Genetics, Genomics, Bioinformatics, and Proteomics

Middle down peptide analysis using an LTQ Orbitrap and ProSightPC 2.0


Authors: Catherine Fenselau, Joe Cannon, Karen Lohnes, Colin Wynne and N.J. Edwards
Department or Program: CHEM
Presented by: Joe Cannon
Abstract: Middle down proteomics using microwave assisted acid cleavage and LC-Orbitrap-MS/MS provides a high throughput method for protein identification. This method achieves high sequence coverage in less time than comparable bottom up methods when used on a complex sample such as the human ribosome. Acid cleavage has been demonstrated as a rapid reaction in which kinetic selectivity is maintained for aspartic acid by holding the temperature above 110¡ C (Swatkoski et al J. Prot. Res. 6: 4525, 2007). These and other longer peptide products offer advantages for protein coverage, chromatographic separation and information on modifications. However, automated analysis by LC-MS/MS strategies has been impaired by the inability of most workflows to identify charge states higher than +3 or +4. We demonstrate here that accurate mass determinations provided by the LC-ion trap Orbitrap can be combined with the computational capabilities of ProSightPC 2.0 to permit high throughput identification of middle mass peptide products and their precursor proteins. The LC-Orbitrap-ProSightPC 2.0 workflow was evaluated based on several criteria: 1). number of unique peptides identified, 2). the percentage of the proteome represented, 3). extent of coverage per protein, 4). and the reliability of identifications as reflected in E-values.

A study of how roasting affects Ara h 2, a major peanut allergen protein


Authors: Jinxi Li, Kevin Shefcheck, John Callahan, Catherine Fenselau
Department or Program: CHEM
Presented by: Jinxi Li
Abstract: About 1% people are hypersensitive to peanuts in the US. Ara h 2 is one of the most abundant allergen proteins that cause the immunological response of patients who are allergic to peanuts. In our previous study of Ara h 2 from the raw peanut, the primary structure and partial secondary structure of the four isoforms of Ara h 2 have been studied using LC-MS/MS methods (J. Protein Science, in press). It has been reported by others that the roasted peanut is more allergenic than the raw peanut. Our present study is focused on the effect of the roasting process on this major peanut allergen protein, Ara h 2. We are looking for structural modifications introduced by roasting. Possible modifications include oxidation of cysteines and glycation of hydroxyprolines, Two-dimensional gel electrophoresis has been utilized to visualize differences between the proteins from raw and dark roasted peanuts. New spots, along with known Ara h 2 spots, have been cut out and digested in-gel by trypsin. The peptide mixtures were analyzed on a MALDI TOF/TOF mass spectrometer.

Top-Down Identification of Erwinia herbicola by LC-Orbitrap MS/MS


Authors: Colin Wynne, Nathan Edwards, Catherine Fenselau
Department or Program: CHEM
Presented by: Colin Wynne
Abstract: Since the anthrax attacks in the fall of 2001, there has been a growing interest in rapid, accurate screns of microorganisms. Many of these screens incorporate mass spectrometry-based proteomics, a technique that provides sensitivity and robustness. However, these mass spectrometry studies are sometimes hindered by the absence of fully sequenced genomes and proteomes in the publicly available databases. Currently, there are only around one thousand fully sequenced bacteria and archea, leaving many hazardous microorganisms with little or no sequence information to support proteomic studies. Therefore, there is a need for rapid identification of proteins in microorganisms that lack sequenced genmes. In this study, we use the primary sequence information of proteins from near-neighbor organisms along with mass spectrometric data to identify proteins from an organism that has few proteins in the public databases. This method of identification is in press in Analytical Chemistry. Top-down mass spectrometry is a growing field in the community due to the increased availability of high resolution, Fourier transform instruments. This approach has three steps. First, the mass to charge ratio of the entire protein is measured. Then, the protein is fragmented using collision induced dissociation with helium gas. Finally, the mass to charge ratios of the fragments are measured. In this study, a resolution of thirty thousand is used to measure the small mass difference between isotopes of highly charged species in order to determine the number of positive charges. Once the charge is known, the molecular weight of the target can be determined through deconvolution software. Erwinia herbicola (or Enterobacter agglomerans) is a bio-safety level 1 bacteria that is used as a simulant for Yersinia pestis (Bubonic plague). Currently, there are only 23 entries in the NCBI RefSeq database for either name of this target bacteria. The bacteria is in the Enterobacteriaceae family, and a custom database of all proteins from that family was constructed. The tandem mass spectra were searched against this database to identify homologous proteins in Erwinia herbicola. Seven of the measured proteins were found to be exact matches to proteins in other Enterobacteriaceae family organisms, while several others were identified with one or two amino acid changes.

