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BACKGROUND: Tandem mass tag spectrometry (TMT labeling-LC-MS/MS) was utilized to examine the global proteomes of Atlantic halibut eggs at the 1-cell-stage post fertilization. Comparisons were made between eggs judged to be of good quality (GQ) versus poor quality (BQ) as evidenced by their subsequent rates of survival for 12 days. Altered abundance of selected proteins in BQ eggs was confirmed by parallel reaction monitoring spectrometry (PRM-LC-MS/MS). Correspondence of protein levels to expression of related gene transcripts was examined via qPCR. Potential mitochondrial differences between GQ and BQ eggs were assessed by transmission electron microscopy (TEM) and measurements of mitochondrial DNA (mtDNA) levels. RESULTS: A total of 115 proteins were found to be differentially abundant between GQ and BQ eggs. Frequency distributions of these proteins indicated higher protein folding activity in GQ eggs compared to higher transcription and protein degradation activities in BQ eggs. BQ eggs were also significantly enriched with proteins related to mitochondrial structure and biogenesis. Quantitative differences in abundance of several proteins with parallel differences in their transcript levels were confirmed in egg samples obtained over three consecutive reproductive seasons. The observed disparities in global proteome profiles suggest impairment of protein and energy homeostasis related to unfolded protein response and mitochondrial stress in BQ eggs. TEM revealed BQ eggs to contain significantly higher numbers of mitochondria, but differences in corresponding genomic mtDNA (mt-nd5 and mt-atp6) levels were not significant. Mitochondria from BQ eggs were significantly smaller with a more irregular shape and a higher number of cristae than those from GQ eggs. CONCLUSION: The results of this study indicate that BQ Atlantic halibut eggs are impaired at both transcription and translation levels leading to endoplasmic reticulum and mitochondrial disorders. Observation of these irregularities over three consecutive reproductive seasons in BQ eggs from females of diverse background, age and reproductive experience indicates that they are a hallmark of poor egg quality. Additional research is needed to discover when in oogenesis and under what circumstances these defects may arise. The prevalence of this suite of markers in BQ eggs of diverse vertebrate species also begs investigation.
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Lenguado , Animales , Cromatografía Liquida , ADN Mitocondrial/genética , Femenino , Lenguado/genética , Homeostasis , Pliegue de Proteína , Proteoma , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Multiple sclerosis (MS) is an autoimmune, neurodegenerative disorder with a strong genetic component that acts in a complex interaction with environmental factors for disease development. CD4+ T cells are pivotal players in MS pathogenesis, where peripherally activated T cells migrate to the central nervous system leading to demyelination and axonal degeneration. Through a proteomic approach, we aim at identifying dysregulated pathways in activated T cells from MS patients as compared to healthy controls. METHODS: CD4+ T cells were purified from peripheral blood from MS patients and healthy controls by magnetic separation. Cells were left unstimulated or stimulated in vitro through the TCR and costimulatory CD28 receptor for 24 h prior to sampling. Electrospray liquid chromatography-tandem mass spectrometry was used to measure protein abundances. RESULTS: Upon T cell activation the abundance of 1801 proteins was changed. Among these proteins, we observed an enrichment of proteins expressed by MS-susceptibility genes. When comparing protein abundances in T cell samples from healthy controls and MS patients, 18 and 33 proteins were differentially expressed in unstimulated and stimulated CD4+ T cells, respectively. Moreover, 353 and 304 proteins were identified as proteins exclusively induced upon T cell activation in healthy controls and MS patients, respectively and dysregulation of the Nur77 pathway was observed only in samples from MS patients. CONCLUSIONS: Our study highlights the importance of CD4+ T cell activation for MS, as proteins that change in abundance upon T cell activation are enriched for proteins encoded by MS susceptibility genes. The results provide evidence for proteomic disturbances in T cell activation in MS, and pinpoint to dysregulation of the Nur77 pathway, a biological pathway known to limit aberrant effector T cell responses.
