Versatile and automated workflow for the analysis of oligodendroglial calcium signals.

Although intracellular Ca2+ signals of oligodendroglia, the myelin-forming cells of the central nervous system, regulate vital cellular processes including myelination, few studies on oligodendroglia Ca2+ signal dynamics have been carried out and existing software solutions are not adapted to the analysis of the complex Ca2+ signal characteristics of these cells. Here, we provide a comprehensive solution to analyze oligodendroglia Ca2+ imaging data at the population and single-cell levels. We describe a new analytical pipeline containing two free, open source and cross-platform software programs, Occam and post-prOccam, that enable the fully automated analysis of one- and two-photon Ca2+ imaging datasets from oligodendroglia obtained by either ex vivo or in vivo Ca2+ imaging techniques. Easily configurable, our software solution is optimized to obtain unbiased results from large datasets acquired with different imaging techniques. Compared to other recent software, our solution proved to be fast, low memory demanding and faithful in the analysis of oligodendroglial Ca2+ signals in all tested imaging conditions. Our versatile and accessible Ca2+ imaging data analysis tool will facilitate the elucidation of Ca2+-mediated mechanisms in oligodendroglia. Its configurability should also ensure its suitability with new use cases such as other glial cell types or even cells outside the CNS.

Click and Release Chemistry for Activity-Based Purification of ß-Lactam Targets.

ß-Lactams, the cornerstone of antibiotherapy, inhibit multiple and partially redundant targets referred to as transpeptidases or penicillin-binding proteins. These enzymes catalyze the essential cross-linking step of the polymerization of cell wall peptidoglycan. The understanding of the mechanisms of action of ß-lactams and of resistance to these drugs requires the development of reliable methods to characterize their targets. Here, we describe an activity-based purification method of ß-lactam targets based on click and release chemistry. We synthesized alkyne-carbapenems with suitable properties with respect to the kinetics of acylation of a model target, the Ldtfm L,D-transpeptidase, the stability of the resulting acylenzyme, and the reactivity of the alkyne for the cycloaddition of an azido probe containing a biotin moiety for affinity purification and a bioorthogonal cleavable linker. The probe provided access to the fluorescent target in a single click and release step.

mineXpert: Biological Mass Spectrometry Data Visualization and Mining with Full JavaScript Ability.

Biological mass spectrometry mainly comprises three fields of endeavor, namely, proteomics, metabolomics, and structural biology. In each of these specialties, the mass spectrometrist needs to access MS1 mass spectral data, although not necessarily on the same basis. For example, the bottom-up proteomics scientist will occasionally access MS1 data to perform data inspection, quality assessments, and quantitation measurements, whereas top-down proteomics, structural biology, or metabolomics scientists will actually spend most of their time mining profile-mode MS1 data. Furthermore, the advent of ion mobility-mass spectrometry imposes new manners of mass spectral data visualization. An open-source MS1-only mass data visualization software for the desktop was developed to allow scientists to visualize conventional and drift time mass data. Various mass data integrations are possible, allowing a thorough mass spectral data scrutiny. Isotopic cluster calculations are easily carried over from the chemical formula up to the display of the mass spectrum. Deconvolution of mass peaks can be achieved with a simple mouse drag. Flexible reporting of data inspection events and of mining discoveries is provided. Very large sparse data sets can be sliced into smaller chunks replicating the original data without data loss. Task automation is achieved in a JavaScript environment. This project allows users of mass spectrometry facilities to inspect and mine their MS1 mass data outside of these facilities without having to resort to the closed-source vendor software shipped with the instruments. mineXpert requires no proprietary software whatsoever once the mass spectrometry data have been converted to mzML. The reference implementation is version 5.8.2 or greater. Reference material, a detailed user manual, and video tutorials are available at http://www.msxpertsuite.org .

The human DNA ends proteome uncovers an unexpected entanglement of functional pathways.

