RESUMO
Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries. In both cases, the resulting spectra are increasingly complex to assign/interpret, and the key to these new directions of native MS research is the ability to perform native top-down analysis, which allows unambiguous peak assignment. To achieve this, detergent removal is necessary prior to MS analyzers, which allow selection of specific m/z values, representing the parent ion for downstream activation. Here, we describe a novel, enhanced declustering (ED) device installed into the first pumping region of a cyclic IMS-enabled mass spectrometry platform. The device enables declustering of ions prior to the quadrupole by imparting collisional activation through an oscillating electric field applied between two parallel plates. The positioning of the device enables liberation of membrane protein ions from detergent micelles. Quadrupole selection can now be utilized to isolate protein-ligand complexes, and downstream collision cells enable the dissociation and identification of binding partners. We demonstrate that ion mobility (IM) significantly aids in the assignment of top-down spectra, aligning fragments to their corresponding parent ions by means of IM drift time. Using this approach, we were able to confidently assign and identify a novel hit compound against PfMATE, obtained from multiplexed ligand libraries.
Assuntos
Espectrometria de Mobilidade Iônica , Proteínas de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/análise , Espectrometria de Mobilidade Iônica/métodos , Micelas , Espectrometria de Massas/métodos , Detergentes/química , Íons/químicaRESUMO
A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.
RESUMO
Unambiguous identification of distinct proteoforms and their biological functions is a significant analytical challenge due to the many combinations of post-translational modifications (PTM) that generate isomeric proteoforms. Resulting chimeric tandem mass spectra hinder detailed structural characterization of individual proteoforms for mixtures with more than two isomers. Large isomeric peptides and intact isomeric proteins are extremely difficult to distinguish with traditional chromatographic separation methods. Gas-phase ion separation techniques such as ion mobility spectrometry (IMS) methods now offer high resolving power that may enable separation of isomeric biomolecules, such as peptides and proteins. We explored novel high-resolution cyclic ion mobility spectrometry (cIM) combined with an electro-magnetostatic cell for "on-the-fly" electron capture dissociation (ECD) for separation and sequencing of large isomeric peptides. We demonstrate the effectiveness of this approach on ternary mixtures of mono- and trimethylated isomers of histone H3 N-tails (â¼5.4 kDa), achieving a complete separation of these isomers with an average resolving power of â¼400 and a resolution of 1.5 and with nearly 100% amino acid sequence coverage. Our results demonstrate the potential of the cIM-MS/MS(ECD) technology to enhance middle-down and top-down proteomics workflows, thereby facilitating the identification of near-identical proteoforms with essential biological functions in complex mixtures.
Assuntos
Elétrons , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Peptídeos/análise , Histonas/química , Sequência de AminoácidosRESUMO
A novel mass spectrometry system is described here comprising a quadrupole-multireflecting time-of-flight design. The new multireflecting time-of-flight analyzer has an effective path length of 48 m and employs planar, gridless ion mirrors providing fourth-order energy focusing resulting in resolving power over 200â¯000 fwhm and sub-ppm mass accuracy. We show how these attributes are maintained with relatively fast acquisition speeds, setting the system apart from other high resolution mass spectrometers. We have integrated this new system into both liquid chromatography-mass spectrometry and mass spectrometry imaging workflows to demonstrate how the instrument characteristics are of benefit to these applications.
RESUMO
Tandem mass spectrometry of denatured, multiply charged high mass protein precursor ions yield extremely dense spectra with hundreds of broad and overlapping product ion isotopic distributions of differing charge states that yield an elevated baseline of unresolved "noise" centered about the precursor ion. Development of mass analyzers and signal processing methods to increase mass resolving power and manipulation of precursor and product ion charge through solution additives or ion-ion reactions have been thoroughly explored as solutions to spectral congestion. Here, we demonstrate the utility of electron capture dissociation (ECD) coupled with high-resolution cyclic ion mobility spectrometry (cIMS) to greatly increase top-down protein characterization capabilities. Congestion of protein ECD spectra was reduced using cIMS of the ECD product ions and "mobility fractions", that is, extracted mass spectra for segments of the 2D mobiligram (m/z versus drift time). For small proteins, such as ubiquitin (8.6 kDa), where mass resolving power was not the limiting factor for characterization, pre-IMS ECD and mobility fractions did not significantly increase protein sequence coverage, but an increase in the number of identified product ions was observed. However, a dramatic increase in performance, measured by protein sequence coverage, was observed for larger and more highly charged species, such as the +35 charge state of carbonic anhydrase (29 kDa). Pre-IMS ECD combined with mobility fractions yielded a 135% increase in the number of annotated isotope clusters and a 75% increase in unique product ions compared to processing without using the IMS dimension. These results yielded 89% sequence coverage for carbonic anhydrase.