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BACKGROUND: Multiple sclerosis (MS) is an autoimmune, neuroinflammatory disease, with an unclear etiology. However, T cells play a central role in the pathogenesis by crossing the blood-brain-barrier, leading to inflammation of the central nervous system and demyelination of the protective sheath surrounding the nerve fibers. MS has a complex inheritance pattern, and several studies indicate that gene interactions with environmental factors contribute to disease onset. METHODS: In the current study, we evaluated T cell dysregulation at the protein level using electrospray liquid chromatography-tandem mass spectrometry to get novel insights into immune-cell processes in MS. We have analyzed the proteomic profiles of CD4+ and CD8+ T cells purified from whole blood from 13 newly diagnosed, treatment-naive female patients with relapsing-remitting MS and 14 age- and sex-matched healthy controls. RESULTS: An overall higher protein abundance was observed in both CD4+ and CD8+ T cells from MS patients when compared to healthy controls. The differentially expressed proteins were enriched for T-cell specific activation pathways, especially CTLA4 and CD28 signaling in CD4+ T cells. When selectively analyzing proteins expressed from the genes most proximal to > 200 non-HLA MS susceptibility polymorphisms, we observed differential expression of eight proteins in T cells between MS patients and healthy controls, and there was a correlation between the genotype at three MS genetic risk loci and protein expressed from proximal genes. CONCLUSION: Our study provides evidence for proteomic differences in T cells from relapsing-remitting MS patients compared to healthy controls and also identifies dysregulation of proteins encoded from MS susceptibility genes.
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BACKGROUND: Satisfactory sample preparation for mass spectrometry-based analysis is a critical step in the proteomics workflow. The quality and reproducibility of sample preparation can determine the coverage and confidence of proteomics results. Up to date, several methodologies have been described to produce suitable peptides for mass spectrometry analysis, followed by strategies for enrichment of post-translational modified peptides, if desired. Among them, the filter-aided sample preparation (FASP) has been introduced as a method to allow for removal of denaturants, reductants, alkylators, lipids and nucleic acids prior to trypsin digestion. Despite the high proteolytic digestion and contaminant removal efficiency described for this method, filter failure and consequently complete sample loss can discourage the use of this approach by the proteomic community. RESULTS: As judged by our quality controls, we were able to perform reliable and reproducible FASP for mass spectrometry analysis that allowed the quantification of 2141 proteins and 3694 phosphopeptides from as little as 20 and 320 µg of protein lysate from acute myeloid leukemia (AML) patients, respectively. Using the immobilized metal ion affinity chromatography (IMAC) method resulted in samples specifically enriched in phosphopeptides and allowed the quantification of a high number of both di- and multi-phosphopeptides in addition to the abundant mono-phosphopeptides. The workflows' high reproducibility from three biological replicates was demonstrated by the similar number of quantified proteins and localized phosphosites, and confirmed by the similar distributions of their molecular functions. We found that the combination of the FASP procedure with StageTip mixed-mode fractionation and IMAC are excellent workflows for the reproducible and deep study of AML proteomes and phosphoproteomes, respectively. CONCLUSIONS: The FASP procedure can be carried out without the risk of filter failure by performing a simple test of the filter quality before adding the protein sample. Herein, we demonstrate an efficient and reproducible FASP-based pipeline for the proteomic and phosphoproteomic analysis of AML patient samples which also can be used for the analysis of any other protein samples.
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As a direct consequence of the high diversity of the aggressive blood cancer acute myeloid leukemia (AML), proteomic samples from patients are strongly heterogeneous, rendering their accurate relative quantification challenging. In the present study, we investigated the benefits of using a super-SILAC mix of AML derived cell lines as internal standard (IS) for quantitative shotgun studies. The Molm-13, NB4, MV4-11, THP-1, and OCI-AML3 cell lines were selected for their complementarity with regard to clinical, cytogenetic, and molecular risk factors used for prognostication of AML patients. The resulting IS presents a high coverage of the AML proteome compared to single cell lines allied with high technical reproducibility, thus enabling its use for AML patient comparison. This was confirmed by comparing the protein regulation between the five cell lines and by applying the IS to patient material; hence, we were able to reproduce specific functional regulations known to be related to disease progression and molecular genetic abnormalities. The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000441.