DNA ends get exposed in cells upon either normal or dysfunctional cellular processes or molecular events. Telomeres need to be protected by the shelterin complex to avoid junctions occurring between chromosomes while failing topoisomerases or clustered DNA damage processing may produce double-strand breaks, thus requiring swift repair to avoid cell death. The rigorous study of the great many proteins involved in the maintenance of DNA integrity is a challenging task because of the innumerous unspecific electrostatic and/or hydrophobic DNA-protein interactions that arise due to the chemical nature of DNA. We devised a technique that discriminates the proteins recruited specifically at DNA ends from those that bind to DNA because of a generic affinity for the double helix. Our study shows that the DNA ends proteome comprises proteins of an unexpectedly wide functional spectrum, ranging from DNA repair to ribosome biogenesis and cytoskeleton, including novel proteins of undocumented function. A global mapping of the identified proteome on published DNA repair protein networks demonstrated the excellent specificity and functional coverage of our purification technique. Finally, the native nucleoproteic complexes that assembled specifically onto DNA ends were shown to be endowed with a highly efficient DNA repair activity.

(p)ppGpp modifies RNAP function to confer ß-lactam resistance in a peptidoglycan-independent manner.

(p)ppGpp is a nucleotide alarmone that controls bacterial response to nutrient deprivation. Since elevated (p)ppGpp levels confer mecillinam resistance and are essential for broad-spectrum ß-lactam resistance as mediated by the ß-lactam-insensitive transpeptidase YcbB (LdtD), we hypothesized that (p)ppGpp might affect cell wall peptidoglycan metabolism. Here we report that (p)ppGpp-dependent ß-lactam resistance does not rely on any modification of peptidoglycan metabolism, as established by analysis of Escherichia coli peptidoglycan structure using high-resolution mass spectrometry. Amino acid substitutions in the ß or ß' RNA polymerase (RNAP) subunits, alone or in combination with the CRISPR interference-mediated downregulation of three of seven ribosomal RNA operons, were sufficient for resistance, although ß-lactams have no known impact on the RNAP or ribosomes. This implies that modifications of RNAP and ribosome functions are critical to prevent downstream effects of the inactivation of peptidoglycan transpeptidases by ß-lactams.

An analytical workflow for the molecular dissection of irreversibly modified fluorescent proteins.

Owing to their ability to be genetically expressed in live cells, fluorescent proteins have become indispensable markers in cellular and biochemical studies. These proteins can undergo a number of covalent chemical modifications that may affect their photophysical properties. Among other mechanisms, such covalent modifications may be induced by reactive oxygen species (ROS), as generated along a variety of biological pathways or through the action of ionizing radiations. In a previous report [1], we showed that the exposure of cyan fluorescent protein (ECFP) to amounts of (•)OH that mimic the conditions of intracellular oxidative bursts (associated with intense ROS production) leads to observable changes in its photophysical properties in the absence of any direct oxidation of the ECFP chromophore. In the present work, we analyzed the associated structural modifications of the protein in depth. Following the quantified production of (•)OH, we devised a complete analytical workflow based on chromatography and mass spectrometry that allowed us to fully characterize the oxidation events. While methionine, tyrosine, and phenylalanine were the only amino acids that were found to be oxidized, semi-quantitative assessment of their oxidation levels showed that the protein is preferentially oxidized at eight residue positions. To account for the preferred oxidation of a few, poorly accessible methionine residues, we propose a multi-step reaction pathway supported by data from pulsed radiolysis experiments. The described experimental workflow is widely generalizable to other fluorescent proteins, and opens the door to the identification of crucial covalent modifications that affect their photophysics.

Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs.

Antibiotics of the ß-lactam (penicillin) family inactivate target enzymes called D,D-transpeptidases or penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. The resulting net-like macromolecule is the essential component of bacterial cell walls that sustains the osmotic pressure of the cytoplasm. In Escherichia coli, bypass of PBPs by the YcbB L,D-transpeptidase leads to resistance to these drugs. We developed a new method based on heavy isotope labeling and mass spectrometry to elucidate PBP- and YcbB-mediated peptidoglycan polymerization. PBPs and YcbB similarly participated in single-strand insertion of glycan chains into the expanding bacterial side wall. This absence of any transpeptidase-specific signature suggests that the peptidoglycan expansion mode is determined by other components of polymerization complexes. YcbB did mediate ß-lactam resistance by insertion of multiple strands that were exclusively cross-linked to existing tripeptide-containing acceptors. We propose that this undocumented mode of polymerization depends upon accumulation of linear glycan chains due to PBP inactivation, formation of tripeptides due to cleavage of existing cross-links by a ß-lactam-insensitive endopeptidase, and concerted cross-linking by YcbB.

