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We introduce a new ionization technique for compact, portable mass spectrometers. It consists of a syringe with sample liquid capped by a self-ionizing spray nozzle containing a microfabricated nozzle chip. Interaction of the sample liquid with the nozzle wall results in electrical charging without the need for electronics. Elaborate cleaning procedures are redundant when disposable syringes and mass-fabricated spray nozzles are used. This self-named electroless spray ionization (ELI) technique shows comparable performance to conventional ionization techniques. In contrast to commonly used electrospray ionization, ELI exhibits excellent ionization efficiency for low-conductive solutions such as water or acetonitrile. Due to its compact size and the absence of high-voltage electronics, it can also be readily integrated in other ionization sources. Besides reviewing the main properties of ELI, we showcase the technique's potential for two on-site, ambient mass spectroscopy applications: perfume fingerprinting and fast screening of fungicides on citrus fruits.
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BACKGROUND: Histopathological assessment of transplant biopsies is currently the standard method to diagnose allograft rejection and can help guide patient management, but it is one of the most challenging areas of pathology, requiring considerable expertise, time, and effort. We aimed to analyse the utility of deep learning to preclassify histology of kidney allograft biopsies into three main broad categories (ie, normal, rejection, and other diseases) as a potential biopsy triage system focusing on transplant rejection. METHODS: We performed a retrospective, multicentre, proof-of-concept study using 5844 digital whole slide images of kidney allograft biopsies from 1948 patients. Kidney allograft biopsy samples were identified by a database search in the Departments of Pathology of the Amsterdam UMC, Amsterdam, Netherlands (1130 patients) and the University Medical Center Utrecht, Utrecht, Netherlands (717 patients). 101 consecutive kidney transplant biopsies were identified in the archive of the Institute of Pathology, RWTH Aachen University Hospital, Aachen, Germany. Convolutional neural networks (CNNs) were trained to classify allograft biopsies as normal, rejection, or other diseases. Three times cross-validation (1847 patients) and deployment on an external real-world cohort (101 patients) were used for validation. Area under the receiver operating characteristic curve (AUROC) was used as the main performance metric (the primary endpoint to assess CNN performance). FINDINGS: Serial CNNs, first classifying kidney allograft biopsies as normal (AUROC 0·87 [ten times bootstrapped CI 0·85-0·88]) and disease (0·87 [0·86-0·88]), followed by a second CNN classifying biopsies classified as disease into rejection (0·75 [0·73-0·76]) and other diseases (0·75 [0·72-0·77]), showed similar AUROC in cross-validation and deployment on independent real-world data (first CNN normal AUROC 0·83 [0·80-0·85], disease 0·83 [0·73-0·91]; second CNN rejection 0·61 [0·51-0·70], other diseases 0·61 [0·50-0·74]). A single CNN classifying biopsies as normal, rejection, or other diseases showed similar performance in cross-validation (normal AUROC 0·80 [0·73-0·84], rejection 0·76 [0·66-0·80], other diseases 0·50 [0·36-0·57]) and generalised well for normal and rejection classes in the real-world data. Visualisation techniques highlighted rejection-relevant areas of biopsies in the tubulointerstitium. INTERPRETATION: This study showed that deep learning-based classification of transplant biopsies could support pathological diagnostics of kidney allograft rejection. FUNDING: European Research Council; German Research Foundation; German Federal Ministries of Education and Research, Health, and Economic Affairs and Energy; Dutch Kidney Foundation; Human(e) AI Research Priority Area of the University of Amsterdam; and Max-Eder Programme of German Cancer Aid.
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Aprendizaje Profundo , Rechazo de Injerto/diagnóstico , Trasplante de Riñón/clasificación , Biopsia , Humanos , Prueba de Estudio Conceptual , Estudios RetrospectivosRESUMEN
Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phosphotargets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A) (to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of nonoverlapping phosphatase inhibition- and activation-responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies.