Eukaryotic Plasma Membrane Proteins: Pellicle enrichment, solubilization and delipidation


Authors: Waeowalee Choksawangkarn, Joe Cannon and Catherine Fenselau
Department or Program: CHEM
Presented by: Waeowalee Choksawangkarn
Abstract: Our objective is the development of a high throughput procedure to enrich and analyze proteins from the eukaryotic plasma membrane. Our approach is built around the use of a nanoparticle pellicle to enrich the plasma membrane from other cellular membranes. After cell lysis and centrifugation, pellicle –bound membrane fragments are washed to remove associated and contaminating proteins. A variety of detergents and chaotropic agents have been evaluated to release the nanoparticles and dissolve the membrane proteins. A variety of methods have been evaluated to separate the proteins from lipids, including gel electrophoresis, solvent extraction, and precipitation/resolubilization. The efficiencies of the different methods are evaluated by LCMSMS analysis of tryptic digests of the recovered proteins, high throughput identification of proteins, and assessment of their cellular origins.

Characterization and Metabolic Regulation of the Vegetative Storage Protein gene family in Populus


Authors: Emily Lunzer and Gary D. Coleman
Department or Program: PSLA
Presented by: Emily Lunzer
Abstract: Vegetative Storage Proteins (VSP) in Populus are involved in the recycling and conservation of both N and C. The poplar VSP gene family consists of 9 genes with 3 genes belonging to the Bark Storage Protein (BSP) subfamily and 4 genes assigned to the win4-like subfamily while the remaining 2 genes do not group with either subfamily. VSP transcript abundance from plants grown in either short-day (SD) or long-day (LD) photoperiods or in LD with various levels of N fertilization indicates that the BSP subfamily is involved in both long-term and short-term N cycling and storage while the win-4 subfamily is associated with short-term cycling. Using a rapid and versatile excised stem assay, we are investigating the metabolic and signaling pathways that regulate BSP gene expression. Results will be presented that demonstrate the role of specific metabolites in regulating BSP gene expression. These results suggest that metabolic pathways and/or metabolic signals are important factors that regulate BSP expression and N cycling. This work illustrates the plasticity of storage in response to metabolic changes.

The Genetic Basis of Convergent Hermaphroditism in Caenorhabditis


Authors: Alana V. Doty and Eric S. Haag
Department or Program: BEES
Presented by: Alana Doty
Abstract:

Though many developmental pathways are conserved across wide   phylogenetic distances, sex determination mechanisms are often   hyperdivergent. To understand the rapid evolution of sex   determination we can examine closely related species, between which   sex determination changes may still be interpretable.    We conducted genetic screens in C. briggsae for Mog   (masculinization of germline) mutants. Our goal is to compare the   identity and regulation of these genes to those of the well- characterized C. elegans sex determination pathway to see how   hermaphroditism has evolved independently in the two species. We   isolated three C. briggsae masculinizing mutant alleles: two from   forward screens, nm41 and nm64, and one from a deletion screen,   nm68. All are alleles of Cbr-gld-1, which encodes the ortholog of   the C. elegans germline RNA-binding protein and translational   repressor, GLD-1. As judged by DIC microscopy and Hoechst staining,   gld-1(lf) hermaphrodite germlines never undergo oogenesis and often   contain excess mature sperm and spermatocytes reaching into their   distal gonad arms. Other mutant animals make germline tumors or have   arrested pachytene cells. The published work of Nayak et al. (2005) and our results show that   gld-1 has an opposite major sex determination role C. elegans vs. C.   briggsae: in C. elegans, it is necessary to allow spermatogenesis in   hermaphrodites, but is instead needed for the switch to oogenesis in   C. briggsae hermaphrodites. Because GLD-1 is well conserved between   species, we hypothesize that GLD-1 has different cofactors and/or   has come to repress different messenger RNA targets in the two   species. To test this, we created transgenic lines via bombardment   in which the null C. elegans gld-1 allele q485 is rescued by the   wild-type C. briggsae gld-1 locus. This demonstrates that Cbr-GLD-1   is capable of acting as a translational repressor as GLD-1 does in   C. elegans, and supports the idea that differential mRNA target   acquisition is the mechanism by which GLD-1 has come to have   opposite sex determination roles in C. briggsae and C. elegans.   Next, to identify these different targets in C. briggsae, we im! ! ! ! munoprecipitated mRNAs bound by wild-type Cbr-GLD-1 in vivo.  We   will report results from microarray analysis using chips containing   probes for all predicted C. briggsae protein-coding genes. By   comparing a validated set of Cbr-GLD-1 targets to what is know of   GLD-1 targets in C. elegans, we can determine which, if any, are   unique to the C. briggsae sex determination pathway and thus may   have been important in the evolution of hermaphroditism in C.   briggsae.



