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1.
Se Pu ; 37(8): 878-886, 2019 Aug 08.
Artigo em Chinês | MEDLINE | ID: mdl-31642259

RESUMO

One of the major shortcomings in top-down proteomics is the lack of efficient separations for intact proteins that can be effectively coupled to mass spectrometry. Capillary zone electrophoresis (CZE) and nanoflow reversed-phase liquid chromatography (nanoRPLC) are two methods that can be coupled to mass spectrometry directly and have been recently advanced in terms of their ability to separate intact proteins in complex biological mixtures. In this work, for the first time, we compared the state-of-the-art nanoRPLC-MS/MS and CZE-MS/MS platforms for top-down characterization of a standard protein mixture and an Escherichia coli (E. coli) proteome sample. CZE-MS produced comparable signals of standard proteins to RPLC-MS with 10-times less sample consumption. Interestingly, the proteins in RPLC-MS tended to have higher charge states than in CZE-MS, most likely due to the high acetonitrile concentration in RPLC mobile phase, leading to the more extensive unfolding of proteins in RPLC compared to in CZE. CZE-MS/MS identified 159 proteins and 513 proteoforms using 1-µg E. coli proteins in a single run and outperformed RPLC-MS/MS using 1-µg E. coli proteins in terms of protein and proteoform identifications (159 vs. 105 proteins and 513 vs. 277 proteoforms). The RPLC-MS/MS using 8-µg E. coli proteins identified 245 proteins and 1004 proteoforms in a single run, and the data was much better than that from CZE-MS/MS (1-µg E. coli proteins) regarding the number of identifications because of the 8-times higher sample loading amount and significantly wider separation window of RPLC-MS/MS compared to CZE-MS/MS.


Assuntos
Cromatografia de Fase Reversa , Eletroforese Capilar , Proteômica/métodos , Espectrometria de Massas em Tandem , Escherichia coli , Proteínas de Escherichia coli/análise
2.
Medicine (Baltimore) ; 98(41): e17478, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31593110

RESUMO

Exosomes are nanometer-sized vesicles with intercellular communication functions, and their encapsulated proteins may participate in the pathological process of neurodegenerative disorders. The aim of this study was to identify the protein changes of serum exosomes in Parkinson disease (PD) patients with different disease progress types, and to identify potential biomarkers. The exosomes of PD patients with different severity and healthy control group were isolated from serum. The exosome proteins were analyzed by mass spectrometry with label-free quantitative proteomics. A total of 429 proteins were identified, of which 14 were significantly different in mild and severe PD patients. The expression levels of 7 proteins, including pigmented epithelium-derived factor, afamin, apolipoprotein D and J, were significantly increased in PD patients. The expression levels of 7 proteins, including complement C1q and protein Immunoglobulin Lambda Variable 1-33 (IGLV1-33)Cluster -33, were decreased in PD patients. These differentially expressed proteins were analyzed by gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, which confirmed that the interaction between prion diseases and ECM receptors was the most significant pathways of enrichment. The changes of proteins and pathways may be related to the pathophysiological mechanism of PD. Therefore, some of these proteins could be considered as potential biomarkers for early PD diagnosis.


Assuntos
Exossomos/genética , Doença de Parkinson/sangue , Doença de Parkinson/genética , Proteômica/métodos , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Progressão da Doença , Feminino , Ontologia Genética , Humanos , Masculino , Índice de Gravidade de Doença
3.
Se Pu ; 37(11): 1135-1141, 2019 Nov 08.
Artigo em Chinês | MEDLINE | ID: mdl-31642265

RESUMO

With the increasing depth of proteomic identification, quantitative accuracy and increasing analytical speed, new challenges are being encountered in the development of proteomics methods. Traditional proteomics methods are time-consuming and have low sensitivity and poor accuracy; hence, they do not satisfy the new demands in proteomics research. Preparation of novel materials with specific functions via chemical and biochemical routes or by methods based on electricity, magnetism, heat, and photoirradiation is the key to overcome the limitations of traditional analytical techniques and promote active research in the field of proteomics. This paper reviews the recent advances in the application of functional materials in proteomics researches.


