Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site‐specific phosphorylation is essential to understand basic and disease biology. In ...vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other “non‐canonical” amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non‐canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry‐based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non‐canonical phosphosites is approximately one‐third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high‐throughput exploration of non‐canonical phosphorylation in all organisms.
Synopsis
Phosphoproteomic analyses in vertebrates have focussed mainly on serine/threonine/tyrosine phosphorylation. A new phosphopeptide enrichment strategy now reveals the extent of non‐canonical phosphorylation in human cells, opening doors to functional investigations.
Unbiased strong anion exchange (SAX)‐based phosphopeptide separation at pH 6.8 (UPAX) permits identification of histidine, aspartate, glutamate, lysine, arginine and cysteine phosphopeptides by tandem mass spectrometry.
Non‐canonical (His, Asp, Glu, Lys, Arg, Cys) protein phosphorylation is extensive in human cell extracts, with the number of non‐canonical phosphosites approximately one‐third that of canonical pSer, pThr and pTyr phosphorylation.
Sequence motifs for non‐canonical phosphorylation sites in human proteins are reported for the first time.
A new phosphopeptide enrichment strategy vastly expands the human phospho proteome beyond Ser/Thr/Tyr phosphorylation.
Batch implementations of support vector regression (SVR) are inefficient when used in an on-line setting because they must be retrained from scratch every time the training set is modified. Following ...an incremental support vector classification algorithm introduced by Cauwenberghs and Poggio (2001), we have developed an accurate on-line support vector regression (AOSVR) that efficiently updates a trained SVR function whenever a sample is added to or removed from the training set. The updated SVR function is identical to that produced by a batch algorithm. Applications of AOSVR in both on-line and cross-validation scenarios are presented.Inbothscenarios, numerical experiments indicate that AOSVR is faster than batch SVR algorithms with both cold and warm start.
A novel data-independent acquisition (DIA) method incorporating a scanning quadrupole in front of a collision cell and orthogonal acceleration time-of-flight mass analyzer is described. The method ...has been characterized for the qualitative and quantitative label-free proteomic analysis of complex biological samples. The principle of the scanning quadrupole DIA method is discussed, and analytical instrument characteristics, such as the quadrupole transmission width, scan/integration time, and chromatographic separation, have been optimized in relation to sample complexity for a number of different model proteomes of varying complexity and dynamic range including human plasma, cell lines, and bacteria. In addition, the technological merits over existing DIA approaches are described and contrasted. The qualitative and semiquantitative performance of the method is illustrated for the analysis of relatively simple protein digest mixtures and a well-characterized human cell line sample using untargeted and targeted search strategies. Finally, the results from a human cell line were compared against publicly available data that used similar chromatographic conditions but were acquired with DDA technology and alternative mass analyzer systems. Qualitative comparison showed excellent concordance of results with >90% overlap of the detected proteins.
ABSTRACT
We present MeqSilhouette v2.0 (MeqSv2), a fully polarimetric, time-and frequency-resolved synthetic data generation software for simulating millimetre (mm) wavelength very long baseline ...interferometry (VLBI) observations with heterogeneous arrays. Synthetic data are a critical component in understanding real observations, testing calibration and imaging algorithms, and predicting performance metrics of existing or proposed sites. MeqSv2 applies physics-based instrumental and atmospheric signal corruptions constrained by empirically derived site and station parameters to the data. The new version is capable of applying instrumental polarization effects and various other spectrally resolved effects using the Radio Interferometry Measurement Equation (RIME) formalism and produces synthetic data compatible with calibration pipelines designed to process real data. We demonstrate the various corruption capabilities of MeqSv2 using different arrays, with a focus on the effect of complex bandpass gains on closure quantities for the EHT at 230 GHz. We validate the frequency-dependent polarization leakage implementation by performing polarization self-calibration of synthetic EHT data using PolSolve. We also note the potential applications for cm-wavelength VLBI array analysis and design and future directions.