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Leucemia Mieloide Aguda/metabolismo , Espectrometría de Masas/métodos , Proteoma/análisis , Proteómica/métodos , Biomarcadores de Tumor , Línea Celular Tumoral , Humanos , Marcaje Isotópico , Proteoma/químicaRESUMEN
Alzheimer's disease (AD) is heterogenous at the molecular level. Understanding this heterogeneity is critical for AD drug development. Here we define AD molecular subtypes using mass spectrometry proteomics in cerebrospinal fluid, based on 1,058 proteins, with different levels in individuals with AD (n = 419) compared to controls (n = 187). These AD subtypes had alterations in protein levels that were associated with distinct molecular processes: subtype 1 was characterized by proteins related to neuronal hyperplasticity; subtype 2 by innate immune activation; subtype 3 by RNA dysregulation; subtype 4 by choroid plexus dysfunction; and subtype 5 by blood-brain barrier impairment. Each subtype was related to specific AD genetic risk variants, for example, subtype 1 was enriched with TREM2 R47H. Subtypes also differed in clinical outcomes, survival times and anatomical patterns of brain atrophy. These results indicate molecular heterogeneity in AD and highlight the need for personalized medicine.
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Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/genética , ProteómicaRESUMEN
This study aimed at exploring the proteomic profile of PBMCs to predict treatment response in pulmonary tuberculosis (PTB). This was a pilot study conducted among 8 adult patients from Zanzibar, Tanzania with confirmed PTB. Blood samples were collected at baseline, at 2 months of treatment, and at the end of treatment at 6 months. Proteins were extracted from PBMCs and analyzed using LC-MS/MS based label free quantitative proteomics. Overall, 3,530 proteins were quantified across the samples, and 12 differentially expressed proteins were identified at both 2 months of treatment and at treatment completion, which were involved in cellular and metabolic processes, as well as binding and catalytic activity. Seven were downregulated proteins (HSPA1B/HSPA1A, HSPH1, HSP90AA1, lipopolysaccharide-binding protein, complement component 9, calcyclin-binding protein, and protein transport protein Sec31A), and 5 proteins were upregulated (SEC14 domain and spectrin repeat-containing protein 1, leucine-rich repeat-containing 8 VRAC subunit D, homogentisate 1,2-dioxygenase, NEDD8-activating enzyme E1 regulatory subunit, and N-acetylserotonin O-methyltransferase-like protein). The results showed that proteome analysis of PBMCs can be used as a novel technique to identify protein abundance change with anti-tuberculosis treatment. The novel proteins elucidated in this work may provide new insights for understanding PTB pathogenesis, treatment, and prognosis.
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Leucocitos Mononucleares , Tuberculosis Pulmonar , Adulto , Humanos , Leucocitos Mononucleares/metabolismo , Proyectos Piloto , Tanzanía , Proteómica/métodos , Cromatografía Liquida , Espectrometría de Masas en Tándem , Proteoma/metabolismo , Tuberculosis Pulmonar/metabolismoRESUMEN
Acute myeloid leukemia (AML) is an aggressive bone marrow malignancy, and non-leukemic stromal cells (including mesenchymal stem cells, MSCs) are involved in leukemogenesis and show AML-supporting effects. We investigated how constitutive extracellular mediator release by primary human AML cells alters proteomic profiles of normal bone marrow MSCs. An average of 6814 proteins (range 6493-6918 proteins) were quantified for 41 MSC cultures supplemented with AML-cell conditioned medium, whereas an average of 6715 proteins (range 6703-6722) were quantified for untreated control MSCs. The AML effect on global MSC proteomic profiles varied between patients. Hierarchical clustering analysis identified 10 patients (5/10 secondary AML) showing more extensive AML-effects on the MSC proteome, whereas the other 31 patients clustered together with the untreated control MSCs and showed less extensive AML-induced effects. These two patient subsets differed especially with regard to MSC levels of extracellular matrix and mitochondrial/metabolic regulatory proteins. Less than 10% of MSC proteins were significantly altered by the exposure to AML-conditioned media; 301 proteins could only be quantified after exposure to conditioned medium and 201 additional proteins were significantly altered compared with the levels in control samples (153 increased, 48 decreased). The AML-modulated MSC proteins formed several interacting networks mainly reflecting intracellular organellar structure/trafficking but also extracellular matrix/cytokine signaling, and a single small network reflecting altered DNA replication. Our results suggest that targeting of intracellular trafficking and/or intercellular communication is a possible therapeutic strategy in AML.