The Genetic Characterization of Toxic Heme Resistant (them) Mutants in C. elegans


Authors: Jonathan Walston, Anita Rao, Iqbal Hamza
Department or Program: ANSC
Presented by: Jonathan Walston
Abstract: Heme is an essential, but cytotoxic cofactor that plays many biological roles within the cell including: respiration, gas sensing, xenobiotic detoxification, cell differentiation, signal transduction, circadian clock control, and microRNA processing. The heme synthesis pathway has been well established, however there is little known about the intracellular trafficking mechanisms involved in heme homeostasis. Most eukaryotic organisms synthesize their own heme, however the nematode Caenorhabditis elegans has been found to be a natural heme auxotroph, requiring heme from environmental sources for proper growth. This provides a tractable animal model to study heme transport which contains no endogenous heme. To examine potential genes involved in intracellular heme homeostasis, a forward genetic screen was used to find mutant C. elegans that could grow at concentrations of heme that normally arrest growth. Thirteen mutants were produced from this screen, dubbed them for Toxic HEMe resistant and were subsequently placed into three phenotypic clusters and five complementation groups. To determine the map position of sample mutants from each of the complementation groups, pyrosequencing was utilized to examine single nucleotide polymorphisms (SNPs). Four of the five sample mutants were able to be mapped. Three of the mutants that mapped to chromosome I share a unique resistance to a toxic heme analog gallium protoporphyrin IX (GaPPIX). Using a combination of RNAi and GaPPIX, the genes around the region to which these mutants mapped to were systematically knocked down to phenocopy the resistance phenotype. Candidate genes will now be explored in them mutants for the genetic alterations that cause the heme resistance phenotype. Identification of these genes will then provide us with the opportunity to explore their role in heme trafficking and allow us to further our understanding of how heme homeostasis is maintained within the cell.

Towards Quantitative Proteomics Analysis – A Test Drive with proteins from Drosophila testis


Authors: Yan Wang, Zifeng Jiang, Carlos Machado
Department or Program: CHEM
Presented by: Yan Wang
Abstract: In this preliminary study, the ability of the Proteomics Core Facility to analyze differentially expressed proteins in different treatments using existing resources is evaluated. Two protein mixtures from testis of Drosophila pseudoobscura were digested with trypsin and labeled with tandem mass tag before being mixed together for fractionation and LCMSMS analysis. Tryptic peptides were separated into 24 fractions by isoelectric focusing. Each fraction was analyzed by LCMSMS. Mass spectrometry experiment was set up to analyze peptides for both sequence information and relative abundance in the two samples (relative abundance of the two tags with different mass). A tryptic digest of standard bovine serum albumin was spiked into the two samples at a ratio of 1:5 prior to labeling experiment. Database search identified 200 proteins with decent confidence. Of the 200 proteins identified, 160 (80%) showed expected ratio in the two samples, including the bovine serum albumin spiked in. Additional 250 proteins were identified with lower confidence, which might be better identified and quantified by increasing the amount of protein loaded.

Screening and characterization of Arabidopsis mutants that are defective in targeting of a resistance protein to the host-pathogen interface.