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Autoantígenos/genética , Hidrolasas de Éster Carboxílico/genética , Proteínas de Unión al ADN/genética , Chaperonas de Histonas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Proteína Fosfatasa 2/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Neoplasias/genética , Neoplasias/patología , Neoplasias/terapia , Lámina Nuclear/efectos de los fármacos , Lámina Nuclear/genética , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 2/genética , Proteoma/efectos de los fármacos , Proto-Oncogenes Mas , ARN Interferente Pequeño/genética , Biología de SistemasRESUMEN
BACKGROUND: Progression of prostate cancer from benign local tumors to metastatic carcinomas is a multistep process. Here we have investigated the signaling pathways that support migration and invasion of prostate cancer cells, focusing on the role of the NFATC1 transcription factor and its post-translational modifications. We have previously identified NFATC1 as a substrate for the PIM1 kinase and shown that PIM1-dependent phosphorylation increases NFATC1 activity without affecting its subcellular localization. Both PIM kinases and NFATC1 have been reported to promote cancer cell migration, invasion and angiogenesis, but it has remained unclear whether the effects of NFATC1 are phosphorylation-dependent and which downstream targets are involved. METHODS: We used mass spectrometry to identify PIM1 phosphorylation target sites in NFATC1, and analysed their functional roles in three prostate cancer cell lines by comparing phosphodeficient mutants to wild-type NFATC1. We used luciferase assays to determine effects of phosphorylation on NFAT-dependent transcriptional activity, and migration and invasion assays to evaluate effects on cell motility. We also performed a microarray analysis to identify novel PIM1/NFATC1 targets, and validated one of them with both cellular expression analyses and in silico in clinical prostate cancer data sets. RESULTS: Here we have identified ten PIM1 target sites in NFATC1 and found that prevention of their phosphorylation significantly decreases the transcriptional activity as well as the pro-migratory and pro-invasive effects of NFATC1 in prostate cancer cells. We observed that also PIM2 and PIM3 can phosphorylate NFATC1, and identified several novel putative PIM1/NFATC1 target genes. These include the ITGA5 integrin, which is differentially expressed in the presence of wild-type versus phosphorylation-deficient NFATC1, and which is coexpressed with PIM1 and NFATC1 in clinical prostate cancer specimens. CONCLUSIONS: Based on our data, phosphorylation of PIM1 target sites stimulates NFATC1 activity and enhances its ability to promote prostate cancer cell migration and invasion. Therefore, inhibition of the interplay between PIM kinases and NFATC1 may have therapeutic implications for patients with metastatic forms of cancer.
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Movimiento Celular , Factores de Transcripción NFATC/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Proliferación Celular , Humanos , Masculino , Espectrometría de Masas , Células PC-3 , Fosforilación , Neoplasias de la Próstata/patología , Transducción de Señal , Células Tumorales CultivadasRESUMEN
When considering incident investigations and security checks focused on energetic materials, there is an ongoing need for rapid, on-scene chemical identification. Currently applied methods are not capable of meeting all requirements, and hence, portable mass spectrometry is an interesting alternative although many instrumental challenges still exist. To be able to analyze explosives with mass spectrometry outside the traditional laboratory, suitable ambient ionization methods need to be developed. Ideally such methods are also easily implemented in the field requiring limited to no power sources, gas supplies, flow controllers, and heating devices. For this reason, the potential of SAWN (surface acoustic wave nebulization) for the ambient ionization and subsequent mass spectrometric (MS) analysis of organic explosives was investigated in this study. Excellent sensitivity was observed for nitrate-based organic explosives when operating the MS in negative mode. No dominant adduct peaks were observed for the peroxides TATP and HMTD with SAWN-MS in positive mode. The MS spectra indicate extensive fragmentation of the peroxide explosives even under the mild ionization conditions provided by SAWN. The potential of SAWN-MS was demonstrated with the correct identification of nitrate-based organic explosives in pre- and post-explosion case samples in only a fraction of the time and effort required for the regular laboratory analysis. Results show that SAWN-MS can convincingly identify intact organic energetic compounds and mixtures but that sensitivity is not always sufficient to detect traces of explosives in post-explosion residues.
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Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh-frozen and formalin-fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue-sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom-up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys-C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue-specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm2 obtained with laser capture microdissection to much larger amounts that weight several milligrams.
