A comparative study of commercially synthesize immobilized pH gradient (IPG) strips for two-dimensional electrophoresis

Abstract only availableTwo-dimensional electrophoresis (2-DE) is a widely used technique for resolving complex protein mixtures. The resurgence in this technique can be attributed to the contemporary approach for isoelectric focusing, which relies on an immobilized pH gradient of ampholines embedded within an acrylamide matrix. Furthermore, the commercial production of these Immobilized pH Gradient (IPG) strips has further simplified this technique to improve consistency of 2-DE. Since IPG strips are primarily purchased from vendors, rather than synthesized by the investigator, we wanted to compare the efficacy of strips from the principal manufacturers. In this study IPG strips selected from three major life science companies, Amersham, Bio-Rad, and Sigma (produced by Proteome Systems) were quantitatively compared for linearity of signal (spot volume) and qualitatively for parameters such as spot shape and same-sample reproducibility. To control for sample variation a Brassica napus protein leaf extract, prepared using an established protocol, was used as the test sample for all experiments. To compare the protein loading capacity and linear quantitative range, IPG strips were loaded with four different concentrations of protein: 50g, 100g, 200g, and 300g using the isoelectric focusing protocol recommended for Amersham strips. However, since the focusing protocol recommended for the Sigma IPG strips was approximately 8,000 Volt hours longer we also tested the strips at a longer focusing protocol. Spot volume was quantified using Phoretix 2D Advanced software. The spot volume was then plotted on graphs to determine the linear quantitative range. The graphs show Sigma to have the best quantitative range for each spot. However, Amersham IPG strips yielded more circular spot shapes and were more reproducible on a lot number and day-to-day basis.Plant Genomics Internship @ M

Protoemics of integral membrane proteins from developing Brassica napus

Abstract only availableAs plant seeds develop the accumulation of natural products, starch, oil, and protein undergo dramatic changes. At the early stages of seed filling in oilseeds starch is the principal component. Oil (triacylglycerol) and protein concentrations do not reach a maximum until the later stages of seed development. This metabolic shift within the seed, from production of starch to production of oil and protein, indicates that seed metabolism is regulated temporally. To better understand these metabolic changes it is important to examine the cognate changes in protein expression. Integral membrane proteins represent one class of proteins which are important for inter-organellar metabolic flow. Current two-dimensional electrophoresis techniques are unsuitable for the profiling of hydrophobic membrane proteins. To specifically characterize this class of proteins, a reproducible protocol for membrane protein isolation that can be used with standard sodium dodecyl sulfate polyacrylamide gel electrophoresis needed to be developed. Alkaline sodium carbonate washing of membranes followed by ultracentrifugation appeared to yield washed membrane fractions distinct from total protein fractions. To quantify relative volume and molecular weights of individual bands, Coomassie stained gels were analyzed with ImageQuant software. Identification of these bands was performed by trypsin digesting each protein (in-gel) and obtaining accurate peptide mass 'tags' using Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry. Peptide mass fingerprinting resulted in twelve conclusive identifications (out of 25 analyzed). Of these, six proteins were involved in the glucosinolate-myrosinase defense pathway. These proteins are suspected to be membrane associated, and are involved in a defense system that protects plant tissues from herbivory and fungal, viral, and bacterial infections. Other proteins were identified as: the RuBisCO large subunit, histone H3, NADH dehydrogenase subunit, pyruvate dehydrogenase E1 alpha subunit, and two types of cruciferins which are seed storage proteins. Of these, only NADH dehydrogenase is an integral membrane protein. Based on this data, the alkaline sodium carbonate wash method did not effectively enrich for integral membrane proteins. This may be due largely to the fact that certain proteins, especially cruciferin seed storage proteins, RuBisCO and myrosinases, are expressed at much higher levels than integral membrane proteins and are not quantitatively removed from membrane fractions by salt washing alone. Future work will include alternative approaches to membrane protein isolation including organic extraction.MU Monsanto Undergraduate Research Fellowshi

Western blot analysis of chloroplast, embryoplast and cytoplasm using sucrose synthase antibodies [abstract]

Abstract only availablePlastids are functionally and structurally diverse organelles and include chloroplasts (found in leaves), leucoplasts (roots), chromoplasts (flower petals), and amyloplasts (tubers). Plant embryos also contain plastids and those present in oilseeds such as rapeseed (Brassica napus) have properties of both chloroplasts and leucoplasts, and are therefore termed embryoplasts. After isolation of plastids from developing embryos of oilseed rape (Brassica napus cv. Reston), embryoplast proteins were identified by liquid chromatography-mass spectrometry. One of the proteins identified was sucrose synthase, a sucrose cleaving enzyme principally located in the cytosol. To confirm that sucrose synthase is associated with isolated embryoplasts, we performed western blots on the protein using four different sucrose synthase antibodies. The western blot results will be presented