Authors: Paymon Nikfarjam and Dr. Shunyuan Xiao
Department or Program: BIOL
Presented by: Paymon Nikfarjam
Abstract: Powdery mildew fungal pathogens develop a structure called the haustorium that can extract nutrition from plant cells. Plants have evolved resistance mechanisms to fight against the robbery by the pathogens. These mechanisms are usually controlled by dominant resistance (R) genes which have products that directly or indirectly detect the pathogen invasion and trigger effective defense responses. One such R gene isolated from the model plant Arabidopsis is RPW8. RPW8 confers broad-spectrum resistance against the haustorium-forming powdery mildew pathogens via the conserved salicylic acid-dependent signaling pathway. It has been recently discovered that RPW8 is specifically targeted to the host-pathogen interface called the extrahaustorial membrane (EHM) whereby it activates defense responses to restrict the haustorium development. To characterize the cellular components that are recruited for targeting RPW8 to the host-pathogen interface, a large-scale of mutagenesis has been conducted. A group of Arabidopsis mutants that are defective in RPW8-targeting (drt) have been isolated and characterized.

A Novel Method to Study Substrate Specificity of Protein Tyrosine Phosphatases


Authors: Puja Giri, Jiangsong Jiang, Shuwei Li
Department or Program: BIOL
Presented by: Puja Giri
Abstract: Protein tyrosine phosphorylation plays a critical role in many biological processes involving cell growth, proliferation, and differentiation. Phosphorylation is regulated by two large families of proteins, protein tyrosine kinases and protein tyrosine phosphatases (PTPs). We are developing a novel method to identify proteins interacting with Protein Tyrosine Phosphatases. Compared to yeast two-hybrid techniques, the generation of readout signals in PCA is faster in that it is determined by the refolding of the two fragments. As a model system to detect protein-protein interactions, we generated a β-lactamase mutant E166NTEM that will fluoresce when it binds to its substrate. We used the β-lactamase gene (TEM-1) from E. coli and introduced a mutation in the TEM-1 end-terminus (E166NTEM). The mutation was confirmed by sequencing of the TEM-1 locus. We next expressed and purified the E166NTEM protein. Finally, we were successful in labeling E166N TEM by using the fluorescent compound (CA). This system based on β-lactamase-designed fluorescent substrate will provide a proof of concept for a novel method to identify and study regulation of protein phosphorylation.

Studying the Pathogenesis of Human Pulmonary Fibrosis in Hermansky-Pudlak Syndrome, a Lysosomal-Related Organelle Disease


Authors: Blanca Gomez, B. Gochuico, C. Yeager, H. Dorward, A Helip-Wooley, K. O’Brien, J. Salas, T. Markello, William Gahl
Department or Program: BIOL
Presented by: Blanca Gomez
Abstract: Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by abnormal biogenesis of lysosome-related organelles (LROs). There are 8 known human HPS genes and disease subtypes. HPS-1 and HPS-4 are notable for development of pulmonary fibrosis, which is the leading cause of mortality in HPS-1. The cause of pulmonary fibrosis in HPS-1 and HPS-4 is unknown. However, type II pneumocytes and alveolar macrophages may contribute to the pathogenesis of disease. We hypothesized that an oncoprotein named galectin-3 contributes to the pathogenesis of HPS-1 pulmonary fibrosis since it is upregulated in other types of human fibrotic diseases. Galectin-3 was analyzed by immunostaining and was measured in the lung tissues from patients with HPS-1 pulmonary fibrosis (HPS-1 PF), idiopathic pulmonary fibrosis (IPF) and normal volunteers. Interestingly, galectin-3 did not co-localize with surfactant protein-C, suggesting that galectin-3 localizes to vesicles distinct from lamellar bodies and lysosomes. However, galectin-3 immunostaining was more intense in HPS-1 PF lung tissue compared normal lung tissues and concentrations of galectin-3 were significantly higher in bronchoalveolar lavage fluid of severe HPS-1 PF patients. Among subjects with HPS-1 PF, galectin-3 levels directly correlated with severity of disease. Taken together, these results strongly suggest that the pathogenesis of HPS-1 Pulmonary Fibrosis may be the result of defective regulation of galectin-3.