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Espectrometría de Masas/métodos , Proteoma/análisis , Proteómica/métodos , Animales , Biopsia , Cromatografía Liquida/métodos , Humanos , Proteínas/análisis , Proteolisis , Conservación de Tejido/métodosRESUMEN
In many applications where small, similar-sized droplets are needed, ultrasonic nebulizers are employed. Little is known about the mechanism of nebulization, for example about what determines the median droplet size. Even less understood, is the droplet size distribution, which is often simply fitted with a log-normal distribution or assumed to be very narrow. We perform the first systematic study of droplet size distributions for different nebulizer technologies, showing that these distributions can be very well fitted with distributions found for sprays, where the size distribution is completely determined by the corrugation of ligaments and the distribution of ligament sizes. In our case, breakup is believed to be due to pinch-off of Faraday instabilities. The droplet size distribution is then set by the distribution of wavelengths of the standing capillary waves and the roughness of the pinch-off ligaments. We show that different nebulizer technologies produce different size distributions, which we relate to (variation in) wavelengths of the waves that contribute to the droplet formation. We further show that the median droplet size scales with the capillary wavelength, with a proportionality constant that depends only slightly on the type of nebulizer, despite order-of-magnitude differences in other parameters.
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Motivation: Mass spectrometry combined with enrichment strategies for phosphorylated peptides has been successfully employed for two decades to identify sites of phosphorylation. However, unambiguous phosphosite assignment is considered challenging. Given that site-specific phosphorylation events function as different molecular switches, validation of phosphorylation sites is of utmost importance. In our earlier study we developed a method based on simulated phosphopeptide spectral libraries, which enables highly sensitive and accurate phosphosite assignments. To promote more widespread use of this method, we here introduce a software implementation with improved usability and performance. Results: We present SimPhospho, a fast and user-friendly tool for accurate simulation of phosphopeptide tandem mass spectra. Simulated phosphopeptide spectral libraries are used to validate and supplement database search results, with a goal to improve reliable phosphoproteome identification and reporting. The presented program can be easily used together with the Trans-Proteomic Pipeline and integrated in a phosphoproteomics data analysis workflow. Availability and implementation: SimPhospho is open source and it is available for Windows, Linux and Mac operating systems. The software and its user's manual with detailed description of data analysis as well as test data can be found at https://sourceforge.net/projects/simphospho/. Supplementary information: Supplementary data are available at Bioinformatics online.
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Fosfopéptidos/análisis , Proteómica/métodos , Programas Informáticos , Espectrometría de Masas en Tándem/métodos , Bases de Datos de Proteínas , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
Human tissues are an important link between organ-specific spatial molecular information, patient pathology, and patient treatment options. However, patient tissues are uniquely obtained by time and location, and limited in their availability and size. Currently, little knowledge exists about appropriate and simplified protocols for routine MS-based analysis of the various types and sizes of tissues. Following standard procedures used in pathology, we selected small fresh frozen uterine tissue samples to investigate how the tissue preparation protocol affected the subsequent proteomics analysis. First, we observed that protein extraction with 0.1% SDS followed by extraction with a 30% ACN/urea resulted in a decrease in the number of identified proteins, when compared to extraction with 30% ACN/urea only. The decrease in the number of proteins was approximately 55% and 20%, for 10 and 16 µm thick tissue, respectively. Interestingly, the relative abundance of the proteins shared between the two methods was higher when SDS/ACN/urea was used, compared to the 30% ACN/urea extraction, indicating the role of SDS to be beneficial for protein solubility. Second, the influence of tissue thickness was investigated by comparing the results obtained for 10, 16, and 20 µm thick (1 mm2) tissue using urea/30% ACN. We observed an increase in the number of identified proteins and corresponding quantity with an increase in the tissue thickness. Finally, by analyzing very small amounts of tissues (â¼0.2 mm2) of 10, 16, and 20 µm thickness, we observed that the increase in tissue thickness resulted in a higher number of protein identifications and corresponding quantitative values.
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Proteínas/análisis , Espectrometría de Masas en Tándem/métodos , Útero/metabolismo , Acetonitrilos/química , Fraccionamiento Químico , Cromatografía Líquida de Alta Presión , Femenino , Congelación , Humanos , Nanotecnología , Proteínas/aislamiento & purificación , Proteómica , Reproducibilidad de los Resultados , Dodecil Sulfato de Sodio/química , Solubilidad , Urea/químicaRESUMEN
Surface acoustic wave nebulisation (SAWN) mass spectrometry (MS) is a method to generate gaseous ions compatible with direct MS of minute samples at femtomole sensitivity. To perform SAWN, acoustic waves are propagated through a LiNbO3 sampling chip, and are conducted to the liquid sample, which ultimately leads to the generation of a fine mist containing droplets of nanometre to micrometre diameter. Through fission and evaporation, the droplets undergo a phase change from liquid to gaseous analyte ions in a non-destructive manner. We have developed SAWN technology for the characterisation of organic colourants in textiles. It generates electrospray-ionisation-like ions in a non-destructive manner during ionisation, as can be observed by the unmodified chemical structure. The sample size is decreased by tenfold to 1000-fold when compared with currently used liquid chromatography-MS methods, with equal or better sensitivity. This work underscores SAWN-MS as an ideal tool for molecular analysis of art objects as it is non-destructive, is rapid, involves minimally invasive sampling and is more sensitive than current MS-based methods. Graphical Abstract á .