Free Open Source Software for Protein and Peptide Mass Spectrometry- based Science.

In the field of biology, and specifically in protein and peptide science, the power of mass spectrometry is that it is applicable to a vast spectrum of applications. Mass spectrometry can be applied to identify proteins and peptides in complex mixtures, to identify and locate post-translational modifications, to characterize the structure of proteins and peptides to the most detailed level or to detect protein-ligand non-covalent interactions. Thanks to the Free and Open Source Software (FOSS) movement, scientists have limitless opportunities to deepen their skills in software development to code software that solves mass spectrometric data analysis problems. After the conversion of raw data files into open standard format files, the entire spectrum of data analysis tasks can now be performed integrally on FOSS platforms, like GNU/Linux, and only with FOSS solutions. This review presents a brief history of mass spectrometry open file formats and goes on with the description of FOSS projects that are commonly used in protein and peptide mass spectrometry fields of endeavor: identification projects that involve mostly automated pipelines, like proteomics and peptidomics, and bio-structural characterization projects that most often involve manual scrutiny of the mass data. Projects of the last kind usually involve software that allows the user to delve into the mass data in an interactive graphics-oriented manner. Software projects are thus categorized on the basis of these criteria: software libraries for software developers vs desktop-based graphical user interface, software for the end-user and automated pipeline-based data processing vs interactive graphics-based mass data scrutiny.

mineXpert2: Full-Depth Visualization and Exploration of MSn Mass Spectrometry Data.

mineXpert is a mass spectrometric data visualization and exploration software supporting only MS1 data that is aimed at proteomics scientists who do rarely require manual MS/MS data visualization and exploration (Rusconi, F. J. Proteome Res. 2019, 18, 2254-2259). In order to adapt it to new use cases in our facility and to widen its user base, mineXpert was entirely rewritten with the main aim of implementing MSn data support. Other feature additions were new data visualization and exploration methods, with an overhaul of the data plotting code to allow more flexible uses of mass data integration results. Further, the whole mass spectral data set can now be explored in a table view where the user may filter the data using a number of criteria that can be logically combined to pinpoint the smallest feature of interest. Ion mobility mass spectrometry is supported with specific data exploration and plotting. With mineXpert2, we provide a software program that will be of use to all mass spectrometrists, without restrictions on the field of endeavor, from pure chemistry to proteomics and metabolomics. As staff members of a mass spectrometry facility, we want to provide all users with a mass spectrometry data visualization and exploration software solution that frees them from the need to use closed-source vendor software. After conversion of the mass data to mzML, mineXpert2 requires no proprietary software whatsoever. The reference implementation is version 7.0.0 or greater. The software, a detailed user manual, and video tutorials are available at http://www.msxpertsuite.org.

massXpert 2: a cross-platform software environment for polymer chemistry modelling and simulation/analysis of mass spectrometric data.

UNLABELLED: Since the middle of the 90s, mass spectrometry has evolved into an almost indispensable tool in structural studies on an ever-growing variety of (bio-)polymers, of which proteins, sugars and nucleic acids are the most prominent. Since the first public release of massXpert, the advances of mass spectrometry have motivated continuous and thorough maintenance of that software, in the form of two full software rewrites, culminating with massXpert 2, which we describe in this report. We shall describe the profound changes in massXpert that were performed so as to keep up with the technical advances in mass spectrometry since a decade. AVAILABILITY: The massXpert 2 software is an open source and free software project hosted at http://www.massxpert.org.

Mass spectrometry-based structural dissection of fluorescent proteins.

Fluorescent proteins (FPs) are essential for live cell studies using fluorescence microscopy. To date, the molecular basis for FPs' irreversible photobleaching and the nature of the associated photoproducts are a matter of debate. Mass spectrometry, which should be an ideal technique for the structural dissection of FPs, cannot be harnessed efficiently due to their extreme resistance to trypsinolysis, due to the compactness of the barrel structure containing the chromopeptide. We devised a mild endoproteolysis procedure that affords a peptide mass fingerprint almost totally covering the sequence, thus allowing high-resolution mass spectrometric investigations of the protein structure.