Quantitative Proteomic Profiling of Prematurely Senescent ARPE-19 Cells


Authors: Ligia Turcios-Flores, Illarion V. Turko, Wei-Li Liao
Department or Program: BIOL
Presented by: Ligia Turcios-Flores
Abstract: Research in senescent cells is a very important topic of discussion today. This in part is due to the fact that we all age in someway and so do our cells. However, premature senescence is not indicative of healthy cells. This is why research in this area is so important to the biomedical industry. It is essential to understand why cells in the body age prematurely and how can we detect these changes at an early stage of disease. Backround Senescent cells or aging cells , express certain proteins that healthy cells do not. Such proteins are called biomarkers and provide us with vital information about what conditions the cells are in and at what stage of senescence they are in. Also, another question that is posed, is what could be causing the cells to age prematurely? In the lab, similar stress was placed on cells that would be observed in the body. Therefore, isolating the specific proteins that appear after a cell, in vitro, is exposed to some type stress is the major finding of any study dealing with aging or diseased cells. Future Research It is of greater importance to find the link between fluctuating levels of these proteins and any correlation with disease. This type of research is crucial in finding treatment options for disease and to impose more preventative measures for patients at risk for cancer and other disease such as AMD (Age-Related Macular Degeneration).

FUNCTIONAL GENOMICS OF HEME HOMEOSTASIS BY RNA INTERFERENCE


Authors: Jason Sinclair, Tamika Samuel, Scott Severance, and Iqbal Hamza
Department or Program: ANSC
Presented by: Jason Sinclair
Abstract: It is thought that as much as 80% of the world’s population is iron-deficient, making nutritional iron deficiency the world’s most common nutritional disorder. Considerable evidence exists to support the idea that heme is an essential bioavailable source of iron in humans, but the pathways for heme absorption and utilization are poorly understood. Heme (iron-protoporphyrin IX) also serves as a prosthetic group in proteins which play a key role in diverse biological processes such as oxygen transport, xenobiotic detoxification, signal transduction, and gene regulation. Since hemes are cytotoxic and insoluble, we hypothesize that specific pathways exist within cells for trafficking heme from the site of synthesis in mitochondria to various intracellular destinations for incorporation into apo-hemoproteins. We have demonstrated that C. elegans is absolutely dependent on heme to sustain metabolic processes because it lacks the ability to synthesize heme. C. elegans, therefore, represents a unique animal model to identify the genetic and cellular pathways for heme transport because they allow external control of heme transport pathways not permissible in other organisms. We have synthesized a transgenic “heme-sensor” strain in C. elegans that expresses green fluorescent protein (GFP) under the control of a heme-responsive gene promoter. In the sensor strain, GFP fluorescence increases under low heme and decreases when the concentration of heme is elevated. We used this strain in a genome-wide, functional reverse genetic RNAi screen using the clones in two commercially available feeding libraries. Changes in GFP fluorescence were monitored as a function of heme levels and gene knock-downs. We knocked down a total of 18,566 genes (≈92% of the C. elegans genome) and identified 1,428 that dramatically altered the levels of GFP. Re-screening the modulators in the “heme-sensor” strain and in a strain that expresses GFP under the control of a promoter that does not respond to heme narrowed the list to 743. Using a COPAS worm sorter, we are currently quantifying the level of GFP in worms fed bacteria producing dsRNA against these 743 genes. We have identified several novel genes which, based on the altered GFP levels they effect, play a role in organismal heme homeostasis. Characterizing these genes simultaneously in worms, yeast, zebrafish, and mammalian cells will provide new mechanistic insights into heme homeostasis in mammals. Information gleaned from this study may aid in the development of heme-based nutritional interventions for human iron deficiency and permit identification of novel drug targets for parasitic worm infestations which exacerbate human iron deficiency.

Allelic expression imbalance in a 3' UTR HLA-DPA1 Single Nucleotide Polymorphism in Human Tissues


Authors: Natalia Orduz, Indu Kohaar, Jennifer L. Hall, Thomas O'Brien, Ludmila Prokunina-Olsson
Department or Program: BIOL
Presented by: Natalia Orduz
Abstract: A study has shown a strong relationship of SNP rs3077 with the expresion of HLA-DPA1 (O'Brien et al, unpublished). HLA-DPA1 is a class II protein that paricipates in the immune response by presenting antigens to T cells. This SNP is located in the 3' untranslated region of HLA-DPA1 in chromosome 6. To assess the validity of this association between rs3077 and HLA-DPA1 expression, we conducted allelic expression imbalance (AEI) experiment. DNA and cDNA from liver tissue (n= 33) and purified peripheral blood monocytes (n= 59) were used. Allelic discrimination genotyping method (Taqman) was used to quantify ratios between alleles in DNA and corresponding cDNA samples, focusing on the heterozygous samples. AEI was observed in liver (p=8.8x10-5) and in blood monocytes (p=7.2x10-7). Based on these results we conclude that rs3077 is associated with difference in expression levels of HLA-DPA1 in microarray experiments and also shows allelic expression imbalance in liver and monocytes.

