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1.
J Proteome Res ; 22(12): 3754-3772, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37939282

RESUMO

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 Zr4+ immobilized metal-ion affinity chromatography (IMAC) and TiO2 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.


Assuntos
Fosfopeptídeos , Tirosina , Humanos , Fosfopeptídeos/análise , Células HEK293 , Fluxo de Trabalho , Tirosina/metabolismo , Proteínas , Fosfotirosina
2.
EMBO J ; 38(21): e100847, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31433507

RESUMO

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.


Assuntos
Ânions/química , Cromatografia por Troca Iônica/métodos , Fosfopeptídeos/análise , Fosfoproteínas/análise , Proteoma/análise , Biologia Computacional , Células HeLa , Humanos , Espectrometria de Massas , Fosforilação
3.
Rapid Commun Mass Spectrom ; 38 Suppl 1: e9523, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37070167

RESUMO

Schistosomes are blood flukes with specialised tissues and organs, each one playing a pivotal role in perpetuating the parasite life cycle. Herein, we describe a detailed methodology for preserving the proteome of adult Schistosoma mansoni worms during manual dissection for enrichment of tissues associated with the parasite's alimentary tract. We provide step-by-step directions for specimen storage and dissection while in preservative solution, tissue homogenisation, protein extraction and digestion using a methodology fully compatible with downstream quantitative liquid chromatography-mass spectrometry analysis. Our methodology uses label-free and QconCAT-based absolute quantification for detection of S. mansoni oesophageal gland products proposed as vaccine candidates. Through stabilisation of the proteome and minimising sample degradation during dissection our approach has allowed us to access the hidden proteome of target tissues not readily available from total lysates because of their small volume. This protocol can be replicated or adapted to other Schistosoma species lacking quantitative proteomics characterisation of specialised tissues for discovery of proteins with potential diagnostic and therapeutic utility.


Assuntos
Espectrometria de Massa com Cromatografia Líquida , Proteômica , Animais , Proteômica/métodos , Cromatografia Líquida , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Schistosoma mansoni/química , Schistosoma mansoni/metabolismo
4.
Biochem J ; 479(19): 2063-2086, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36240066

RESUMO

Previously, we discovered that deletion of c-Rel in the Eµ-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-κB subunit in B-cell malignancies. Here we report that Eµ-Myc/cRel-/- cells have an unexpected and major defect in the CHK1 pathway. Total and phospho proteomic analysis revealed that Eµ-Myc/cRel-/- lymphomas highly resemble wild-type (WT) Eµ-Myc lymphomas treated with an acute dose of the CHK1 inhibitor (CHK1i) CCT244747. Further analysis demonstrated that this is a consequence of Eµ-Myc/cRel-/- lymphomas having lost expression of CHK1 protein itself, an effect that also results in resistance to CCT244747 treatment in vivo. Similar down-regulation of CHK1 protein levels was also seen in CHK1i resistant U2OS osteosarcoma and Huh7 hepatocellular carcinoma cells. Further investigation revealed that the deubiquitinase USP1 regulates CHK1 proteolytic degradation and that its down-regulation in our model systems is responsible, at least in part, for these effects. We demonstrate that treating WT Eµ-Myc lymphoma cells with the USP1 inhibitor ML323 was highly effective at reducing tumour burden in vivo. Targeting USP1 activity may thus be an alternative therapeutic strategy in MYC-driven tumours.


Assuntos
Linfoma , Proteínas Proto-Oncogênicas c-myc , Aminopiridinas , Animais , Enzimas Desubiquitinantes , Linfoma/metabolismo , Linfoma/patologia , Camundongos , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirimidinas
5.
Biochem J ; 479(19): 2131-2151, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36240067