Analytical biochemistry of DNA--protein assemblies from crude cell extracts.

Purification of specific DNA-protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA-protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA-protein complexes, showing the benefits to uncouple the DNA-protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA-protein assemblies.

High sensitivity of human centrin 2 toward radiolytical oxidation: C-terminal tyrosinyl residue as the main target.

Centrins are calcium-binding proteins that play a significant role in the maintenance of the centrosomal organization, mainly in the continuity between centrosome and microtubular network. Recent data showed that centrosome duplication abnormalities, like overduplication for example, could be due to hydrogen peroxide, suggesting an important impact of oxidative stress. To challenge this hypothesis, we performed one-electron oxidation experiments with human centrin 2, starting from azide radicals. Our results first revealed several intermolecular cross-links generating dimers, tetramers, hexamers, and higher molecular mass species. Dimers result from covalent bond linking the C-terminal tyrosines of each monomer. Second, the methionyl residue at position 19 was oxidized on the monomeric centrin. Further, electron microscopy experiments on centrin 2 showed a preexisting hexameric organization that was stabilized by covalent bonds as a result of irradiation. Overall, these results show that centrin 2 is highly sensitive to ionizing radiation, which could have important consequences on its biological functions.

Superoxide radical anions protect enkephalin from oxidation if the amine group is blocked.

The pentapeptide methionine-enkephalin (Met-enk) is a natural opiate that inhibits signals of pain. The N-terminal tyrosyl residue is important in the recognition of the peptide by its receptor. In oxidative stress, this residue can be oxidized by reactive oxygen species. The one-electron oxidation of Met-enk and of tert-butoxycarbonyl-methionine-enkephalin (Boc-Met-enk) was studied by gamma- and pulse radiolysis in the absence and in the presence of superoxide radical anions (O(2)(.-)) and oxygen, using azidyl radicals as oxidants. Without oxygen, both peptides behaved similarly. The tyrosyl radical resulting from the oxidation of tyrosyl residue produced the dimer linked by dityrosines. Methionine was also oxidized to its sulfoxide; however, this reaction is of minor importance. When O(2)(.-) was present, it added to tyrosyl radical giving a hydroperoxide. For Met-enk, this adduct cyclized via an intramolecular Michael addition of the amine on the aromatic ring. Conversely, for Boc-Met-enk, the adduct eliminated oxygen which led to 97% regeneration of the nonmodified peptide. Blocking the terminal amine group had thus a key role in protection of the tyrosyl residue. This finding might be exploited in the search for new pain inhibitors.

Tubulin glycylation controls primary cilia length.

As essential components of the eukaryotic cytoskeleton, microtubules fulfill a variety of functions that can be temporally and spatially controlled by tubulin posttranslational modifications. Tubulin glycylation has so far been mostly found on motile cilia and flagella, where it is involved in the stabilization of the axoneme. In contrast, barely anything is known about the role of glycylation in primary cilia because of limitations in detecting this modification in these organelles. We thus developed novel glycylation-specific antibodies with which we detected glycylation in many primary cilia. Glycylation accumulates in primary cilia in a length-dependent manner, and depletion or overexpression of glycylating enzymes modulates the length of primary cilia in cultured cells. This strongly suggests that glycylation is essential for the homeostasis of primary cilia, which has important implications for human disorders related to primary cilia dysfunctions, such as ciliopathies and certain types of cancer.

GNU polyxmass: a software framework for mass spectrometric simulations of linear (bio-)polymeric analytes.

