Blumeria graminis is an economically important obligate plant-pathogenic fungus, whose entire genome was recently sequenced and manually annotated using ab initio in silico predictions (Spanu et al. ...2010, Science 330, 1543-1546). Employing large scale proteogenomic analysis we are now able to verify independently the existence of proteins predicted by ∼24% of open reading frame models. We compared the haustoria and sporulating hyphae proteomes and identified 71 proteins exclusively in haustoria, the feeding and effector-delivery organs of the pathogen. These proteins are significantly smaller than the rest of the protein pool and predicted to be secreted. Most do not share any similarities with Swiss–Prot or Trembl entries nor possess any identifiable Pfam domains. We used a novel automated prediction pipeline to model the 3D structures of the proteins, identify putative ligand binding sites and predict regions of intrinsic disorder. This revealed that the protein set found exclusively in haustoria is significantly less disordered than the rest of the identified Blumeria proteins or random (and representative) protein sets generated from the yeast proteome. For most of the haustorial proteins with unknown functions no good templates could be found, from which to generate high quality models. Thus, these unknown proteins present potentially new protein folds that can be specific to the interaction of the pathogen with its host.
New liquid atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) matrices that produce predominantly multiply charged ions have been developed and evaluated with respect to ...their performance for peptide and protein analysis by mass spectrometry (MS). Both the chromophore and the viscous support liquid in these matrices were optimized for highest MS signal intensity, S/N values and maximum charge state. The best performance in both protein and peptide analysis was achieved employing light diols as matrix support liquids (e.g. ethylene glycol and propylene glycol). Investigating the influence of the chromophore, it was found that 2,5-dihydroxybenzoic acid resulted in a higher analyte ion signal intensity for the analysis of small peptides; however, larger molecules (>17 kDa) were undetectable. For larger molecules, a sample preparation based on α-cyano-4-hydroxycinnammic acid as the chromophore was developed and multiply protonated analytes with charge states of more than 50 were detected. Thus, for the first time it was possible to detect with MALDI MS proteins as large as ∼80 kDa with a high number of charge states, i.e. m/z values below 2000. Systematic investigations of various matrix support liquids have revealed a linear dependency between laser threshold energy and surface tension of the liquid MALDI sample.
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•New liquid AP-MALDI matrices for protein and peptide analysis were developed.•Electrospray-like MS spectra of proteins as high as ∼80 kDa were detected by liquid AP-UV-MALDI MS.•Chromophores, support liquids and plume desolvation conditions play a crucial role in protein detection by liquid MALDI MS.•Laser threshold energy for analyte ionization is linearly dependent on the surface tension of the liquid MALDI sample.
Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ...ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle.
Wiskott-Aldrich Syndrome protein (WASp) is a key regulator of the Arp2/3 complex and the actin cytoskeleton in hematopoietic
cells. WASp is capable of forming an auto-inhibited conformation, which ...can be disrupted by binding of Cdc42 and phosphatidylinositol
4,5-bisphosphate, leading to its activation. Stimulation of the collagen receptor on platelets and crosslinking the B-cell
receptor induce tyrosine phosphorylation of WASp. Here we show that the Src family kinase Hck induces phosphorylation of WASp-Tyr 291 independently of Cdc42 and that this causes a shift in the mobility of WASp upon SDS-PAGE. A phospho-mimicking mutant, WASp-Y291E,
exhibited an enhanced ability to stimulate actin polymerization in a cell-free system and when microinjected into primary
macrophages induced extensive filopodium formation with greater efficiency than wild-type WASp or a Y291F mutant. We propose
that phosphorylation of Tyr 291 directly regulates WASp function.