RESUMO

The development of resistance and the activation of bypass pathway signalling represents a major problem for the clinical application of protein kinase inhibitors. While investigating the effect of either a c-Rel deletion or RelAT505A phosphosite knockin on the Eµ-Myc mouse model of B-cell lymphoma, we discovered that both NF-κB subunit mutations resulted in CHK1 inhibitor resistance, arising from either loss or alteration of CHK1 activity, respectively. However, since Eµ-Myc lymphomas depend on CHK1 activity to cope with high levels of DNA replication stress and consequent genomic instability, it was not clear how these mutant NF-κB subunit lymphomas were able to survive. To understand these survival mechanisms and to identify potential compensatory bypass signalling pathways in these lymphomas, we applied a multi-omics strategy. With c-Rel-/- Eµ-Myc lymphomas we observed high levels of Phosphatidyl-inositol 3-kinase (PI3K) and AKT pathway activation. Moreover, treatment with the PI3K inhibitor Pictilisib (GDC-0941) selectively inhibited the growth of reimplanted c-Rel-/- and RelAT505A, but not wild type (WT) Eµ-Myc lymphomas. We also observed up-regulation of a RHO/RAC pathway gene expression signature in both Eµ-Myc NF-κB subunit mutation models. Further investigation demonstrated activation of the RHO/RAC effector p21-activated kinase (PAK) 2. Here, the PAK inhibitor, PF-3758309 successfully overcame resistance of RelAT505A but not WT lymphomas. These findings demonstrate that up-regulation of multiple bypass pathways occurs in CHK1 inhibitor resistant Eµ-Myc lymphomas. Consequently, drugs targeting these pathways could potentially be used as either second line or combinatorial therapies to aid the successful clinical application of CHK1 inhibitors.


Assuntos
Linfoma , Fosfatidilinositol 3-Quinases , Animais , Inositol , Linfoma/tratamento farmacológico , Linfoma/genética , Linfoma/metabolismo , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Regulação para Cima , Quinases Ativadas por p21/genética
6.
Proc Natl Acad Sci U S A ; 117(44): 27465-27473, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33077605

RESUMO

Mating plugs are produced by many sexually reproducing animals and are hypothesized to promote male fertilization success under promiscuous mating. However, tests of this hypothesis have been constrained by an inability to discriminate ejaculates of different males in direct competition. Here, we use stable isotope labeling in vivo and proteomics to achieve this in a promiscuous rodent, Myodes glareolus We show that, although the first male's plug is usually dislodged, it can be retained throughout the second male's copulation. Retained plugs did not completely block rival sperm but did significantly limit their numbers. Differences in the number of each male's sperm progressing through the female reproductive tract were also explained by natural variation in the size of mating plugs and reproductive accessory glands from which major plug proteins originate. Relative sperm numbers in turn predicted the relative fertilization success of rival males. Our application of stable isotopes to label ejaculates resolves a longstanding debate by revealing how rodent mating plugs promote fertilization success under competitive conditions. This approach opens new opportunities to reveal cryptic mechanisms of postcopulatory sexual selection among diverse animal taxa.


Assuntos
Arvicolinae/fisiologia , Copulação/fisiologia , Proteínas de Plasma Seminal/metabolismo , Seleção Sexual/fisiologia , Transporte Espermático/fisiologia , Animais , Feminino , Masculino , Preferência de Acasalamento Animal , Proteômica , Glândulas Seminais/metabolismo , Contagem de Espermatozoides , Motilidade dos Espermatozoides
7.
PLoS Biol ; 17(1): e3000059, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30645593

RESUMO

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.


Assuntos
Infecções por Salmonella/genética , Salmonella typhimurium/genética , Animais , Gastroenterite/microbiologia , Perfilação da Expressão Gênica/métodos , Variação Genética/genética , Humanos , Macrófagos , Camundongos , Infecções por Salmonella/microbiologia , Virulência
8.
Biochem J ; 478(24): 4153-4167, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34661239

RESUMO

Dietary restriction (DR) has been shown to increase lifespan in organisms ranging from yeast to mammals. This suggests that the underlying mechanisms may be evolutionarily conserved. Indeed, upstream signalling pathways, such as TOR, are strongly linked to DR-induced longevity in various organisms. However, the downstream effector proteins that ultimately mediate lifespan extension are less clear. To shed light on this, we used a proteomic approach on budding yeast. Our reasoning was that analysis of proteome-wide changes in response to DR might enable the identification of proteins that mediate its physiological effects, including replicative lifespan extension. Of over 2500 proteins we identified by liquid chromatography-mass spectrometry, 183 were significantly altered in expression by at least 3-fold in response to DR. Most of these proteins were mitochondrial and/or had clear links to respiration and metabolism. Indeed, direct analysis of oxygen consumption confirmed that mitochondrial respiration was increased several-fold in response to DR. In addition, several key proteins involved in mating, including Ste2 and Ste6, were down-regulated by DR. Consistent with this, shmoo formation in response to α-factor pheromone was reduced by DR, thus confirming the inhibitory effect of DR on yeast mating. Finally, we found that Hsp26, a member of the conserved small heat shock protein (sHSP) family, was up-regulated by DR and that overexpression of Hsp26 extended yeast replicative lifespan. As overexpression of sHSPs in Caenorhabditis elegans and Drosophila has previously been shown to extend lifespan, our data on yeast Hsp26 suggest that sHSPs may be universally conserved effectors of longevity.


Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteoma
9.
Biochem J ; 478(3): 533-551, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33438746

RESUMO

Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3ε binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA.


Assuntos
Dano ao DNA , Processamento de Proteína Pós-Traducional , Fator de Transcrição RelA/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Cromatografia Líquida , Sequência Consenso , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Etoposídeo/farmacologia , Humanos , Hidroxiureia/farmacologia , Osteossarcoma/patologia , Fosforilação , Mapas de Interação de Proteínas , Proteínas Quinases/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem , Fatores de Tempo
10.
Biochem J ; 477(13): 2451-2475, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32501498

RESUMO

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely 'non-canonical' substrates. Surprisingly, sequence interrogation of ∼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/farmacologia , Sulfonas/farmacologia , Linhagem Celular Tumoral , Citometria de Fluxo , Fluorometria , Humanos , Imunoprecipitação , Leupeptinas/farmacologia , Microscopia de Fluorescência , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Espectrometria de Massas em Tandem
11.
J Proteome Res ; 19(1): 314-326, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31729880

RESUMO

Schistosomes are blood-dwelling helminth parasites that cause schistosomiasis, a debilitating disease resulting in inflammation and, in extreme cases, multiple organ damage. Major challenges to control the transmission persist, and the discovery of protective antigens remains of critical importance for vaccine development. Rhesus macaques can self-cure following schistosome infection, generating antibodies that target proteins from the tegument, gut, and esophagus, the last of which is the least investigated. We developed a dissection technique that permitted increased sensitivity in a comparative proteomics profiling of schistosome esophagus and gut. Proteome analysis of the male schistosome esophagus identified 13 proteins encoded by microexon genes (MEGs), 11 of which were uniquely located in the esophageal glands. Based on this and transcriptome information, a QconCAT was designed for the absolute quantification of selected targets. MEGs 12, 4.2, and 4.1 and venom allergen-like protein 7 were the most abundant, spanning over 245 million to 6 million copies per cell, while aspartyl protease, palmitoyl thioesterase, and galactosyl transferase were present at <1 million copies. Antigenic variation by alternative splicing of MEG proteins was confirmed together with a specialized machinery for protein glycosylation/secretion in the esophagus. Moreover, some gastrodermal secretions were highly enriched in the gut, while others were more uniformly distributed throughout the parasite, potentially indicating lysosomal activity. Collectively, our findings provide a more rational, better-oriented selection of schistosome vaccine candidates in the context of a proven model of protective immunity.


Assuntos
Trato Gastrointestinal/metabolismo , Proteínas de Helminto/metabolismo , Proteômica/métodos , Schistosoma mansoni/metabolismo , Animais , Esôfago/metabolismo , Ontologia Genética , Proteínas de Helminto/análise , Proteínas de Helminto/genética , Masculino , Camundongos , Schistosoma mansoni/patogenicidade , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
12.
J Proteome Res ; 19(9): 3779-3791, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32538093

RESUMO

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.


Assuntos
Resinas Acrílicas , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas
13.
Mol Cell Proteomics ; 15(4): 1309-22, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26750110

RESUMO

Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800Saccharomyces cerevisiaeproteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ∼100 million molecules-per-cell, a median of ∼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a "gold-standard" reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies.


Assuntos
Espectrometria de Massas/métodos , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Perfilação da Expressão Gênica/métodos , Marcação por Isótopo , Modelos Lineares , Espectrometria de Massas/normas , Proteômica/normas , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência de RNA/métodos
15.
J Proteome Res ; 16(9): 3448-3459, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28741359

RESUMO

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.