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Na+/H+ Exchanger 9 (NHE9) is an endosomal membrane protein encoded by the Solute Carrier 9A, member 9 gene (SLC9A9). SLC9A9 has been implicated in attention deficit hyperactivity disorder (ADHD), autism spectrum disorders (ASDs), epilepsy, multiple sclerosis and cancers. To better understand the function of NHE9 and the effects of disease-associated variants on protein-protein interactions, we conducted a quantitative analysis of the NHE9 interactome using co-immunoprecipitation and isobaric labeling-based quantitative mass spectrometry. We identified 100 proteins that interact with NHE9. These proteins were enriched in known functional pathways for NHE9: the endocytosis, protein ubiquitination and phagosome pathways, as well as some novel pathways including oxidative stress, mitochondrial dysfunction, mTOR signaling, cell death and RNA processing pathways. An ADHD-associated mutation (A409P) significantly altered NHE9's interactions with a subset of proteins involved in caveolae-mediated endocytosis and MAP2K2-mediated downstream signaling. An ASD nonsense mutation in SLC9A9, R423X, produced no-detectable amount of NHE9, suggesting the overall loss of NHE9 functional networks. In addition, seven of the NHE9 interactors are products of known autism candidate genes (Simons Foundation Autism Research Initiative, SFARI Gene) and 90% of the NHE9 interactome overlap with SFARI protein interaction network PIN (p < 0.0001), supporting the role of NHE9 interactome in ASDs molecular mechanisms. Our results provide a detailed understanding of the functions of protein NHE9 and its disrupted interactions, possibly underlying ADHD and ASDs. Furthermore, our methodological framework proved useful for functional characterization of disease-associated genetic variants and suggestion of druggable targets.
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Trastorno por Déficit de Atención con Hiperactividad/genética , Trastorno del Espectro Autista/genética , Predisposición Genética a la Enfermedad/genética , Mutación , Mapas de Interacción de Proteínas/genética , Intercambiadores de Sodio-Hidrógeno/genética , HumanosRESUMEN
UNLABELLED: MS-based proteomic studies aiming for the discovery of acute myeloid leukemia (AML) biomarkers require sample processing that can assure an optimal proteome coverage and identification of PTMs. We evaluated different in-solution and filter-aided sample preparation (FASP) proteomic workflows and different enrichment strategies of phosphorylated peptides. The FASP protocols in the label-free and SILAC (stable isotope labelling with amino acids in cell culture) approaches were selected for producing the highest number of quantified proteins with reduced number of missed cleavages. The IMAC method was selected for producing the highest number of quantified phosphopeptides from SILAC-labelled peptides prepared with FASP. Using these selected workflows, we studied the effect of liquid nitrogen storage on the proteome and phosphoproteome of four AML patients. Our results showed that although there was not a major global proteome and phosphoproteome change when compared to their freshly processed counterparts, the freezing appeared to influence the abundance of mitochondrial proteins involved in the respiratory chain transport and affect the phosphorylation of apoptosis related proteins, cell surface interactors, ERK/MAPK targets and proteins involved in thrombin signalling. Our results encourage the assessment of current procedures of AML sample collection and preservation that could be used in future AML biomarker discovery studies. BIOLOGICAL SIGNIFICANCE: Proteomic studies aiming to identify potential cancer biomarkers need to utilize the best sample preparation workflows on the samples of interest to achieve maximal proteome coverage. We have tested the most popular and recent proteomic and phosphoproteomic methods on cell lysates from patients with AML and systematically evaluated their performance. Our study shows the relevance of selecting the patient sample procedure giving the highest protein and PTM coverage. Moreover, we assessed how the proteome and phosphoproteome were affected by the conventional liquid nitrogen storage compared to cell lysis of fresh material, using the methods that worked best in our hands. For potential biomarkers that could be used for AML diagnostic and prognostic, it is of great importance to study the behaviour during sample conservation in order to avoid artefactual findings. Our results recommend caution in data interpretation when using different protocols of sample collection and conservation for proteomic and phosphosproteomic research.