The first stable version of the Proteomics Standards Initiative mzIdentML open data standard (version 1.1) was published in 2012—capturing the outputs of peptide and protein identification software. ...In the intervening years, the standard has become well-supported in both commercial and open software, as well as a submission and download format for public repositories. Here we report a new release of mzIdentML (version 1.2) that is required to keep pace with emerging practice in proteome informatics. New features have been added to support: (1) scores associated with localization of modifications on peptides; (2) statistics performed at the level of peptides; (3) identification of cross-linked peptides; and (4) support for proteogenomics approaches. In addition, there is now improved support for the encoding of de novo sequencing of peptides, spectral library searches, and protein inference. As a key point, the underlying XML schema has only undergone very minor modifications to simplify as much as possible the transition from version 1.1 to version 1.2 for implementers, but there have been several notable updates to the format specification, implementation guidelines, controlled vocabularies and validation software. mzIdentML 1.2 can be described as backwards compatible, in that reading software designed for mzIdentML 1.1 should function in most cases without adaptation. We anticipate that these developments will provide a continued stable base for software teams working to implement the standard. All the related documentation is accessible at http://www.psidev.info/mzidentml.
We analyse very long baseline interferometry (VLBI) observations of the blazar CGRaBS J0809+5341 using Bayesian inference methods. The observation was carried out at 5 GHz using eight telescopes ...which form part of the European VLBI Network. Imaging and deconvolution using traditional methods imply that the blazar is unresolved. To search for source structure beyond the diffraction limit, we perform Bayesian model selection between three source models (point, elliptical Gaussian and circular Gaussian). Our modelling jointly accounts for antenna-dependent gains and system equivalent flux densities. We obtain posterior distributions for the various source and instrumental parameters along with the corresponding uncertainties and correlations between them. We find that there is very strong evidence (>10 super( 9):1) in favour of an elliptical Gaussian structure and using this model derive the apparent brightness temperature distribution of the blazar, accounting for uncertainties in the shape estimates. To test the integrity of our method, we also perform model selection on synthetic observations and use this to develop a Bayesian criterion for the minimum resolvable source size and consequently the maximum measurable brightness temperature for a given interferometer, dependent on the signal-to-noise ratio (SNR) of the data incorporating the aforementioned systematics. We find that calibration errors play an increasingly important role in determining the over-resolution limit for SNR...100. We show that it is possible to exploit the resolving power of future VLBI arrays down to about 5 per cent of the size of the naturally weighted restoring beam, if the gain calibration is precise to 1 per cent or less. (ProQuest: ... denotes formulae/symbols omitted.)
MeerKAT-16 H i observation of the dIrr galaxy WLM Ianjamasimanana, Roger; Namumba, Brenda; Ramaila, Athanaseus J T ...
Monthly notices of the Royal Astronomical Society,
10/2020, Volume:
497, Issue:
4
Journal Article
Peer reviewed
Open access
ABSTRACT
We present observations and models of the kinematics and the distribution of the neutral hydrogen (H i) in the isolated dwarf irregular galaxy, Wolf-Lundmark-Melotte (WLM). We observed WLM ...with the Green Bank Telescope (GBT) and as part of the MeerKAT Early Science Programme, where 16 dishes were available. The H i disc of WLM extends out to a major axis diameter of 30 arcmin (8.5 kpc), and a minor axis diameter of 20 arcmin (5.6 kpc) as measured by the GBT. We use the MeerKAT data to model WLM using the tirific software suite, allowing us to fit different tilted-ring models and select the one that best matches the observation. Our final best-fitting model is a flat disc with a vertical thickness, a constant inclination and dispersion, and a radially varying surface brightness with harmonic distortions. To simulate bar-like motions, we include second-order harmonic distortions in velocity in the tangential and vertical directions. We present a model with only circular motions included and a model with non-circular motions. The latter describes the data better. Overall, the models reproduce the global distribution and the kinematics of the gas, except for some faint emission at the 2σ level. We model the mass distribution of WLM with pseudo-isothermal (ISO) and Navarro–Frenk–White (NFW) dark matter halo models. The NFW and the ISO models fit the derived rotation curves within the formal errors, but with the ISO model giving better reduced chi-square values. The mass distribution in WLM is dominated by dark matter at all radii.
