procurement (experimental cold ischemic time (CIT)-dependent tissue degradation model). Using two-dimensional fluorescence difference gel electrophoresis in conjunction with mass spectrometry, we performed comparative proteomic analyses on cells at different CIT exposures and identified proteins with CIT-dependent changes. The results were validated by testing clinical specimens with western blot analysis. We identified 26 proteins that underwent dynamic changes (characterized by continuous quantitative changes, isoelectric changes, and/or proteolytic cleavages) in our degradation model. These changes are strongly associated with the length of CIT. We demonstrate these proteins to represent universal tissue degradation indicators (TDIs) in clinical specimens. We also devised and implemented a unique degradation measure by calculating the quantitative ratio between TDIs’ intact forms and their respective degradation-modified products. For the first time, we have identified protein TDIs for quantitative measurement of specimen degradation. Implementing these indicators may yield a potentially transformative platform dedicated to quality control in clinical specimen analyses.