Assuntos
Proteômica/métodos , Proteômica/tendências , Proteínas/química
4.
Braz Oral Res ; 33: e043, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31508727

RESUMO

Proteomic techniques have become popular in medicine and dentistry because of their widespread use in analyzing bodily fluids such as blood, saliva, urine, and gingival crevicular fluids as well as hard tissues such as enamel, dentine, and cementum. This review is a guide to proteomic techniques in general dentistry, summarizing techniques and their clinical application in understanding and diagnosing diseases and their use in identifying biomarkers of various diseases.


Assuntos
Proteoma , Proteômica/métodos , Saliva/química , Proteínas e Peptídeos Salivares/química , Biomarcadores/química , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Espectrometria de Massas/métodos , Neoplasias Bucais/diagnóstico , Síndrome de Sjogren/diagnóstico
5.
Adv Exp Med Biol ; 1158: 101-117, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452138

RESUMO

Targeted mass spectrometry in the selected or parallel reaction monitoring (SRM or PRM) mode is a widely used methodology to quantify proteins based on so-called signature or proteotypic peptides. SRM has the advantage of being able to quantify a range of proteins in a single analysis, for example, to measure the level of enzymes comprising a biochemical pathway. In this chapter, we will detail how to set up an SRM assay on the example of the mitochondrial protein succinate dehydrogenase [ubiquinone] flavoprotein subunit (mouse UniProt-code Q8K2B3). First, we will outline the in silico assay design including the choice of peptides based on a range of properties. We will further delineate different quantification strategies and introduce the reader to LC-MS assay development including the selection of the optimal peptide charge state and fragment ions as well as a discussion of the dynamic range of detection. The chapter will close with an application from the area of mitochondrial biology related to the quantification of a set of proteins isolated from mouse liver mitochondria in a study on mitochondrial respiratory flux decline in aging mouse muscle.


Assuntos
Mitocôndrias , Proteômica , Animais , Cromatografia Líquida , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Peptídeos/química , Proteômica/instrumentação , Proteômica/métodos , Espectrometria de Massas em Tandem
6.
Mol Biol (Mosk) ; 53(4): 685-691, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397442

RESUMO

In the past decade, mass spectrometry studies of skeletal muscles have become common. In this tissue, the abundance of several contractile proteins significantly limits the depth of the panoramic proteome analysis. The use of isobaric labels allows improving assessment of the changes in the protein content, while analyzing up to 10 samples in a single run. Here we present the results of a comparative study of various methods for the fractionation of skeletal muscle peptides labeled with an isobaric label iTRAQ. Samples from m. vastus lateralis of eight young males were collected with a needle biopsy. After digestion into peptides and labeling, the preparations were carried out according to three different protocols: (1) peptide purification, HPLC-MS/MS; (2) peptide purification, isoelectric focusing, HPLC-MS/MS; (3) high pH reverse-phase LC fractionation, HPLC-MS/MS. Fractionation of labeled peptides by high pH reverse-phase LC was the optimal strategy for increasing the depth of the proteome analysis. This approach, in addition to contractile and mitochondrial proteins, allowed us to detect a variety of regulatory molecules, including the nucleic acids binding the proteins, chaperones, receptors, and transcription factors.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Proteoma/análise , Proteômica/métodos , Coloração e Rotulagem/métodos , Humanos , Focalização Isoelétrica , Proteoma/química , Espectrometria de Massas em Tandem
7.
BMC Ophthalmol ; 19(1): 168, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375076

RESUMO

BACKGROUND: Age-related macular degeneration (AMD) is the primary cause of blindness and severe vision loss in developed countries and is responsible for 8.7% of blindness globally. Ultraviolet radiation can induce DNA breakdown, produce reactive oxygen species, and has been implicated as a risk factor for AMD. This study investigated the effects of UVA radiation on Human retinal pigment epithelial cell (ARPE-19) growth and protein expression. METHODS: ARPE-19 cells were irradiated with a UVA lamp at different doses (5, 10, 20, 30 and 40 J/cm2) from 10 cm. Cell viability was determined by MTT assay. Visual inspection was first achieved with inverted light microscopy and then the DeadEnd™ Fluorometric TUNEL System was used to observe nuclear DNA fragmentation. Flow cytometry based-Annexin V-FITC/PI double-staining was used to further quantify cellular viability. Mitochondrial membrane potential was assessed with JC-1 staining. 2D electrophoresis maps of exposed cells were compared to nonexposed cells and gel images analyzed with PDQuest 2-D Analysis Software. Spots with greater than a 1.5-fold difference were selected for LC-MS/MS analysis and some confirmed by western blot. We further investigated whether caspase activation, apoptotic-related mitochondrial proteins, and regulators of ER stress sensors were involved in UVA-induced apoptosis. RESULTS: We detected 29 differentially expressed proteins (9 up-regulated and 20 down-regulated) in the exposed cells. Some of these proteins such as CALR, GRP78, NPM, Hsp27, PDI, ATP synthase subunit alpha, PRDX1, and GAPDH are associated with anti-proliferation, induction of apoptosis, and oxidative-stress protection. We also detected altered protein expression levels among caspases (caspase 3 and 9) and in the mitochondrial (cytosolic cytochrome C, AIF, Mcl-1, Bcl-2, Bcl-xl, Bax, Bad, and p-Bad) and ER stress-related (p-PERK, p-eIF2α, ATF4 and CHOP) apoptotic pathways. CONCLUSIONS: UVA irradiation suppressed the proliferation of ARPE-19 cells in a dose-dependent manner, caused quantitative loses in transmembrane potential (ΔΨm), and induced both early and late apoptosis.


Assuntos
Degeneração Macular/patologia , Estresse Oxidativo , Proteômica/métodos , Epitélio Pigmentado da Retina/metabolismo , Raios Ultravioleta , Apoptose , Sobrevivência Celular , Células Cultivadas , Cromatografia Líquida , Citocromos c/metabolismo , Humanos , Degeneração Macular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/efeitos da radiação , Transdução de Sinais , Espectrometria de Massas em Tandem
8.
Clin Exp Rheumatol ; 37 Suppl 118(3): 240-248, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31464680

RESUMO

In the era of personalised medicine new biomarkers are required to early diagnose Sjögren's syndrome (SS), to define different disease subsets and to direct patients' clinical management and therapeutic intervention. In the last few years, several efforts have evaluated saliva proteome to detect and monitor primary SS. Although clinically valuable, these studies presented some limitations that have partially prevented the use of salivary biomarkers in clinical practice. Nowadays, proteomic of extracellular vesicle (EV) represents an emerging and promising field in the discovery of -omic biomarkers for pSS. EV is a relatively new term that includes exosomes, microvesicles and apoptotic body. EVs are packed with proteins, growth factors, cytokines, bioactive lipids, but also nucleic acids and in particular: mRNA, microRNA, long non-coding RNA, tRNA and rRNA. Therefore, they may represent a useful source for diagnostic, prognostic and therapeutic biomarkers in several conditions. In this review we will specifically focus on EV proteomics as a tool for the identification of novel biomarkers for pSS. In the first part we focused on the state of the art of the studies on proteomics in SS existing in the literature. In the second part we provided a definition of EV with an update on biological sample collection and processing for EV proteomic studies. Finally, we summarised the state of the art of EV -omics in SS highlighting the potential advantages of this novel approach compared to the overall traditional concept of analysing the proteome of blood or saliva.


Assuntos
Vesículas Extracelulares , Proteômica/métodos , Síndrome de Sjogren , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Proteoma/metabolismo , Saliva/metabolismo , Síndrome de Sjogren/metabolismo
9.
Food Chem ; 298: 124973, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31261005

RESUMO

The aim of the present study was to explore the underlying mechanisms involved in anthocyanin biosynthesis in purple, blue, and white barley using quantitative proteomics analysis. We identified the differences in protein expression and related functions involved in anthocyanin biosynthesis in purple, blue, and white barley (named H, M, and L groups, respectively, based on their anthocyanin content) using TMT-liquid chromatography/mass spectroscopy-based proteomic methods. Totally, 297, 300, 254, and 1421 differentially expressed proteins (DEPs) were found in H vs. L, H vs. M, L vs. M, and H vs. L vs. M groups, respectively. Six clusters of proteins from the 1421 DEPs were mainly involved in carbon metabolism, amino acid and secondary metabolite biosynthesis, and metabolic pathways. Several proteins were validated using parallel reaction monitoring. The proteins involved in amino acid biosynthesis, carbon metabolism, metabolic pathways, and phenylpropanoid biosynthesis were responsible for the color differences in the three barley varieties.


Assuntos
Antocianinas/metabolismo , Hordeum/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Cromatografia Líquida , Pigmentação , Proteômica/métodos , Espectrometria de Massas em Tandem
10.
Gene ; 714: 143996, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348980

RESUMO

The uniquely human α7-nAChR gene (CHRFAM7A) is evolved from the fusion of two partially duplicated genes, FAM7 and α7-nAChR gene (CHRNA7), and is inserted on same chromosome 15, 5' end of the CHRNA7 gene. Transcription of CHRFAM7A gene produces a 1256-bp open reading frame encoding dup-α7-nAChR, where a 27-aminoacid residues from FAM7 replaced the 146-aminoacid residues of the N-terminal extracellular ligand binding domain of α7-nAChR. In vitro, dup-α7-nAChR has been shown to form hetero-pentamer with α7-nAChR and dominant-negatively regulates the channel functions of α7-nAChR. However, the contribution of CHRFAM7A gene to the biology of α7-nAChR in the brain in vivo remains largely a matter of conjecture. CHRFAM7A transgenic mouse was created and differentially expressed proteins were profiled from the whole brain using iTRAQ-2D-LC-MS/MS proteomic technology. Proteins with a fold change of ≥1.2 or ≤0.83 and p < 0.05 were considered to be significant. Bioinformatics analysis showed that over-expression of the CHRFAM7A gene significantly modulated the proteins commonly involved in the signaling pathways of α7-nAChR-mediated neuropsychiatric disorders including Parkinson's disease, Alzheimer's disease, Huntington's disease, and alcoholism, suggesting that the CHRFAM7A gene contributes to the pathogenesis of neuropsychiatric disorders mostly likely through fine-tuning the functions of α7-nAChR in the brain.


Assuntos
Camundongos Transgênicos/genética , Receptor Nicotínico de Acetilcolina alfa7/genética , Animais , Encéfalo/metabolismo , Cromatografia Líquida/métodos , Cromossomos Humanos Par 15/genética , Perfilação da Expressão Gênica/métodos , Genes Duplicados/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteômica/métodos , Transdução de Sinais/genética , Espectrometria de Massas em Tandem/métodos
11.
J Cancer Res Clin Oncol ; 145(9): 2211-2225, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31297606

RESUMO

PURPOSE: To investigate the expression of biological markers in primary vulvar Paget's disease (VPD). METHODS: Forty-one patients referred to a single major Center for Gynecologic Oncology from January 2008 to June 2018 were enrolled retrospectively: 30 non-invasive-VPD and 11 invasive-VPD. A total number of 60 samples, from all the 41 vulvar sites (VS), 8 metastatic lymph node sites (MLS) and 11 successive recurrent disease in vulvar site (RVS), were tested for an immunohistochemical panel, including the following markers: PD-L1, CD3, MSH2, MSH6, MLH1, PMS2, HER2/neu, EGFR, p16, p53, Ki67, ER, PR, AR, VEGF and CD31. RESULTS: We found a positive PD-L1 in 10% of non-invasive-VPD and 27% of invasive-VPD (18% VS; 38% MLS). ER and AR were expressed respectively in more than 70% and 75% of all specimens. HER2/neu amplification was found in 21% of non-invasive-VPD and 45% of invasive-VPD (40% VS; 38% MLS). A machine learning cluster analysis identified three groups among non- invasive-VPD: cluster-1 with higher median ER expression (40%); cluster-3 with more frequent HER2/neu overexpression (46%). Among invasive-VPD, two clusters were found: the second with more frequent HER2/neu overexpression (67% vs. 0%) and nodal metastases (100% vs. 25%). Repeating the IHC panel on the correspondent MLS and RVS, it significantly changed, respectively, in 50% and 27%. CONCLUSIONS: This study reveals the expression of PDL-1 and ER and confirms the expression of HER2/AR in VPD; new bases are provided to design multicenter clinical trials on personalized target therapies.


Assuntos
Metaboloma , Proteínas de Neoplasias/metabolismo , Doença de Paget Extramamária/diagnóstico , Doença de Paget Extramamária/metabolismo , Neoplasias Vulvares/diagnóstico , Neoplasias Vulvares/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imuno-Histoquímica/métodos , Metástase Linfática , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Doença de Paget Extramamária/patologia , Proteômica/métodos , Estudos Retrospectivos , Neoplasias Vulvares/patologia
12.
BMC Bioinformatics ; 20(1): 397, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315562

RESUMO

BACKGROUND: Tandem mass spectrometry (MS/MS)-based database searching is a widely acknowledged and widely used method for peptide identification in shotgun proteomics. However, due to the rapid growth of spectra data produced by advanced mass spectrometry and the greatly increased number of modified and digested peptides identified in recent years, the current methods for peptide database searching cannot rapidly and thoroughly process large MS/MS spectra datasets. A breakthrough in efficient database search algorithms is crucial for peptide identification in computational proteomics. RESULTS: This paper presents MCtandem, an efficient tool for large-scale peptide identification on Intel Many Integrated Core (MIC) architecture. To support big data processing capability, a novel parallel match scoring algorithm, named MIC-SDP (spectrum dot product), and its two-level parallelization are presented in MCtandem's design. In addition, a series of optimization strategies on both the host CPU side and the MIC side, which includes pre-fetching, optimized communication overlapping scheme, multithreading and hyper-threading, are exploited to improve the execution performance. CONCLUSIONS: For fair comparisons, we first set up experiments and verified the 28 fold times speedup on a single MIC against the original CPU-based implementation. We then execute the MCtandem for a very large dataset on an MIC cluster (a component of the Tianhe-2 supercomputer) and achieved much higher scalability than in a benchmark MapReduce-based programs, MR-Tandem. MCtandem is an open-source software tool implemented in C++. The source code and the parameter settings are available at https://github.com/LogicZY/MCtandem .


Assuntos
Peptídeos/química , Software , Espectrometria de Massas em Tandem , Algoritmos , Bases de Dados de Proteínas , Humanos , Proteômica/métodos
13.
Anal Bioanal Chem ; 411(21): 5351-5361, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31267193

RESUMO

Exosomes are membrane-bound vesicles secreted by cells, and contain various important biological molecules, such as lipids, proteins, messenger RNAs, microRNAs, and noncoding RNAs. Emerging evidence demonstrates that proteomic analysis of exosomes is of great significance in studying metabolic diseases, tumor metastasis, immune regulation, and so forth. However, exosome proteomic analysis has high requirements with regard to the purity of collected exosomes. Here recent advances in the methods for isolating exosomes and their applications in proteomic analysis are summarized. Graphical abstract.