Assuntos
Elétrons , Espectrometria de Mobilidade Iônica , Sequência de Aminoácidos , Proteínas/análise , Espectrometria de Massas em Tandem/métodosRESUMO
Cancer is a disease of cellular evolution where single base changes in the genetic code can have significant impact on the translation of proteins and their activity. Thus, in cancer research there is significant interest in methods that can determine mutations and identify the significant binding sites (epitopes) of antibodies to proteins in order to develop novel therapies. Nano molecularly imprinted polymers (nanoMIPs) provide an alternative to antibodies as reagents capable of specifically capturing target molecules depending on their structure. In this study, we used nanoMIPs to capture KRAS, a critical oncogene, to identify mutations which when present are indicative of oncological progress. Herein, coupling nanoMIPs (capture) and liquid chromatography-mass spectrometry (detection), LC-MS has allowed us to investigate mutational assignment and epitope discovery. Specifically, we have shown epitope discovery by generating nanoMIPs to a recombinant KRAS protein and identifying three regions of the protein which have been previously assigned as epitopes using much more time-consuming protocols. The mutation status of the released tryptic peptide was identified by LC-MS following capture of the conserved region of KRAS using nanoMIPS, which were tryptically digested, thus releasing the sequence of a non-conserved (mutated) region. This approach was tested in cell lines where we showed the effective genotyping of a KRAS cell line and in the plasma of cancer patients, thus demonstrating its ability to diagnose precisely the mutational status of a patient. This work provides a clear line-of-sight for the use of nanoMIPs to its translation from research into diagnostic and clinical utility.
Assuntos
Impressão Molecular , Nanopartículas , Humanos , Espectrometria de Massas , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
We describe the implementation of a simple three-electrode surface-induced dissociation (SID) cell on a cyclic ion mobility spectrometer (cIMS) and demonstrate the utility of multipass mobility separations for resolving multiple conformations of protein complexes generated during collision-induced and surface-induced unfolding (CIU & SIU) experiments. In addition to CIU and SIU, SID of protein complexes is readily accomplished within the native instrument software and with no additional external power supplies by entering a single SID collision energy, a simplification in user experience compared to prior implementations. A set of cyclic homomeric protein complexes and a heterohexamer with known CID and SID behavior were analyzed to investigate mass and mobility resolution improvements, the latter of which improved by 20-50% (median: 33%) compared to a linear travelling wave device. Multiple passes of intact complexes, or their SID fragments, increased the mobility resolution by an average of 15% per pass, with the racetrack effect being observed after â¼3 or 4 passes, depending on the drift time spread of the analytes. Even with modest improvements to apparent mobility resolving power, multipass experiments were particularly useful for separating conformations produced from CIU and SIU experiments. We illustrate several examples where either (1) multipass experiments revealed multiple overlapping conformations previously unobserved or obscured due to limited mobility resolution, or (2) CIU or SIU conformations that appeared 'native' in a single pass experiment were actually slightly compacted or expanded, with the change only being measurable through multipass experiments. The work conducted here, the first utilization of multipass cyclic ion mobility for CIU, SIU, and SID of protein assemblies and a demonstration of a wholly integrated SIU/SID workflow, paves the way for widespread adoption of SID technology for native mass spectrometry and also improves our understanding of gas-phase protein complex CIU and SIU conformationomes.