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Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram-positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique interprotein cross-linked peptides with <1% false discovery rate by mass spectrometry and genome-wide database searching. Nearly 60% of the interprotein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and ß' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth. Data are available via ProteomeXchange with identifier PXD006287.
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Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Glutaratos/química , Mapeo de Interacción de Proteínas/métodos , Succinimidas/química , Secuencia de Aminoácidos , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Reactivos de Enlaces Cruzados/química , Medios de Cultivo/química , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glutamato Deshidrogenasa/química , Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Biogénesis de Organelos , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Especificidad de la Especie , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
A critical step in the bottom-up characterization of proteomes is the conversion of proteins to peptides, by means of endoprotease digestion. Nowadays this method typically uses overnight digestion and as such represents a considerable bottleneck for high-throughput analysis. This report describes protein digestion using an immobilized-enzyme reactor (IMER), which enables accelerated digestion times that are completed within seconds to minutes. For rapid digestion to occur, a cyclic-olefin-copolymer microfluidic reactor was constructed containing trypsin immobilized on a polymer monolithic material through a 2-vinyl-4,4-dimethylazlactone linker. The IMER was applied for the rapid offline digestion of both singular protein standards and a complex protein mixture prior to liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The effects of protein concentration and residence time in the IMER were assessed for protein standards of varying molecular weight between 11 and 240kDa. Compared to traditional in-solution digestion, IMER-facilitated protein digestion at room temperature for 5min yielded similar results in terms of sequence coverage and number of identified peptides. Good repeatability was demonstrated with a relative standard deviation of 6% for protein-sequence coverage. The potential of the IMER was also demonstrated for a complex protein mixture in the analysis of dried blood spots. Compared to a traditional workflow a similar number of proteins could be identified, while reducing the total analysis time from 22.5h to 4h and importantly omitting the sample-pre-treatment steps (denaturation, reduction, and alkylation). The identified proteins from two workflows showed similar distributions in terms of molecular weight and hydrophobic character.
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Proteínas Sanguíneas/análisis , Cicloparafinas/química , Pruebas con Sangre Seca , Enzimas Inmovilizadas , Técnicas Analíticas Microfluídicas/instrumentación , Polímeros/química , Pruebas con Sangre Seca/instrumentación , Pruebas con Sangre Seca/métodos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , HumanosRESUMEN
Recent metagenomic studies have demonstrated that the overall functional potential of the intestinal microbiome is rather conserved between healthy individuals. Here we assessed the biological processes undertaken in-vivo by microbes and the host in the intestinal tract by conducting a metaproteome analysis from a total of 48 faecal samples of 16 healthy adults participating in a placebo-controlled probiotic intervention trial. Half of the subjects received placebo and the other half consumed Lactobacillus rhamnosus GG for three weeks (1010 cfu per day). Faecal samples were collected just before and at the end of the consumption phase as well as after a three-week follow-up period, and were processed for microbial composition and metaproteome analysis. A common core of shared microbial protein functions could be identified in all subjects. Furthermore, we observed marked differences in expressed proteins between subjects that resulted in the definition of a stable and personalized microbiome both at the mass-spectrometry-based proteome level and the functional level based on the KEGG pathway analysis. No significant changes in the metaproteome were attributable to the probiotic intervention. A detailed taxonomic assignment of peptides and comparison to phylogenetic microarray data made it possible to evaluate the activity of the main phyla as well as key species, including Faecalibacterium prausnitzii. Several correlations were identified between human and bacterial proteins. Proteins of the human host accounted for approximately 14% of the identified metaproteome and displayed variations both between and within individuals. The individually different human intestinal proteomes point to personalized host-microbiota interactions. Our findings indicate that analysis of the intestinal metaproteome can complement gene-based analysis and contributes to a thorough understanding of the activities of the microbiome and the relevant pathways in health and disease.
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Microbioma Gastrointestinal , Probióticos/uso terapéutico , Adulto , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Estudios de Cohortes , Método Doble Ciego , Heces/microbiología , Femenino , Microbioma Gastrointestinal/genética , Genoma Bacteriano , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Mapeo Peptídico , Filogenia , Medicina de Precisión , Proteómica , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Adulto JovenRESUMEN
The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed.
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Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Transducción de Señal , Trichoderma/metabolismo , Sitios de Unión , Celulasas/metabolismo , Cromatografía Liquida , Glucanos/metabolismo , Glucólisis , Glicósido Hidrolasas/metabolismo , Hidrólisis , Fosforilación , Sorbitol/metabolismo , Espectrometría de Masas en TándemRESUMEN
The cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) is a well-established model species in oxygenic photosynthesis research and a potential host for biotechnological applications. Despite recent advances in genome sequencing and microarray techniques applied in systems biology, quantitative proteomics approaches with corresponding accuracy and depth are scarce for S. 6803. In this study, we developed a protocol to screen changes in the expression of 106 proteins representing central metabolic pathways in S. 6803 with a targeted mass spectrometry method, selected reaction monitoring (SRM). We evaluated the response to the exposure of both short- and long-term iron deprivation. The experimental setup enabled the relative quantification of 96 proteins, with 87 and 92 proteins showing adjusted p-values <0.01 under short- and long-term iron deficiency, respectively. The high sensitivity of the SRM method for S. 6803 was demonstrated by providing quantitative data for altogether 64 proteins that previously could not be detected with the classical data-dependent MS approach under similar conditions. This highlights the effectiveness of SRM for quantification and extends the analytical capability to low-abundance proteins in unfractionated samples of S. 6803. The SRM assays and other generated information are now publicly available via PASSEL and Panorama.
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Proteínas Bacterianas/química , Hierro/metabolismo , Proteoma/química , Proteómica/métodos , Synechocystis/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Cromatografía Líquida de Alta Presión , Fotosíntesis , Proteoma/aislamiento & purificación , Proteoma/metabolismo , Espectrometría de Masas en TándemRESUMEN
The expression of proteins can be quantified in high-throughput means using different types of mass spectrometers. In recent years, there have emerged label-free methods for determining protein abundance. Although the expression is initially measured at the peptide level, a common approach is to combine the peptide-level measurements into protein-level values before differential expression analysis. However, this simple combination is prone to inconsistencies between peptides and may lose valuable information. To this end, we introduce here a method for detecting differentially expressed proteins by combining peptide-level expression-change statistics. Using controlled spike-in experiments, we show that the approach of averaging peptide-level expression changes yields more accurate lists of differentially expressed proteins than does the conventional protein-level approach. This is particularly true when there are only few replicate samples or the differences between the sample groups are small. The proposed technique is implemented in the Bioconductor package PECA, and it can be downloaded from http://www.bioconductor.org.
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Fragmentos de Péptidos/genética , Proteínas/genética , Proteómica/métodos , Programas Informáticos , Regulación de la Expresión Génica , Internet , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/metabolismo , Proteínas/metabolismo , Proteolisis , Sensibilidad y Especificidad , Tripsina/químicaRESUMEN
As tools for quantitative label-free mass spectrometry (MS) rapidly develop, a consensus about the best practices is not apparent. In the work described here we compared popular statistical methods for detecting differential protein expression from quantitative MS data using both controlled experiments with known quantitative differences for specific proteins used as standards as well as "real" experiments where differences in protein abundance are not known a priori. Our results suggest that data-driven reproducibility-optimization can consistently produce reliable differential expression rankings for label-free proteome tools and are straightforward in their application.
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Fragmentos de Péptidos/análisis , Proteoma/aislamiento & purificación , Proteómica/estadística & datos numéricos , Programas Informáticos , Espectrometría de Masas en Tándem/estadística & datos numéricos , Algoritmos , Animales , Interpretación Estadística de Datos , Bases de Datos de Proteínas , Conjuntos de Datos como Asunto , Humanos , Hígado/química , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Tripsina/químicaRESUMEN
OBJECTIVE: We aimed to decipher the molecular genetic basis of disease in a cohort of children with a uniform clinical presentation of neonatal irritability, spastic or dystonic quadriplegia, virtually absent psychomotor development, axonal neuropathy, and elevated blood/CSF lactate. METHODS: We performed whole-exome sequencing of blood DNA from the index patients. Detected compound heterozygous mutations were confirmed by Sanger sequencing. Structural predictions and a bacterial activity assay were performed to evaluate the functional consequences of the mutations. Mass spectrometry, Western blotting, and protein oxidation detection were used to analyze the effects of selenoprotein deficiency. RESULTS: Neuropathology indicated laminar necrosis and severe loss of myelin, with neuron loss and astrogliosis. In 3 families, we identified a missense (p.Thr325Ser) and a nonsense (p.Tyr429*) mutation in SEPSECS, encoding the O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, which was previously associated with progressive cerebellocerebral atrophy. We show that the mutations do not completely abolish the activity of SEPSECS, but lead to decreased selenoprotein levels, with demonstrated increase in oxidative protein damage in the patient brain. CONCLUSIONS: These results extend the phenotypes caused by defective selenocysteine biosynthesis, and suggest SEPSECS as a candidate gene for progressive encephalopathies with lactate elevation.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Encefalopatías Metabólicas Innatas/genética , Encefalopatías Metabólicas Innatas/metabolismo , Ácido Láctico/sangre , Ácido Láctico/líquido cefalorraquídeo , Selenoproteínas/deficiencia , Adolescente , Encéfalo/metabolismo , Encéfalo/patología , Encefalopatías Metabólicas Innatas/sangre , Encefalopatías Metabólicas Innatas/líquido cefalorraquídeo , Niño , Preescolar , Femenino , Humanos , Masculino , Mutación , Estrés Oxidativo/genética , Selenoproteínas/biosíntesisRESUMEN
We have investigated if phosphopeptide identification and simultaneous site localization can be achieved by spectral library searching. This allows taking advantage of comparison of specific spectral features, which would lead to improved discrimination of differential localizations. For building a library, we propose a spectral simulation strategy where all possible single phosphorylations can be simply and accurately (re)constructed on enzymatically dephosphorylated peptides, by predicting the diagnostic fragmentation events produced in beam-type CID. To demonstrate the performance of our approach, enriched HeLa phosphopeptides were dephosphorylated with alkaline phosphatase and analyzed with higher energy collisional dissociation (HCD), which were then used for creating a spectral library of simulated phosphopeptides. Spectral library searching using SpectraST was performed on data sets of synthetic phosphopeptides and the HeLa phosphopeptides, and subsequently compared to Mascot and Sequest database searching followed by phosphoRS and Ascore afforded localization, respectively. Our approach successfully led to accurate localization, and it outperformed other methods, when phosphopeptides were covered by the library. These results suggest that the searching with simulated spectral libraries serves as a crucial approach for both supplementing and validating the phosphorylation sites obtained by database searching and localization tools. For future development, simulation of multiply phosphorylated peptides remains to be implemented.
Asunto(s)
Algoritmos , Modelos Químicos , Biblioteca de Péptidos , Fosfopéptidos/análisis , Programas Informáticos , Secuencia de Aminoácidos , Cromatografía Liquida , Simulación por Computador , Células HeLa , Humanos , Datos de Secuencia Molecular , Fosfopéptidos/síntesis química , Fosforilación , Proteómica/métodos , Espectrometría de Masas en TándemRESUMEN
Organellar reactive oxygen species (ROS) signalling is a key mechanism that promotes the onset of defensive measures in stress-exposed plants. The underlying molecular mechanisms and feedback regulation loops, however, still remain poorly understood. Our previous work has shown that a specific regulatory B'γ subunit of protein phosphatase 2A (PP2A) is required to control organellar ROS signalling and associated metabolic adjustments in Arabidopsis thaliana. Here, we addressed the mechanisms through which PP2A-B'γ impacts on organellar metabolic crosstalk and ROS homeostasis in leaves. Genetic, biochemical and pharmacological approaches, together with a combination of data-dependent acquisition (DDA) and selected reaction monitoring (SRM) MS techniques, were utilized to assess PP2A-B'γ-dependent adjustments in Arabidopsis thaliana. We show that PP2A-B'γ physically interacts with the cytoplasmic form of aconitase, a central metabolic enzyme functionally connected with mitochondrial respiration, oxidative stress responses and regulation of cell death in plants. Furthermore, PP2A-B'γ impacts ROS homeostasis by controlling the abundance of specific alternative oxidase isoforms, AOX1A and AOX1D, in leaf mitochondria. We conclude that PP2A-B'γ-dependent regulatory actions modulate the functional status of metabolic enzymes that essentially contribute to intracellular ROS signalling and metabolic homeostasis in plants.