Project description:The presence of lunasin was investigated in two soybean products, raw soy seeds and a commercial soybean beverage powder. Lunasin from the commercial soybean derivative was purified by reverse phase high pressure liquid chromatography and bystrong anion exchange solid phase extraction. Lunasin was further characterized by Top-down mass spectrometry (MS). The top-down characterization of lunasin unraveled a wide range of post-translationally modified proteoforms.
Project description:Protein posttranslational methylation and acetylations have been reported to occur in archaea, including members of the genus Sulfolobus, but have not been characterized on a proteome-wide scale. Sulfolobus chromatin proteins are known to be methylated and acetylated on lysine side chains, resembling eukaryotic histones in this respect. We utilized bottom-up and top-down proteomic approaches to perform a global and deep methylation study in the hyperthermoacidophylic archaeon S. islandicus with particular interest in chromatin proteins. Without specific enrichment, 731 protein were found by bottom-up proteomic analysis. The methylation sites on >400 proteins were monitored throughout 3 cell culture growth stages: early exponential, late exponential and stationary. (The previously described aKMT4 is found to be a plausible methyltransferase responsible for the massive methylation.) The proteome-wide top-down study/approach revealed 3778 proteoforms of 681 proteins, including 292 methylated proteoforms, of which 85 were comprehensively characterized by high-resolution MS/MS. Methylated proteoforms of the five chromatin proteins were characterized in detail by combination of bottom-up and top-down data showing the differences between the closely related Sul7d proteins. The two Alba chromatin proteins are, for the first time, reported to be methylated in this work. Alba1 protein is shown to be mono-, di- and trimethylated at the lysine-16 side chain. Stage-wise top-down analysis shows that the relative abundance of the methylated proteoforms versus non-methylated ones significantly grows/increases for Alba1 and Cren7 chromatin proteins throughout the cell growth. These findings highlight the ubiquitous lysine methylation throughout the S. islandicus proteome and singificantly enrich our knowledge related aboutarchaeal chromatin proteins.
Project description:We report the discovery of a photo-cleavable anionic surfactant, 4-hexylphenylazosulfonate (Azo) that can be rapidly degraded upon UV irradiation, for top-down proteomics. Azo can effectively solubilize proteins with performance comparable to SDS and is mass spectrometry (MS)-compatible. Azo-aided top-down proteomics allowed the detection of 100-fold more unique proteoforms and enabled the solubilization of membrane proteins for comprehensive characterization of post-translational modifications. Azo is simple to synthesize in large quantity for general use as an SDS replacement.
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:In mammals, the capacity of the female germ cell, the oocyte, to develop into embryo is acquired throughout meiotic maturation. Immature oocyte cannot be fertilized while mature oocyte is apt to accept spermatozoa and to develop an embryo. In a follicle, the oocyte is surrounded by mural granulosa cells (GC) and is physically and metabolically coupled with specialized granulosa cumulus cells (CC) which play an important role in oocyte maturation and fertilization. Factors expressing in GC and CC during maturation may reflect the oocyte quality, i.e. its capacity to be fertilized and assure early embryo development. However, the modifications of the content and the amount of peptide/proteins in the oocyte and the surrounding CC during oocyte maturation are mostly unknown and so there is not an accurate way of evaluating/monitoring how different in vitro maturation (IVM) protocols being in use in assisted reproduction technologies, can affect the process. In this context, Intact Cell MALDI-TOF Mass Spectrometry (ICM-MS) was applied to bovine follicular cells (bovine single oocytes, cumulus cells and granulosa cells) in order to characterize proteomic changes that occur in the follicle during female gamete development. In order to characterize finely endogenous molecular species observed on ICM-MS profiles and to identify markers of interest with their post-translational modifications, we carried out top-down proteomic on the different follicular cells from oocyte, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts. Prior to top-down MS using a dual linear ion trap Fourier Transform Mass Spectrometer LTQ Orbitrap Velos, depending on the amount of available biological material, we employed three analytic strategies as a direct infusion, a mono-dimensional liquid chromatography (µLC-1D-MS/MS) and an off-line multi-dimensional liquid chromatography combining four fractionations (based on reverse phase or gel filtration LC) to µLC-MS/MS. Here, we deposited our dataset from µLC-1D-MS/MS (analyses of oocytes, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts) and MDLC-MS/MS (analyses of granulosa cells protein extract).
Project description:Reduction in the cellular levels of the cyclin kinase inhibitor p27kip1 are frequently found in many human cancers and correlate directly with patient prognosis. Specifically ubiquitin dependent proteasomal turnover has been shown to cause reduced p27 expression in many human cancers. We recently demonstated that expression of a stabilized version of p27kip1 (p27kip1T187A) in a genetically modified mouse significantly reduced the number of intestinal adenomatous polyps which progressed to invasive carcinomas. Based on this work we set out to identify compounds which lead to a re-expression of p27 in cancer tissues. In this work we identify Argyrin A a compound derived from myxobacterium archangium gephyra as a potent inducer of p27kip1 expression. Argyrin A induces apoptosis in human colon cancer xenografts and tumor vasculature in vivo leading to a profound reduction in tumor size at well tolerated levels. Argyrin A functions are strictly dependent on the expression of p27kip1 as neither tumor cells nor endothelial cells which do not express p27kip1 respond to this compound. Surprisingly the molecular mechanism by which Argyrin A exerts its p27 dependent biological function is through a potent inhibition of the 20S proteasome. Experiment Overall Design: MCF7 cells were incubated with proteasome inhibitors bortezomib or Argyrin A or proteasome activity was reduced by treatment with siRNA against proteasomal subunits and gene expression profiles were determined at different timepoints thereafter.