Assuntos
Proteínas , Software , Espectrometria de MassasRESUMO
INTRODUCTION: Preterm birth (PTB) is defined as birth occurring before 37 weeks' gestation, affects 5-9% of all pregnancies in developed countries, and is the leading cause of perinatal mortality. Spontaneous preterm birth (sPTB) accounts for 31-50% of all PTB, but the underlying pathophysiology is poorly understood. OBJECTIVE: This study aimed to decipher the lipidomics pathways involved in pathophysiology of sPTB. METHODS: Blood samples were taken from SCreening fOr Pregnancy Endpoints (SCOPE), an international study that recruited 5628 nulliparous women, with a singleton low-risk pregnancy. Our analysis focused on plasma from SCOPE in Cork. Discovery profiling of the samples was undertaken using liquid chromatography-mass spectrometry Lipidomics, and features significantly altered between sPTB (n = 16) and Control (n = 32) groups were identified using empirical Bayes testing, adjusting for multiple comparisons. RESULTS: Twenty-six lipids showed lower levels in plasma of sPTB compared to controls (adjusted p < 0.05), including 20 glycerophospholipids (12 phosphatidylcholines, 7 phosphatidylethanolamines, 1 phosphatidylinositol) and 6 sphingolipids (2 ceramides and 4 sphingomyelines). In addition, a diaglyceride, DG (34:4), was detected in higher levels in sPTB compared to controls. CONCLUSIONS: We report reduced levels of plasma phospholipids in sPTB. Phospholipid integrity is linked to biological membrane stability and inflammation, while storage and breakdown of lipids have previously been implicated in pregnancy complications. The contribution of phospholipids to sPTB as a cause or effect is still unclear; however, our results of differential plasma phospholipid expression represent another step in advancing our understanding of the aetiology of sPTB. Further work is needed to validate these findings in independent pregnancy cohorts.
Assuntos
Lipidômica , Fosfolipídeos/metabolismo , Nascimento Prematuro/metabolismo , Adulto , Teorema de Bayes , Estudos de Coortes , Feminino , Humanos , Recém-Nascido , Fosfolipídeos/sangue , Gravidez , Nascimento Prematuro/sangue , Fatores de RiscoRESUMO
RATIONALE: There is a considerable clinical demand to determine key mutations in genes involved with cancer which necessitates the deployment of highly specific and robust analytical methods. Multiplex liquid chromatography with selected reaction monitoring (LC/SRM) assays offer the ability to achieve quantitation down to levels expected to be present in clinical samples. Ion mobility mass spectrometry (IMS/MS) assays can provide increased peak capacity and hence separation in an extremely short time frame, and in addition provide physicochemical data regarding the collision cross-section of an analyte which can be used in conjunction with the m/z value of an ion to increase detection specificity. METHODS: For LC/SRM, unlabelled peptides and corresponding stable-isotope-labelled standards were spiked into digested human plasma and analysed using ultrahigh-performance liquid chromatography (UHPLC) coupled to a triple quadrupole mass spectrometer to enable the generation of analyte-specific calibration lines. Synthetic unlabelled peptides were infused into a Synapt G2 mass spectrometer for travelling wave ion mobility separation and TW CCSN2 values were derived from comparison with previously generated TW CCSN2 calibration values. RESULTS: Linear calibration lines (0.125 to 25 fmol/µL) were established for each of the KRAS peptides. UHPLC separated the peptides and hence enabled them to be split into different retention time functions/windows. This separation enabled detection of three or four transitions for each light and heavy peptide with at least 10 points per peak for accurate quantitation. All six KRAS G12 peptides were separated using IMS/MS, enabling precise TW CCSN2 values to be determined. Although some of the G12 peptides chromatographically co-eluted, all the peptides were distinguished by m/z, retention time and/or drift time. CONCLUSIONS: This study advocates that LC/SRM and IMS/MS could both be used to identify single amino acid substitutions in KRAS as an alternative to commonly used methods such as circulating tumour DNA analysis.
