Bottom-up LC-MS/MS and top-down LC-MS analysis of B-phycoerythrin
ABSTRACT: Analysis of fluorescent assembly B-phycoerythrin with a combination of bottom-up and top-down mass spectrometry to reveal heterogeneity of proteoforms and characterize their chromophorylations.
Project description:This study aims at addressing soybean seeds (variety Absolute RR) germination, at 48h, during optimal and salt stressed condition when treated with bacterial signal compounds lipo-chitooligosaccharide (LCO) and thuricin 17 (Th17). Soybean growth is negatively affected when exposed to 40 mM NaCl and exposure to 80 mM NaCl is often lethal. When treated with the bacterial signal compounds lipo-chitooligosaccharide (LCO) and thuricin 17 (Th17), soybean seeds (variety Absolute RR) responded positively at salt stress of up to 150 mM NaCl. Shotgun proteomics of unstressed and 100 mM NaCl stressed seeds (48 h) in combination with the LCO and Th17 revealed many known, predicted, hypothetical and unknown proteins. In all, carbon, nitrogen and energy metabolic pathways were affected under both unstressed and salt stressed conditions when treated with signals. PEP carboxylase, Rubisco oxygenase large subunit, pyruvate kinase, and isocitrate lyase were some of the noteworthy proteins enhanced by the signals, along with antioxidant glutathione-S-transferase and other stress related proteins. These findings suggest that the germinating seeds alter their proteome based on bacterial signals and on stress, the specificity of this response plays a crucial role in organ maturation and transition from one stage to another in the plants life cycle; understanding this response is of fundamental importance in agriculture and, as a result, global food security.
Project description:Ribosomal purifications of spinach 70S ribosome and human 40S and 60S ribosomal subunits were analyzed with bottom-up LC-MS/MS to identify proteins comprising purified complexes as well as co-purified proteins. In order to assess and quantify proteoforms of ribosomal proteins in ribosomal purifications we employed top-down LC-MS/MS techniques with optimized methods, wherein apart from standard parameters (e.g. collision voltage and dynamic exclusion time) high-resolution and low-resolution were alternately employed in full MS mode.
Project description:Abstract Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regeneration, regulating gene expression especially via interaction with Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analysed Taz in vivo and ex vivo in comparison to Yap. Taz was expressed in activated satellite cells. siRNA knockdown or constitutive expression of wildtype or constitutively active TAZ mutants showed that TAZ promoted proliferation, a function that was shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while muscle growth was mildly affected in Taz (gene symbol Wwtr1-/-) knockout mice, there were no overt effect on regeneration. However, conditional knockout of Yap in satellite cells of Pax7Cre-ERT2/+ : Yapflox/flox : Rosa26Lacz mice produced a marked regeneration deficit. To identify potential mechanisms, microarray analysis showed many common Taz/Yap targets, but Taz also regulates some genes independently of Yap, including myogenic genes such as Pax7, Myf5 and Myod1. Proteomic analysis of Yap/Taz revealed many common binding partners, but Taz also interacts with proteins distinct from Yap, that are mainly involved in myogenesis and aspects of cytoskeleton organization. Neither TAZ nor YAP bind members of the Wnt destruction complex but both extensively changed expression of Wnt and Wnt-cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in promoting myoblast proliferation but Taz then switches to promote myogenic differentiation.
Project description:Lipo-chitooligosaccharide (LCO) and thuricin 17 (Th17) are two bacterial compounds that have been shown to positively influence plant growth of both legumes and non-legumes. Arabidopsis thaliana responded positively to treatment with the bacterial signal compounds LCO and Th17 in the presence of salt stress (up to 250 mM NaCl). Shotgun proteomics of unstressed and 250 mM NaCl stressed A. thaliana rosettes (7 days post stress) in combination with the LCO and Th17 revealed many known, putative, hypothetical and unknown proteins. Overall, carbon and energy metabolic pathways were affected under both unstressed and salt stressed conditions when treated with these signals. PEP carboxylase, Rubisco-oxygenase large subunit, pyruvate kinase, and proteins of photosystem I and II were some of the noteworthy proteins enhanced by the signals, along with other stress related proteins. These findings suggest that the proteome of A. thaliana rosettes is altered by the bacterial signals tested, and more so under salt stress, thereby imparting a positive effect on plant growth under high salt stress. The roles of the identified proteins are discussed here in relation to salt stress adaptation, which, when translated to field grown crops can be a crucial component and of significant importance in agriculture and global food production.
Project description:To investigate effects of Helical multi-walled carbon nanotubes (Helical MWCNTs) on total expression profile of tomato seeds and young sedlings we introduced Helical MWCNTs in MS agar medium in concentrationd of 25 ug/ml. Objectives for this study included the identification of genes that were up or down-regulated at the transcriptional level in tomato seeds and seedlings in response to application of Helical MWCNTs Tomato seeds (1 day after placement of medium) and 11-days old seedlings growing on regular MS agar medium or MS medium supplemented with Helical MWCNTs (25 ug/ml) were selected for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Multiple Sclerosis (MS) is an immune-mediated chronic inflammatory disease affecting the central nervous system. The cause of MS is not known and the mechanism of IFN-beta, a disease-modifying treatment (DMT) approved for MS, is not well-understood. Oligonucleotide microarrays were used to study gene expression in plasmacytoid denditic cells (pDCs) which are antigen-presenting cells implicated in MS pathogenesis. We analyzed gene expression in pDCs of healthy controls, untreated and IFN-beta treated MS patients. We were able to identify 60 genes which were abnormally expressed in untreated MS patients and were corrected after treatment with IFN-beta. PDCs were separated from healthy donors and MS patients at two time points: before and 3 months after initiation of treatment with IFN-beta for RNA extraction and hybridization on Affymetrix microarrays. Gene expression data analysis of was done by GeneSpring software (Agilent Technologies). An unpaired t-test was applied to select genes with significant difference in expression between healthy donors and untreated MS patients. A paired t-test was applied to select genes with significant difference in expression in MS patients before and after IFN-beta treatment. To select differentially expressed/regulated genes, the cut-off criteria consisted of a P value < 0.05 and fold change >1.5.
Project description:Verticillium dahliae is a soilborne fungus that causes wilt disease in plants. The microsclerotia of V. dahliae produce infectious hyphae that give rise to primary infections. In this study, RNA-seq libraries were prepared from microsclerotia (MS)-producing cultures of V. dahliae (ave = 52.23 million reads), and those not producing microsclerotia (NoMS, ave = 50.58 million reads) and analyzed for differential gene expression.