Assuntos
Espectrometria de Massas/métodos , Osteoblastos/metabolismo , Fragmentos de Peptídeos/análise , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Algoritmos , Benchmarking , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas/instrumentação , Osteoblastos/citologia , Fosfoproteínas/química , Fosforilação , Software
16.
Biochem J ; 473(19): 3159-75, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27444646

RESUMO

cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility-mass spectrometry (IM-MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R2C2 holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a 'kinase-dead' PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM-MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fluorometria/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Sondas Moleculares , Fosforilação
17.
BMC Biol ; 13: 87, 2015 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-26507392

RESUMO

BACKGROUND: Ejaculates contain a diverse mixture of sperm and seminal fluid proteins, the combination of which is crucial to male reproductive success under competitive conditions. Males should therefore tailor the production of different ejaculate components according to their social environment, with particular sensitivity to cues of sperm competition risk (i.e. how likely it is that females will mate promiscuously). Here we test this hypothesis using an established vertebrate model system, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our study tests for the first time how both sperm and seminal fluid components of the ejaculate are tailored to the social environment. RESULTS: Our quantitative proteomics analysis reveals that the relative production of different proteins found in seminal fluid--i.e. seminal fluid proteome composition--differs significantly according to cues of sperm competition risk. Using a conservative analytical approach to identify differential expression of individual seminal fluid components, at least seven of 31 secreted seminal fluid proteins examined showed consistent differences in relative abundance under high versus low sperm competition conditions. Notably three important proteins with potential roles in sperm competition--SVS 6, SVS 5 and CEACAM 10--were more abundant in the high competition treatment groups. Total investment in both sperm and seminal fluid production also increased with cues of heightened sperm competition risk in the social environment. By contrast, relative investment in different ejaculate components was unaffected by cues of mating opportunities. CONCLUSIONS: Our study reveals significant plasticity in different ejaculate components, with the production of both sperm and non-sperm fractions of the ejaculate strongly influenced by the social environment. Sperm competition risk is thus shown to be a key factor in male ejaculate production decisions, including driving plasticity in seminal fluid composition.


Assuntos
Camundongos/fisiologia , Proteoma , Sêmen/fisiologia , Comportamento Sexual Animal , Meio Social , Espermatozoides/fisiologia , Animais , Comportamento Competitivo , Masculino
18.
J Proteome Res ; 13(11): 5008-21, 2014 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-25181601

RESUMO

The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054.


Assuntos
Envelhecimento/metabolismo , Cisteína/química , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Proteômica/métodos , Acetilação , Aconitato Hidratase/análise , Aconitato Hidratase/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Chaperona BiP do Retículo Endoplasmático , Frutose-Bifosfato Aldolase/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Musculares/análise , Músculo Esquelético/metabolismo , Oxirredução , Estresse Oxidativo , Espectrometria de Massas em Tandem/métodos
19.
J Proteome Res ; 13(12): 5635-47, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25250876

RESUMO

The aim of this study was the development of a quantitative assay that could support future studies of a panel of acute phase proteins (APPs) in the horse. The assay was based on a quantification concatamer (QconCAT) coupled to selected reaction monitoring methodology. Thirty-two peptides, corresponding to 13 putative or confirmed APPs for the Equus caballus (equine) species were selected for the design of a QconCAT construct. The gene encoding the QconCAT was synthesized and expressed as an isotope-labeled chimaeric protein in Escherichia coli. The QconCAT tryptic peptides were analyzed on a triple-quadrupole instrument, and the quantotypic properties were assessed in equine serum, wound tissue, and wound interstitial fluid. Reasonable quantotypic performance was found for 12, 14, and 14 peptides in serum, wound tissue, and interstitial fluid, respectively. Seven proteins were quantified in absolute terms in serum collected from a horse before and after the onset of a systemic inflammatory condition, and the observed protein concentrations were in close agreement with previous data. We conclude, that this QconCAT is applicable for concurrent quantitative analysis of multiple APPs in serum and may also support future studies of these proteins in other types of tissues and body fluids from the horse.


Assuntos
Proteínas de Fase Aguda/metabolismo , Sequência de Aminoácidos , Animais , Calibragem , Cavalos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/normas , Cicatrização
20.
bioRxiv ; 2024 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-39345498

RESUMO

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria. This self-assembling proteinaceous organelle encapsulates the key CO2-fixing enzymes, Rubisco and carbonic anhydrase, using a polyhedral shell constructed by hundreds of shell protein paralogs. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding the formation process and overall functionality of carboxysomes. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within ß-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 that were grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the ß-carboxysome lumen. We also identified the binding of Rubisco with the scaffolding protein CcmM in ß-carboxysomes, which is instrumental for Rubisco encapsulation and ß-carboxysome assembly. Using QconCAT-based quantitative mass spectrometry, we further determined the absolute stoichiometric composition of the entire ß-carboxysome. This study and recent findings on the ß-carboxysome structure provide insights into the assembly principles and structural variation of ß-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.

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