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Congelación , Leucemia Mieloide Aguda/patología , Proteoma/análisis , Proteínas Reguladoras de la Apoptosis/metabolismo , Biomarcadores de Tumor , Transporte de Electrón , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Marcaje Isotópico , Leucemia Mieloide Aguda/metabolismo , Fosfopéptidos/análisis , Fosfopéptidos/metabolismo , Fosforilación , Proteoma/metabolismo , Trombina/metabolismo , Flujo de TrabajoRESUMEN
BACKGROUND: Survival time within a disabled submarine (SUBSUNK) is dependant on atmospheric composition and proper design and use of emergency atmospheric control systems. The objective of this study was to investigate atmospheric changes and physiological responses during a SUBSUNK trial. METHODS: There were 18 volunteers who were restrained within a 250 m3 front compartment of an Ula-class submarine submerged in 8 degrees C seawater for 6 d, 18 h. Atmospheric control was maintained according to emergency procedures using non-electrically powered chemical CO2 absorption, and O2 was replenished using chlorate candles. Atmospheric parameters, skin and body temperatures, weight, urine, and drinking volume were measured. Subjective responses to cold were measured on a visual analog scale (VAS), and symptoms were logged on the environmental symptoms questionnaire (ESQ). RESULTS: Atmospheric temperature gradually decreased to a minimum of 14.1 degrees C. Toe, heel, and finger temperatures decreased significantly. Subjects reported inferior subjective thermal comfort on the VAS and increased cold stress on the ESQ. Except for CO2, no inorganic or volatile organic compounds exceeded occupational exposure limits. The PO2 and PCO2 ranged from 17.4-20.3 and 1.9-2.8 kPa, respectively, during the first 5 d. During the last 2 d, PO2 and PCO2 were deliberately maintained at about 15.8 and 3.1 kPa, respectively. Mean oxygen consumption and CO2 production were 23.8 and 19.8 L standard temperature and pressure (STP) x man(-1) x h(-1), respectively. Soda lime and lithium hydroxide CO2 absorption capacities were 126 and 405 L STP x kg(-1) respectively. CONCLUSIONS: Atmospheric conditions can be controlled acceptably for 6 d, 18 h within the front compartment of an Ula-class submarine operating according to emergency SUBSUNK procedures.
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Atmósfera , Dióxido de Carbono/análisis , Ergonomía , Medicina Submarina , Adulto , Presión Atmosférica , Temperatura Corporal , Humanos , Hipercapnia , Hipoxia , Masculino , Temperatura Cutánea , Sobrevida , TemperaturaRESUMEN
Several issues need to be considered concerning chemical labeling strategies in proteomics. Some of these are labeling specificity, possible side reactions, completeness of reaction, recovery rate, conserving integrity of sample, hydrolysis of peptide bonds at high pH, and signal suppression in mass spectrometry (MS). We tested the effects of different reaction conditions for 2-methoxy-4,5-dihydro-1H-imidazole (Lys Tag) derivatization of the ε-amine group of lysine (K) residues. By using nanoflow LC-electrospray ionization-MS (LC-ESI-MS) and MS/MS in combination with MSight 2-D image analysis, we found that standard Lys Tag derivatization processes and conditions induce side reactions such as (i) Lys Tag labeling of the N-terminus, (ii) methylation of internal aspartic acid (D), glutamic acid (E) and C- and N-peptide termini and (iii) deamidation of asparagine (N) and glutamine (Q). We found temperature and pH to be the main variables to control side reactions. Lowering the reaction temperature from 55°C to room temperature reduced deamidation from 22.8±1.4% (SEM) to 7.7±5.5% (SEM) and almost totally blocked methylation (7.0±1.2% (SEM) to 0.4±0.4% (SEM) of the internal acidic amino acids (D and E) at high pH. We conclude that lowering the reaction temperature minimizes undesired side reactions during Lys Tag derivatization in solution.