BACKGROUND: Nowadays, a variety of (bio-)polymers can be analyzed by mass spectrometry. The detailed interpretation of the spectra requires a huge number of "hypothesis cycles", comprising the following three actions 1) put forth a structural hypothesis, 2) test it, 3) (in)validate it. This time-consuming and painstaking data scrutiny is alleviated by using specialized software tools. However, all the software tools available to date are polymer chemistry-specific. This imposes a heavy overhead to researchers who do mass spectrometry on a variety of (bio-)polymers, as each polymer type will require a different software tool to perform data simulations and analyses. We developed a software to address the lack of an integrated software framework able to deal with different polymer chemistries. RESULTS: The GNU polyxmass software framework performs common (bio-)chemical simulations-along with simultaneous mass spectrometric calculations-for any kind of linear bio-polymeric analyte (DNA, RNA, saccharides or proteins). The framework is organized into three modules, all accessible from one single binary program. The modules let the user to 1) define brand new polymer chemistries, 2) perform quick mass calculations using a desktop calculator paradigm, 3) graphically edit polymer sequences and perform (bio-)chemical/mass spectrometric simulations. Any aspect of the mass calculations, polymer chemistry reactions or graphical polymer sequence editing is configurable. CONCLUSION: The scientist who uses mass spectrometry to characterize (bio-)polymeric analytes of different chemistries is provided with a single software framework for his data prediction/analysis needs, whatever the polymer chemistry being involved.

Seasonal dynamics and toxicity of Cylindrospermopsis raciborskii in Lake Guiers (Senegal, West Africa).

Cylindrospermopsis raciborskii is a toxic bloom-forming cyanobacterium that occurs at tropical and temperate latitudes. Despite several reports from Africa, no data were previously available about its dynamics or toxic potential there. We therefore carried out a 1-year survey of the dynamics of C. raciborskii in the main water reservoir in Senegal, Lake Guiers. Cylindrospermopsis raciborskii never formed a bloom in this lake during the period studied, but was dominant during the dry season. The only observed bloom-forming species was a diatom, Fragilaria sp., which displayed a seasonal pattern contrary to that exhibited by C. raciborskii. Principal component analysis applied to environmental and phytoplankton data showed that high C. raciborskii biomasses were mainly related to high temperature and water column stability. Tests for C. raciborskii species-related toxicity and/or toxin synthesis were performed on 21 isolated clones. All the strains isolated tested negative in mouse toxicity bioassays, toxin analysis (MS/MS) and tests for known cylindrospermopsin genes (ps, pks). The limited number of isolates studied, and the occurrence of toxic and nontoxic clones in natural cyanobacterial populations, mean that we cannot conclude that there is no C. raciborskii-associated health risk in this drinking water reservoir.

Identification of low molecular weight molecules as new components of the nacre organic matrix.

Nacre of Pinctada margaritifera displays a number of interesting biological activities on bone, mainly concentrated in a water-soluble organic matrix representing 0.24% of the nacre weight. Dialysis of that matrix through 8 kDa and 1 kDa cut-off membranes showed that 60% of it is made of small molecules of molecular masses below 1 kDa. Reversed-phase high-performance liquid chromatography of the small molecule fractions and subsequent electrospray ionization mass spectrometric analysis of 19 fractions thereof indicated the presence of at least 110 different molecules, in the range 100 Da-700 Da. Evidence for aggregate-forming properties of the small molecules was given. Amino acid analysis revealed that most of the small molecules were not peptides and tandem mass spectrometric gas-phase fragmentations clearly indicated a structural relationship between several molecules. Intriguingly, differences of a single Dalton between mono-charged ions peaks were observed. Further, approximately 40 analytes could be arranged in a ladder-like manner with mass spaces of 57 Da. Some of the water-soluble peptide sequences obtained after MS/MS fragmentation revealed that the 57 Da shift corresponds to the repetition of glycine residues. Furthermore, the exchange of glycine against alanine explains the 14 Da shift observed between some peptides. These data show for the first time that small molecules, especially peptides, are prevalent components of nacre. The molecular species described in this report might have a functional role in nacre.

Functional complementation of RNA interference mutants in trypanosomes.

BACKGROUND: In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments. RESULTS: Using the protozoan Trypanosoma brucei as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 (TbPFR2), whose product is essential for flagellar motility. We demonstrate the complementation of TbPFR2, silenced via dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant TbPFR2 encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of TbPFR2, silenced via dsRNA targeting its coding sequence, through heterologous expression of the TbPFR2 orthologue gene from Trypanosoma cruzi: the flagellum regained its motility. CONCLUSIONS: This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms.