Online Weak Cation Exchange-HILIC × Reversed Phase LC UVPD-HRMS of intact HeLa Histones
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Andrea Gargano
LAB HEAD: Ljiljana Paša-Tolić
PROVIDER: PXD008978 | Pride | 2018-09-19
REPOSITORIES: Pride
Publications
Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS.
Gargano Andrea F G AFG Shaw Jared B JB Zhou Mowei M Wilkins Christopher S CS Fillmore Thomas L TL Moore Ronald J RJ Somsen Govert W GW Paša-Tolić Ljiljana L
Journal of proteome research 20181008 11
Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, ...[more]
Similar Datasets
Project description:Hydrogen/deuterium exchange mass spectrometric methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in the deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues.
2016-08-15 | PXD004630 | Pride
Project description:Spermatozoa Top-down analyses of different epididymal regions to identify endogenous biomolecules.
2014-12-16 | PXD001303 | Pride
Project description:Mass spectrometry-based proteomics has emerged as a powerful technique in the field of clinical diagnostics but requires a multistep process for sample preparation. The purpose of clinical mass spectrometry is to detect and quantify protein targets using well-established methods. Here, we found that the osmotic shock (OS) process allowed us to reduce the salt concentration and simultaneously enrich periplasmic proteins, specifically target proteins associated with antibiotic resistance in Gram-negative bacteria. Furthermore, we demonstrated that an optimized condition of the OS lysis allows the highest sensitivity in the MS analysis, and the OS lysate containing the proteins of interest (e.g., KPC-2) can be directly injected into multiple mass spectrometers without desalting process. Our study provides a detergent-free, straightforward method for periplasmic protein analysis using mass spectrometry.
2023-09-06 | PXD025999 | Pride
Project description:Here we present a high-performance software for proteome analysis that combines different mass spectrometric approaches, such as, top-down for intact protein analyses and both bottom-up and middle-down, for proteolytic fragment characterization.
2020-11-02 | PXD017618 | Pride
Project description:Tissue Top-down microproteomic was performed on 3 brain regions. 156 references proteins from different cellular compartments were characterized. Moreover, 11 Alternative proteins issued from alternative open reading frames (AltORF) were identified and were relied to the brain regions and associated to the function of their reference proteins. Some proteins display specific post-translational modifications profiles or truncation linked the brain regions and their functions. Systemic biology performed on microproteome identified in each region allowed to associate sub-networks with functional physiology of each brain region. Back correlation of the local extracted and identified microproteome with tissue cellular localization was then performed by MALDI mass spectrometry imaging. 40 proteins including 4 Altproteins have been back correlated in MALDI MSI and are in line with their tissue extracted region and physiological function. Taken together, we established the molecular physiome of 3 rat brain regions through reference and hidden proteome characterization.
2017-11-14 | PXD005424 | Pride
Project description:Top-down tissue microproteomic was performed in order to investigate tumoral tissue microenvironment. 289 proteins with post-translational modifications have been isolated from biospies including Tumor, Borderline and healthy regions of serous ovarian cancers. Tumor tissue displays a majority of proteins from the nucleus and extracellular vesicles. By contrast proteins identified in the healthy tissue or bordeline are related to cellular trafficking and membranes. Global subnetwork analyses confirmed that tumor pathways displayed proteins involved in neoplasm, autophagy, apoptosis, cell proliferation, tumor immunity whereas in healthy tissue pathways are mainly in cell survival, growth, motion, adhesion, differentiation and vascularization. Interestingly, truncated proteins are in majority observed in tumor region reflecting a high level expression of enzymes in this region. Besides the reference proteome, a deeper analysis of the hidden proteome issued from Alternative ORF, led a total of 14 alternative proteins identified i.e. Six Altprot from healthy tissues, 4 from borderline tissues and 4 from tumors. Expression of one of the 4 tumoral alternative protein, AltGNL1 which is translated from an AltORF nested within the GNL1 canonical coding sequence have been performed. Co-expression of annotated and alternative proteins from the same gene, was realized by transfection in HEK 293 and HeLa cells with an expression plasmid containing a GNL1-Flag(V5) construct. Western blot and immunofluorescence experiments with an anti-FLAG confirmed that AltGNL1 is constitutively co-expressed with GNL1. AltGNL1 displays a nuclear localization whereas GNL1 is present in the cytosol. Thus were report here for the first time the co-localization of two proteomes: the one issued from the kozak code and the one from Alternative ORF, so called the Hidden Proteome.
2017-06-23 | PXD005420 | Pride
Project description:The current technique used for microbial identification in hospitals is MALDI-TOF MS. However, it suffers from important limitations, in particular for closely-related species or when the database used for the identification lacks the appropriate reference. In this work, we set up a high throughput LC-MS/MS top-down proteomics platform dedicated to intact bacterial protein analysis. Using Escherichia coli as a model, all steps of the workflow were optimized: protein extraction, on-line liquid chromatographic separation, MS/MS fragmentation and data analysis. Using the optimized parameters, about 220 proteins, corresponding to more than 500 proteoforms, could be identified in a single run. We then demonstrated the suitability of the developed platform for the characterization and discrimination of enterobacterial pathogens rather undistinguishable by MALDI-TOF although leading to very different clinical outcomes. For each pathogen, we identified specific proteoforms that could potentially be used as biomarkers. We also improved the characterization of poorly described bacterial strains. Our results highlight the advantage of targeting proteoforms vs peptides for accurate bacterial characterization, and qualify top-down proteomics as a promising tool in clinical microbiology.
2020-09-24 | PXD019247 | Pride
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.
2016-09-13 | PXD003074 | Pride
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).
2017-04-10 | PXD004892 | Pride
Project description:Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric L-amino acid oxidase (LAAO, ~130 kDa) and the multi-chain toxin cobra venom factor (~147 kDa). The LAAO complex exhibited two clusters of multi-proteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new anti-venoms or other biotherapeutics.
2016-05-18 | PXD003403 | Pride