Assuntos
Exossomos , Proteômica/métodos , Cromatografia de Afinidade/métodos , Humanos , MicroRNAs/metabolismo , Polietilenoglicóis/química , Proteínas/metabolismo , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo
14.
Adv Exp Med Biol ; 1140: 169-198, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347048

RESUMO

Mass Spectrometry (MS) has revolutionized the way we study biomolecules, especially proteins, their interactions and posttranslational modifications (PTM). As such MS has established itself as the leading tool for the analysis of PTMs mainly because this approach is highly sensitive, amenable to high throughput and is capable of assigning PTMs to specific sites in the amino acid sequence of proteins and peptides. Along with the advances in MS methodology there have been improvements in biochemical, genetic and cell biological approaches to mapping the interactome which are discussed with consideration for both the practical and technical considerations of these techniques. The interactome of a species is generally understood to represent the sum of all potential protein-protein interactions. There are still a number of barriers to the elucidation of the human interactome or any other species as physical contact between protein pairs that occur by selective molecular docking in a particular spatiotemporal biological context are not easily captured and measured.PTMs massively increase the complexity of organismal proteomes and play a role in almost all aspects of cell biology, allowing for fine-tuning of protein structure, function and localization. There are an estimated 300 PTMS with a predicted 5% of the eukaryotic genome coding for enzymes involved in protein modification, however we have not yet been able to reliably map PTM proteomes due to limitations in sample preparation, analytical techniques, data analysis, and the substoichiometric and transient nature of some PTMs. Improvements in proteomic and mass spectrometry methods, as well as sample preparation, have been exploited in a large number of proteome-wide surveys of PTMs in many different organisms. Here we focus on previously published global PTM proteome studies in the Apicomplexan parasites T. gondii and P. falciparum which offer numerous insights into the abundance and function of each of the studied PTM in the Apicomplexa. Integration of these datasets provide a more complete picture of the relative importance of PTM and crosstalk between them and how together PTM globally change the cellular biology of the Apicomplexan protozoa. A multitude of techniques used to investigate PTMs, mostly techniques in MS-based proteomics, are discussed for their ability to uncover relevant biological function.


Assuntos
Espectrometria de Massas , Mapeamento de Interação de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Humanos , Simulação de Acoplamento Molecular , Proteoma
15.
Adv Exp Med Biol ; 1140: 501-513, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347067

RESUMO

The diversity of biological samples and dynamic range of analytes being analyzed can prove to be an analytical challenge and is particularly prevalent to proteomic studies. Maximizing the peak capacity of the workflow employed can extend the dynamic range and increase identification rates. The focus of this chapter is to present means of achieving this for various analytical techniques such as liquid chromatography, mass spectrometry and ion mobility. A combination of these methods can be used as part of a data independent acquisition strategy, thereby limiting issues such as chimericy when analyzing regions of extreme analyte density.


Assuntos
Cromatografia Líquida , Espectrometria de Massas , Proteoma , Proteômica/métodos , Fluxo de Trabalho
16.
Adv Exp Med Biol ; 1140: 531-539, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347069

RESUMO

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach for high-throughput quantitative proteomics. SILAC allows highly accurate protein quantitation through metabolic encoding of whole cell proteomes using stable isotope labeled amino acids. Since its introduction in 2002, SILAC has become increasingly popular. In this chapter we review the methodology and application of SILAC, with an emphasis on three research areas: dynamics of posttranslational modifications, protein-protein interactions, and protein turnover.


Assuntos
Aminoácidos/química , Técnicas de Cultura de Células , Marcação por Isótopo , Proteômica/métodos , Proteoma
17.
Adv Exp Med Biol ; 1140: 563-574, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347071

RESUMO

Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS PAGE) is a method that separates proteins according to their isoelectric points in the first dimension and molecular masses in the second dimension. Evidence is provided that 2D SDS PAGE is reproducible, robust and compatible with SDS in both dimensions including isoelectric focusing in tube gels, the first dimension. The 2D gel pattern of rat liver microsomes shows more detail and sharper spot outlines when dissolved in SDS buffer with heating than in urea buffer and is better yet when dissolved in a mixture of both buffers. Quantification of 60 proteins in rat liver cytosol over a wide range of pI and MW gave linear plots of spot density versus total protein for loads of 200, 400 and 600 µg protein dissolved in SDS buffer and run in triplicate on 2D gels (Average R2 = 0.987). Examples of biomedical applications are provided in which 2D proteins of interest found by comparing stained or western blotted 2D gel patterns were identified by mass spectrometry (MS).