Two MAP kinases, MPK9 and MPK12, are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling


Authors: F. Jammes, C. Song, D. Shin, S. Munemasa, K. Takeda, D. Gu, Leonhardt, B. E. Ellis, Y. Murata and J. M. Kwak
Department or Program: CBMG
Presented by: Fabien Jammes
Abstract: Abscisic acid (ABA) controls stomatal apertures to limit water loss by modulating ion fluxes across membranes. Reactive oxygen species (ROS) mediate ABA signaling in guard cells. Using cell-type-specific functional genomics approach to genetically dissect guard cell ABA-ROS signaling, we identified two MPK genes, MPK9 and MPK12, that are preferentially expressed in guard cells and positively regulate ABA signaling by working downstream of ROS. Mutations in both MPK9 and MPK12 lead to impaired stomatal response to ABA and ABA activation of anion channels. We also show that MPK12 kinase activity is increased by ABA and H2O2 treatment.

Characterization of Betelgeuse, an ENU induced mouse mutant


Authors: Ray Mullen, Dawn Watkins-Chow, William Pavan
Department or Program: BIOL
Presented by: Raymond Mullen
Abstract: Mouse models are used to study many human diseases including ones that result from abnormal development of the neural crest in humans, for example Waardenburg syndrome. Waardenburg syndrome is an inherited disorder that is often characterized by changes in hair and skin color as well as varying degrees of hearing loss; people with this disorder may have differently colored eyes. The symptoms of Waardenburg syndrome can vary and some individuals also display severe aganglianosis. One mouse model of Waardenburg syndrome, the Betelgeuse (Btg) phenotype, was identified through an ENU screen designed to identify additional genes affecting the severity of Waardenburg syndrome. The homozygous Betelgeuse mice are embryonic lethal, whereas the heterozygous mutant mice are viable. In order to identify the gene responsible for the Btg phenotype, the gene was mapped to a region of mouse chromosome 3 containing the mouse homologue of the FSA gene. Currently, PCR is being used to amplify specific target fragments of this cDNA for sequencing. Some double peaks were identified in an amplicon of Btg, but sequencing of a Balb/c control revealed the same double peaks. This suggests use of an alternate splice site in both the wild-type and mutant mice. The future plans of the project are to determine if this gene is normally expressed in a Btg heterozygote as well as finish the amplification and sequencing of the gene to determine if the FSA homologue is causing the Btg phenotype. Identification of the causative mutation will add to our understanding of neural crest development and Waardenburg syndrome.

Autosomal Dominant Diseases: Characterization of Protein Class and Mutant Locations


Authors: Naim Haque, John Moult, Lipika Ray
Department or Program: BIOL
Presented by: Naim Haque
Abstract: Autosomal dominant diseases, such as Huntington disease and congenital cataracts, can be passed down easily from parent to child through the inheritance of just one mutant gene in a pair of autosomal chromosomes. Currently, there is still not much known about the molecular aspects of this disease, in terms of how a protein structure is affected after given mutation, resulting in a dominant phenotype. Therefore, deciphering the molecular mechanisms of this disease is of great importance. The objective of our study was to examine the various types of the protein classes present in both autosomal dominant and autosomal recessive diseases, and to determine the frequency of oligomeric structures in each. We also examined the locations of mutants in both autosomal dominant and recessive gene products using a program called Pymol and the website SNPs 3D. We found that autosomal dominant genes had significantly more non-oligomeric structures compared to autosomal recessive genes. Also, most mutations (around 50%) were found to be located in the interior of the protein structure, while most of the interface mutations were located on tetrameric structures in both autosomal dominant and recessive genes. Using this knowledge, we may be able to design better drugs and gene therapy in the future to treat autosomal dominant diseases.