Cloning and recombinant expression of two Arabidopsis 14-3-3 binding proteins expressed during seed development [abstract]

Abstract only availableAs a participant in cellular signaling, the 14-3-3 binding protein is essential to cellular function in both plants and animals. Two Arabidopsis thaliana 14-3-3 genes from phylogenetically different families were studied: the GF14-chi and GF14-231, 14-3-3 epsilon which were previously shown to be expressed in developing Arabidopsis seed. One of the principal functions of these proteins is to mediate signals by binding specific client proteins. One strategy to characterize these interacting client proteins is to use purified recombinant 14-3-3 proteins as bait in pull-down assays. The objective of this thesis was to create the expression constructs and purify the recombinant protein. To obtain a cloned gene, isolation of plasmid DNA was followed by PCR amplification. After a ligation reaction and transformation into E.coli Top 10, products were digested. The digested products were sent for sequencing. The GF14-231, 14-3-3 epsilon was successfully cloned. To express the 14-3-3 protein in E.coli, the construct was transformed in BL21 Star (DE3) One Shot cells. After transformation, IPTG was added to induce protein expression. Recombinant 14-3-3 protein was then purified by Ni-NTA column chromatography and analyzed by SDS-PAGE analysis. After obtaining purified protein an antibody will be developed. Future work will include a similar analysis of the GF14-chi.MU Monsanto Undergraduate Research Fellowshi

Accurate and affordable allergen quantification for the seed biotech industry

This presentation was made as part of the Life Science Elevator Pitch session.Plant seeds provide a significant portion of the protein present in the human diet, but are also the major contributors of allergenic proteins that cause a majority of the reported cases of food-induced anaphylaxis in the U.S. It is estimated that as many as 12 million Americans have food allergies, and there is a need for better methods for analytical screening of foods, or protein phenotyping, particularly for the seed industry. The current invention developed by researchers at the University of Missouri is a high-throughput, inexpensive workflow for quantifying prominent plant seed proteins. This was done by developing a mass spectrometry-based workflow beginning with intact, whole plant seed. The method does not require gel electrophoresis, antibodies, chemical labeling or a priori information about the seed to be analyzed

Development of a simplified, economical polyacrylamide gel staining protocol for phosphoproteins

Pro-Q Diamond (Pro-Q DPS) is a commercially available stain that binds the phosphate moiety of phosphoproteins with high sensitivity and linearity. To conserve consumable costs we demonstrate that threefold diluted Pro-Q DPS offers the same sensitivity and linearity of signal to that obtained with undiluted Pro-Q DPS. The optimal conditions for Pro-Q DPS indicate that fixation, staining, and destaining of gels longer than 1 h, 2 h, and four 30-min incubations, respectively, are not required. The fixation and destaining solutions, but not the threefold diluted Pro-Q DPS, can be re-used without compromising the signal intensity or linear dynamic range. This modified protocol of Pro-Q DPS reduces the cost at least by fourfold, making the stain economically attractive for large-scale analysis of phosphoproteins

Oil bodies isolated from Brassica napus mature seed

Abstract only availablePlants store seed triacylglycerols in discrete lipid monolayer storage organelles called oil bodies. Only two proteins have been characterized from oil bodies, namely oleosin and caleosin, which are both integral membrane proteins. To better understand the protein composition this organelle, oil bodies were isolated from Brassica napus (cultivar westar) mature seed. Oil bodies were isolated using two published methods that utilize phase separation in aqueous media. Method 1 employed iterative washes in aqueous media containing sucrose, and 2M NaCl while method 2 made use of only one type of aqueous media (minus NaCl) put through multiple washes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed method 1 to yield isolated oil bodies with higher purity based on the absence of the storage protein napin that was present in the total protein of isolated oil bodies from method 2. The oil bodies isolated with method 1 were subjected to washes in 2M NaCl or 8M urea to determine the nature of protein association to oil bodies. The isolated oil bodies were fractionated through petroleum ether to extract neutral lipids (triacylglycerols) that are contained by the monolayer membrane. Polar lipids were then extracted with chloroform/methanol. The interfacial pad which contained the associated proteins was suspended in water, sonicated, and subjected to acetone precipitation. Analysis of salt and urea washed oil body proteins by SDS-PAGE revealed abundant bands of the proper molecular weight for oleosins as well as at least 10 other additional proteins. Identification of these proteins by mass spectrometry will reveal novel proteins associated with oil bodies.Plant Genomics Internship @ M