Assuntos
Western Blotting , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Proteômica/métodos , Animais , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Microssomos Hepáticos , Ratos
18.
Adv Exp Med Biol ; 1140: 753-769, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347083

RESUMO

Mass spectrometry (MS) is the core for advanced methods in proteomic experiments. When effectively used, proteomics may provide extensive information about proteins and their post-translational modifications, as well as their interaction partners. However, there are also many problems that one can encounter during a proteomic experiment, including, but not limited to sample preparation, sample fractionation, sample analysis, data analysis & interpretation and biological significance. Here we discuss some of the problems that researchers should be aware of when performing a proteomic experiment.


Assuntos
Espectrometria de Massas , Proteínas/análise , Proteômica/métodos , Processamento de Proteína Pós-Traducional
19.
BMC Bioinformatics ; 20(1): 398, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315557

RESUMO

BACKGROUND: Utilization of quantitative proteomics data on the network level is still a challenge in proteomics data analysis. Currently existing models use sophisticated, sometimes hard to implement analysis techniques. Our aim was to generate a relatively simple strategy for quantitative proteomics data analysis in order to utilize as much of the data generated in a proteomics experiment as possible. RESULTS: In this study, we applied label-free proteomics, and generated a network model utilizing both qualitative, and quantitative data, in order to examine the early host response to Human Immunodeficiency Virus type 1 (HIV-1). A weighted network model was generated based on the amount of proteins measured by mass spectrometry, and analysis of weighted networks and functional sub-networks revealed upregulation of proteins involved in translation, transcription, and DNA condensation in the early phase of the viral life-cycle. CONCLUSION: A relatively simple strategy for network analysis was created and applied to examine the effect of HIV-1 on host cellular proteome. We believe that our model may prove beneficial in creating algorithms, allowing for both quantitative and qualitative studies of proteome change in various biological and pathological processes by quantitative mass spectrometry.


Assuntos
HIV-1/fisiologia , Proteômica/métodos , HIV-1/genética , Humanos , Espectrometria de Massas , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Transdução Genética
20.
Toxicol Lett ; 313: 150-158, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276768

RESUMO

Ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins, is a possible carcinogen to humans. We previously demonstrated that long-term (40 weeks) OTA exposure induces the malignant transformation of human gastric epithelium cells (GES-1) in vitro. However, the specific mechanism underlying OTA-induced gastric carcinogenesis is complex. In the present study, we used 2-DE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF MS) combined with bioinformatics and immunoblotting to investigate the differentially expressed proteins between GES-1 and OTA-malignant transformed GES-1 cells (OTA-GES-1T cells) in vitro. We found that four differentially expressed proteins were identified after malignant transformation, including actin, cytoplasmic 1 (ACTB), F-actin-capping protein subunit alpha-1 (CAPZA1), Annexin A3 (ANXA3), thioredoxin peroxidase B from red blood cells (TPx-B) and Fibrinogen beta B (Fibrinogen ß). Among the differentially expressed proteins, the effect of Annexin A3 was analyzed by MTT assay, western blot, cell cycle analysis, wound healing assay, Transwell assay, and colony formation assay in OTA-GES-1T cells. The results showed that inhibition of Annexin A3 by siRNA effectively prevented the proliferation, migration, and invasion abilities of OTA-GES-1T cells. Collectively, the results of this study will guide future research on OTA carcinogenicity.


Assuntos
Anexina A3/metabolismo , Carcinógenos/toxicidade , Transformação Celular Neoplásica/induzido quimicamente , Células Epiteliais/efeitos dos fármacos , Mucosa Gástrica/efeitos dos fármacos , Ocratoxinas/toxicidade , Neoplasias Gástricas/induzido quimicamente , Anexina A3/genética , Western Blotting , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Biologia Computacional , Eletroforese em Gel Bidimensional , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Humanos , Invasividade Neoplásica , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
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