Assuntos
Espectrometria de Massas/métodos , Mutação , Proteínas Proto-Oncogênicas p21(ras) , Substituição de Aminoácidos , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Mobilidade Iônica , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/sangue , Proteínas Proto-Oncogênicas p21(ras)/químicaRESUMO
Matrix-Assisted Laser Desorption Ionization (MALDI) and Desorption Electrospray Ionization (DESI) are two complementary ionization techniques that have transformed the field of biomolecular analysis, enabling the measurement of a wide range of biomolecules by mass spectrometry. These techniques have also been applied to imaging mass spectrometry where the spatial localization of molecules is determined. Coupling this with Ion Mobility Spectrometry (IM) allows an additional level of separation and specificity to be obtained. Here, we describe the coupling of the technologies and the practical advantages of these combinations, highlighting specific examples.
Assuntos
Espectrometria de Mobilidade Iônica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Mobilidade Iônica/métodos , Lipídeos/análise , Peptídeos/análise , Polissacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The fucosylation of glycans leads to diverse structures and is associated with many biological and disease processes. The exact determination of fucoside positions by tandem mass spectrometry (MS/MS) is complicated because rearrangements in the gas phase lead to erroneous structural assignments. Here, we demonstrate that the combined use of ion-mobility MS and well-defined synthetic glycan standards can prevent misinterpretation of MS/MS spectra and incorrect structural assignments of fucosylated glycans. We show that fucosyl residues do not migrate to hydroxyl groups but to acetamido moieties of N-acetylneuraminic acid as well as N-acetylglucosamine residues and nucleophilic sites of an anomeric tag, yielding specific isomeric fragment ions. This mechanistic insight enables the characterization of unique IMS arrival-time distributions of the isomers which can be used to accurately determine fucosyl positions in glycans.
Assuntos
Fucose/química , Polissacarídeos/química , Bibliotecas de Moléculas Pequenas/química , Acetilglucosamina/química , Gases/química , Íons/química , Isomerismo , Espectrometria de Massas , Estrutura Molecular , Ácido N-Acetilneuramínico/químicaRESUMO
A comprehensive Collision Cross Section (CCS) library was obtained via Travelling Wave Ion Guide mobility measurements through direct infusion (DI). The library consists of CCS and Mass Spectral (MS) data in negative and positive ElectroSpray Ionisation (ESI) mode for 463 and 479 endogenous metabolites, respectively. For both ionisation modes combined, TWCCSN2 data were obtained for 542 non-redundant metabolites. These data were acquired on two different ion mobility enabled orthogonal acceleration QToF MS systems in two different laboratories, with the majority of the resulting TWCCSN2 values (from detected compounds) found to be within 1% of one another. Validation of these results against two independent, external TWCCSN2 data sources and predicted TWCCSN2 values indicated to be within 1-2% of these other values. The same metabolites were then analysed using a rapid reversed-phase ultra (high) performance liquid chromatographic (U(H)PLC) separation combined with IM and MS (IM-MS) thus providing retention time (tr), m/z and TWCCSN2 values (with the latter compared with the DI-IM-MS data). Analytes for which TWCCSN2 values were obtained by U(H)PLC-IM-MS showed good agreement with the results obtained from DI-IM-MS. The repeatability of the TWCCSN2 values obtained for these metabolites on the different ion mobility QToF systems, using either DI or LC, encouraged the further evaluation of the U(H)PLC-IM-MS approach via the analysis of samples of rat urine, from control and methotrexate-treated animals, in order to assess the potential of the approach for metabolite identification and profiling in metabolic phenotyping studies. Based on the database derived from the standards 63 metabolites were identified in rat urine, using positive ESI, based on the combination of tr, TWCCSN2 and MS data.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Metaboloma , Urina/química , Aminas/análise , Animais , Calibragem , Aprendizado de Máquina , Ratos , Padrões de ReferênciaRESUMO
Stress adaptation is critical for the survival of microbes in dynamic environments, and in particular, for fungal pathogens to survive in and colonise host niches. Proteomic analyses have the potential to significantly enhance our understanding of these adaptive responses by providing insight into post-transcriptional regulatory mechanisms that contribute to the outputs, as well as testing presumptions about the regulation of protein levels based on transcript profiling. Here, we used label-free, quantitative mass spectrometry to re-examine the response of the major fungal pathogen of humans, Candida albicans, to osmotic stress. Of the 1,262 proteins that were identified, 84 were down-regulated in response to 1M NaCl, reflecting the decrease in ribosome biogenesis and translation that often accompanies stress. The 64 up-regulated proteins included central metabolic enzymes required for glycerol synthesis, a key osmolyte for this yeast, as well as proteins with functions during stress. These data reinforce the view that adaptation to salt stress involves a transient reduction in ribosome biogenesis and translation together with the accumulation of the osmolyte, glycerol. The specificity of the response to salt stress is highlighted by the small proportion of quantified C. albicans proteins (5%) whose relative elevated abundances were statistically significant.
Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica , Pressão Osmótica , Proteômica , Candida albicans/genética , Proteínas Fúngicas/genética , HumanosRESUMO
Mass Spectrometry Imaging (MSI) holds significant promise in augmenting digital histopathologic analysis by generating highly robust big data about the metabolic, lipidomic and proteomic molecular content of the samples. In the process, a vast quantity of unrefined data, that can amount to several hundred gigabytes per tissue section, is produced. Managing, analysing and interpreting this data is a significant challenge and represents a major barrier to the translational application of MSI. Existing data analysis solutions for MSI rely on a set of heterogeneous bioinformatics packages that are not scalable for the reproducible processing of large-scale (hundreds to thousands) biological sample sets. Here, we present a computational platform (pyBASIS) capable of optimized and scalable processing of MSI data for improved information recovery and comparative analysis across tissue specimens using machine learning and related pattern recognition approaches. The proposed solution also provides a means of seamlessly integrating experimental laboratory data with downstream bioinformatics interpretation/analyses, resulting in a truly integrated system for translational MSI.
Assuntos
Biologia Computacional/métodos , Histocitoquímica/métodos , Processamento de Imagem Assistida por Computador/métodos , Espectrometria de Massas/métodos , Aprendizado de Máquina , Metabolômica/métodos , Reconhecimento Automatizado de Padrão , Proteômica/métodosRESUMO
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.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Escherichia coli/química , Proteoma/isolamento & purificação , Proteômica/métodos , Sequência de Aminoácidos , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Misturas Complexas/química , Células HeLa , Humanos , Células K562 , Proteólise , Proteômica/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Peanut is an important food allergen, but it cannot currently be reliably detected and quantified in processed foods at low levels. A level of 3 mg protein/kg is increasingly being used as a reference dose above which precautionary allergen labeling is applied to food products. Two exemplar matrices (chocolate dessert and chocolate bar) were prepared and incurred with 0, 3, 10, or 50 mg/kg peanut protein using a commercially available lightly roasted peanut flour ingredient. After simple buffer extraction employing an acid-labile detergent, multiple reaction monitoring (MRM) experiments were used to assess matrix effects on the detection of a set of seven peptide targets derived from peanut allergens using either conventional or microfluidic chromatographic separation prior to mass spectrometry. Microfluidic separation provided greater sensitivity and increased ionization efficiency at low levels. Individual monitored transitions were detected in consistent ratios across the dilution series, independent of matrix. The peanut protein content of each sample was then determined using ELISA and the optimized MRM method. Although other peptide targets were detected with three transitions at the 50 mg/kg peanut protein level in both matrices, only Arah2(Q6PSU2)147-155 could be quantified reliably and only in the chocolate dessert at 10 mg/kg peanut protein. Recoveries were consistent with ELISA analysis returning around 30-50% of the incurred dose. MS coupled with microfluidic separation shows great promise as a complementary analytical tool for allergen detection and quantification in complex foods using a simple extraction methodology.
Assuntos
Alérgenos/análise , Arachis/imunologia , Espectrometria de Massas/métodos , Microfluídica/métodos , Arachis/química , Análise de Alimentos/métodos , Hipersensibilidade a Amendoim/etiologia , Proteínas de Plantas/análise , Proteínas de Plantas/imunologiaRESUMO
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.