ATP-Affinity chromatography to enrich protein kinases and associated proteins expressed in developing oilseed rape [abstract]

Abstract only availableFaculty Mentor: Dr. Jay J. Thelen, BiochemistryProtein kinases and associated proteins like 14-3-3 regulate many cellular processes.  These proteins are usually expressed at very low levels depending on development and environmental factors.  Identification of these proteins, which are largely under-represented in widely used gel-based (such as two-dimensional gel electrophoresis) and gel-free (multi-dimensional protein identification technology) proteomics approaches, will help in understanding the signaling and metabolic pathways involved in regulating the levels of storage reserves in oilseed plants.  Here, we have applied an affinity-based ATP-binders resin purification strategy in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography tandem mass spectrometry on developing seeds (four week after flowering)  of oilseed rape (Brassica napus L. Reston) to enrich and identify protein kinases and associated proteins.  A total of 32 high-confident proteins were identified.  Many of these proteins are known ATP-binding proteins including fructokinase and heat shock proteins as well as 14-3-3 proteins (known to interact with and modulate the activity of many kinases) and components of the 20S proteasome.  Bioinformatic analysis of identified proteins and application of this approach to other oilseed plants, such as Arabidopsis and soybean, are in progress.  This research was supported by NSF grants DBI-03324418 and DBI-044528

Multi-targeted trehalose-6-phosphate phosphatase I harbors a novel peroxisomal targeting signal 1 and is essential for flowering and development

A putative, non-canonical peroxisomal targeting signal type 1 (PTS1) Pro-Arg-Met > was identified in the extreme C-terminus of trehalose-6-phosphate phosphatase (TPP)I. TPP catalyzes the final step of trehalose synthesis, and the enzyme was previously characterized to be nuclear only (Krasensky et al. in Antioxid Redox Signal 21(9):1289–1304, 2014). Here we show that the TPPI C-terminal decapeptide ending with Pro-Arg-Met > or Pro-Lys-Met > can indeed function as a PTS1. Upon transient expression in two plant expression systems, the free C- or N-terminal end led to the full-length TPPI targeting to peroxisomes and plastids, respectively. The nucleus and nucleolus targeting of the full-length TPPI was observed in both cases. The homozygous T-DNA insertion line of TPPI showed a pleiotropic phenotype including smaller leaves, shorter roots, delayed flowering, hypersensitivity to salt, and a sucrose dependent seedling development. Our results identify novel PTS1s, and TPPI as a protein multi-targeted to peroxisomes, plastids, nucleus, and nucleolus. Altogether our findings implicate an essential role for TPPI in development, reproduction, and cell signaling.publishedVersio

The response of Asterochloris erici (Ahmadjian) Skaloud et Peksa to desiccation: a proteomic approach

18 p.The study of desiccation tolerance of lichens, and of their chlorobionts in particular, has frequently focused on the anti-oxidant system that protects the cell against photo-oxidative stress during dehydration/rehydration cycles. In this study, we used proteomic and transcript analyses to assess the changes associated with desiccation in the isolated phycobiont Aste-rochloris erici. Algae were dried either slowly (5?6 h) or rapidly (<60 min), and rehydrated after 24 h in the desiccated state. To identify proteins that accumulated during the drying or rehydration processes, we employed two-dimensional (2D) difference gel electrophoresis (DIGE) coupled with individual protein identi?cation using trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic analyses revealed that desiccation caused an increase in relative abundance of only 11?13 proteins, regard-less of drying rate, involved in glycolysis, cellular protection, cytoskeleton, cell cycle, and targeting and degradation. Tran-scripts of ?ve Hsp90 and two b-tubulin genes accumulated primarily at the end of the dehydration process. In addition, transmission electron microscopy (TEM) images indicate that ultrastructural cell injuries, perhaps resulting from physical or mechanical stress rather than metabolic damage, were more intense after rapid dehydration. This occurred with no major change in the proteome. These results suggest that desiccation tolerance of A. erici is achieved by constitu-tive mechanisms.Ministerio de Ciencia e InnovaciónGeneralitat ValencianaUnited States Department of Agricultur