Calcineurin is a critical cell-signaling protein that orchestrates growth, stress response, virulence, and antifungal drug resistance in several fungal pathogens. Blocking calcineurin signaling ...increases the efficacy of several currently available antifungals and suppresses drug resistance. We demonstrate the application of a novel scanning quadrupole DIA method for the analysis of changes in the proteins coimmunoprecipitated with calcineurin during therapeutic antifungal drug treatments of the deadly human fungal pathogen Aspergillus fumigatus. Our experimental design afforded an assessment of the precision of the method as demonstrated by peptide- and protein-centric analysis from eight replicates of the study pool QC samples. Two distinct classes of clinically relevant antifungal drugs that are guideline recommended for the treatment of invasive “aspergillosis” caused by Aspergillus fumigatus, the azoles (voriconazole) and the echinocandins (caspofungin and micafungin), which specifically target the fungal plasma membrane and the fungal cell wall, respectively, were chosen to distinguish variations occurring in the proteins coimmunoprecipitated with calcineurin. Novel potential interactors were identified in response to the different drug treatments that are indicative of the possible role for calcineurin in regulating these effectors. Notably, treatment with voriconazole showed increased immunoprecipitation of key proteins involved in membrane ergosterol biosynthesis with calcineurin. In contrast, echinocandin (caspofungin or micafungin) treatments caused increased immunoprecipitation of proteins involved in cell-wall biosynthesis and septation. Furthermore, abundant coimmunoprecipitation of ribosomal proteins with calcineurin occurred exclusively in echinocandins treatment, indicating reprogramming of cellular growth mechanisms during different antifungal drug treatments. While variations in the observed calcineurin immunoprecipitated proteins may also be due to changes in their expression levels under different drug treatments, this study suggests an important role for calcineurin-dependent cellular mechanisms in response to antifungal treatment of A. fumigatus that warrants future studies.
Confident identification of sites of protein phosphorylation by mass spectrometry (MS) is essential to advance understanding of phosphorylation-mediated signaling events. However, the development of ...novel instrumentation requires that methods for MS data acquisition and its interrogation be evaluated and optimized for high-throughput phosphoproteomics. Here we compare and contrast eight MS acquisition methods on the novel tribrid Orbitrap Fusion MS platform using both a synthetic phosphopeptide library and a complex phosphopeptide-enriched cell lysate. In addition to evaluating multiple fragmentation regimes (HCD, EThcD, and neutral-loss-triggered ET(ca/hc)D) and analyzers for MS/MS (orbitrap (OT) versus ion trap (IT)), we also compare two commonly used bioinformatics platforms, Andromeda with PTM-score, and MASCOT with ptmRS for confident phosphopeptide identification and, crucially, phosphosite localization. Our findings demonstrate that optimal phosphosite identification is achieved using HCD fragmentation and high-resolution orbitrap-based MS/MS analysis, employing MASCOT/ptmRS for data interrogation. Although EThcD is optimal for confident site localization for a given PSM, the increased duty cycle compared with HCD compromises the numbers of phosphosites identified. Finally, our data highlight that a charge-state-dependent fragmentation regime and a multiple algorithm search strategy are likely to be of benefit for confident large-scale phosphosite localization.
Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalyzed by Golgi-resident tyrosyl protein sulfo transferases (TPSTs). Information on sY in humans is currently limited to ...∼50 proteins, with only a handful having verified sites of sulfation. As such, the contribution of sulfation to the regulation of biological processes remains poorly defined. Mass spectrometry (MS)-based proteomics is the method of choice for PTM analysis but has yet to be applied for systematic investigation of the "sulfome", primarily due to issues associated with discrimination of sY-containing from phosphotyrosine (pY)-containing peptides. In this study, we developed an MS-based workflow for sY-peptide characterization, incorporating optimized Zr
immobilized metal-ion affinity chromatography (IMAC) and TiO
enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of fragmentation regimes (CID, HCD, EThcD, ETciD, UVPD) highlighted differences in the generation of site-determining product ions and allowed us to develop a strategy for differentiating sulfated peptides from nominally isobaric phosphopeptides based on low collision energy-induced neutral loss. Application of our "sulfomics" workflow to a HEK-293 cell extracellular secretome facilitated identification of 21 new sulfotyrosine-containing proteins, several of which we validate enzymatically, and reveals new interplay between enzymes relevant to both protein and glycan sulfation.
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