Rapid profiling of the biomolecular components of milk can be useful for food quality assessment and for food fraud detection. Differences in commercial value and availability of milk from specific ...species are often the reasons for the illicit and fraudulent sale of milk whose species origin is wrongly declared. In this study, a fast, MS-based speciation method is presented to distinguish sheep from goat milk and sheep colostrum at different phases. Using liquid atmospheric pressure (AP)-matrix-assisted laser desorption/ionisation (MALDI) MS, it was possible to classify samples of goat and sheep milk with 100% accuracy in one minute of data acquisition per sample. Moreover, an accuracy of 98% was achieved in classifying pure sheep milk samples and sheep milk samples containing 10% goat milk. Evaluating colostrum quality and postnatal stages represents another possible application of this technology. Classification of sheep colostrum samples that were collected within 6 hours after parturition and 48 hours later was achieved with an accuracy of 84.4%. Our data show that substantial changes in the lipid profile can account for the accurate classification of colostrum collected at the early and late time points. This method applied to the analysis of protein orthologs of different species can, as in this case, allow unequivocal speciation analysis.
The environment, including animals and animal products, is colonized by bacterial species that are typical and specific of every different ecological niche. Natural and human-related ecological ...pressure promotes the selection and expression of genes related to antimicrobial resistance (AMR). These genes might be present in a bacterial consortium but might not necessarily be expressed. Their expression could be induced by the presence of antimicrobial compounds that could originate from a given ecological niche or from human activity. In this work, we applied (meta)proteomics analysis of bacterial compartment of raw milk in order to obtain a method that provides a measurement of circulating AMR involved proteins and gathers information about the whole bacterial composition. Results from milk analysis revealed the presence of 29 proteins/proteoforms linked to AMR. The detection of mainly β-lactamases suggests the possibility of using the milk microbiome as a bioindicator for the investigation of AMR. Moreover, it was possible to achieve a culture-free qualitative and functional analysis of raw milk bacterial consortia.
With its highly fluctuating ion production matrix-assisted laser desorption/ionization (MALDI) poses many practical challenges for its application in mass spectrometry. Instrument tuning and ...quantitative ion abundance measurements using ion signal alone depend on a stable ion beam. Liquid MALDI matrices have been shown to be a promising alternative to the commonly used solid matrices. Their application in areas where a stable ion current is essential has been discussed but only limited data have been provided to demonstrate their practical use and advantages in the formation of stable MALDI ion beams. In this article we present experimental data showing high MALDI ion beam stability over more than two orders of magnitude at high analytical sensitivity (low femtomole amount prepared) for quantitative peptide abundance measurements and instrument tuning in a MALDI Q-TOF mass spectrometer. Samples were deposited on an inexpensive conductive hydrophobic surface and shrunk to droplets <10 nL in size. By using a sample droplet <10 nL it was possible to acquire data from a single irradiated spot for roughly 10,000 shots with little variation in ion signal intensity at a laser repetition rate of 5–20 Hz.
MS is an important analytical tool in clinical proteomics, primarily in the disease‐specific discovery, identification and characterisation of proteomic biomarkers and patterns. MS‐based proteomics ...is increasingly used in clinical validation and diagnostic method development. The latter departs from the typical application of MS‐based proteomics by exchanging some of the high performance of analysis for the throughput, robustness and simplicity required for clinical diagnostics. Although conventional MS‐based proteomics has become an important field in clinical applications, some of the most recent MS technologies have not yet been extensively applied in clinical proteomics. In this review, we will describe the current state of MS in clinical proteomics and look to the future of this field.
This article contains raw and processed data related to research published by Bryant et al.1. Data was obtained by MS-based proteomics, analysing trichome-enriched, trichome-depleted and whole leaf ...samples taken from the medicinal plant Artemisia annua and searching the acquired MS/MS data against a recently published contig database 2 and other genomic and proteomic sequence databases for comparison. The processed data shows that an order-of-magnitude more proteins have been identified from trichome-enriched Artemisia annua samples in comparison to previously published data. Proteins known to have a role in the biosynthesis of artemisinin and other highly abundant proteins were found which imply additional enzymatically driven processes occurring within the trichomes that are significant for the biosynthesis of artemisinin.
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