Assuntos
Aspergillus fumigatus/efeitos dos fármacos , Calcineurina/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Proteínas Ribossômicas/isolamento & purificação , Voriconazol/farmacologia , Antifúngicos/farmacologia , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Caspofungina , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Cromatografia Líquida/métodos , Equinocandinas/farmacologia , Ergosterol/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Ontologia Genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação , Lipopeptídeos/farmacologia , Micafungina , Anotação de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The need for rapid and efficient high throughput metabolic phenotyping (metabotyping) in metabolomic/metabonomic studies often requires compromises to be made between analytical speed and metabolome coverage. Here the effect of column length (150, 75 and 30 mm) and gradient duration (15, 7.5 and 3 min respectively) on the number of features detected when untargeted metabolic profiling of human urine using reversed-phase gradient ultra performance chromatography with, and without, ion mobility spectrometry, has been examined. As would be expected, reducing column length from 150 to 30 mm, and gradient duration, from 15 to 3 min, resulted in a reduction in peak capacity from 311 to 63 and a similar reduction in the number of features detected from over ca. 16,000 to ca. 6500. Under the same chromatographic conditions employing UPLC/IMS/MS to provide an additional orthogonal separation resulted in an increase in the number of MS features detected to nearly 20,000 and ca. 7500 for the 150 mm and the 30 mm columns respectively. Based on this limited study the potential of LC/IMS/MS as a tool for improving throughput and increasing metabolome coverage clearly merits further in depth study.
Assuntos
Cromatografia Líquida de Alta Pressão , Espectrometria de Mobilidade Iônica , Metaboloma , Urina/química , HumanosRESUMO
RATIONALE: A novel data-independent acquisition method is detailed that incorporates a scanning quadrupole in front of an orthogonal acceleration time-of-flight (TOF) mass analyser. This approach is described and the attributes are compared and contrasted to other DIA approaches. METHODS: Specific application of the method to both targeted and untargeted lipidomic identification strategies is discussed, with data from both shotgun and LC separated lipidomics experiments presented. RESULTS: The benefits of the fast quadrupole scanning technique are highlighted, and include improvements in speed and specificity for complex mixtures providing high quality qualitative and quantitative data. CONCLUSIONS: In particular the high specificity afforded by the scanning quadrupole improves qualitative information for lipid identification.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Lipídeos/análise , Espectrometria de Massas/métodos , Animais , Bovinos , Masculino , Metabolômica/métodos , Camundongos , Miocárdio/química , Testículo/químicaRESUMO
The unique physiochemical properties of wheat gluten enable a diverse range of food products to be manufactured. However, gluten triggers coeliac disease, a condition which is treated using a gluten-free diet. Analytical methods are required to confirm if foods are gluten-free, but current immunoassay-based methods can unreliable and proteomic methods offer an alternative but require comprehensive and well annotated sequence databases which are lacking for gluten. A manually a curated database (GluPro V1.0) of gluten proteins, comprising 630 discrete unique full length protein sequences has been compiled. It is representative of the different types of gliadin and glutenin components found in gluten. An in silico comparison of their coeliac toxicity was undertaken by analysing the distribution of coeliac toxic motifs. This demonstrated that whilst the α-gliadin proteins contained more toxic motifs, these were distributed across all gluten protein sub-types. Comparison of annotations observed using a discovery proteomics dataset acquired using ion mobility MS/MS showed that more reliable identifications were obtained using the GluPro V1.0 database compared to the complete reviewed Viridiplantae database. This highlights the value of a curated sequence database specifically designed to support the proteomic workflows and the development of methods to detect and quantify gluten. SIGNIFICANCE: We have constructed the first manually curated open-source wheat gluten protein sequence database (GluPro V1.0) in a FASTA format to support the application of proteomic methods for gluten protein detection and quantification. We have also analysed the manually verified sequences to give the first comprehensive overview of the distribution of sequences able to elicit a reaction in coeliac disease, the prevalent form of gluten intolerance. Provision of this database will improve the reliability of gluten protein identification by proteomic analysis, and aid the development of targeted mass spectrometry methods in line with Codex Alimentarius Commission requirements for foods designed to meet the needs of gluten intolerant individuals.