Two-dimensional gel electrophoresis in proteomics: Past, present and future

Rabilloud, Thierry; Chevallet, Mireille; Luche, Sylvie; Lelong, Cécile

doi:10.1016/j.jprot.2010.05.016

Cited by 393 publications

(274 citation statements)

References 207 publications

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“…Disorder-specific protein markers play a central diagnostic, prognostic, and therapeutic role in skeletal muscle pathology and the systematic application of proteomics has greatly expanded the range of biomarkers for neuromuscular disorders [6]. Proteome-wide studies combine protein separation methods, such as high-resolution two-dimensional gel electrophoresis [7][8][9] and liquid chromatography [10], with sophisticated mass spectrometric techniques to determine potential changes in protein concentration, isoform expression patterns, protein-protein interactions and posttranslational modifications [11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle

Carberry

Zweyer

Swandulla

et al. 2014

Analytical Biochemistry

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a b s t r a c tIn basic and applied myology, gel-based proteomics is routinely used for studying global changes in the protein constellation of contractile fibers during myogenesis, physiological adaptations, neuromuscular degeneration, and the natural aging process. Since the main proteins of the actomyosin apparatus and its auxiliary sarcomeric components often negate weak signals from minor muscle proteins during proteomic investigations, we have here evaluated whether a simple prefractionation step can be employed to eliminate certain aspects of this analytical obstacle. To remove a large portion of highly abundant contractile proteins from skeletal muscle homogenates without the usage of major manipulative steps, differential centrifugation was used to decisively reduce the sample complexity of crude muscle tissue extracts. The resulting protein fraction was separated by two-dimensional gel electrophoresis, and 2D-landmark proteins were identified by mass spectrometry. To evaluate the suitability of the contractileprotein-depleted fraction for comparative proteomics, normal versus dystrophic muscle preparations were examined. The mass spectrometric analysis of differentially expressed proteins, as determined by fluorescence difference in-gel electrophoresis, identified 10 protein species in dystrophic mdx hindlimb muscles. Interesting new biomarker candidates included Hsp70, transferrin, and ferritin, whereby their altered concentration levels in dystrophin-deficient muscle were confirmed by immunoblotting.Ó 2013 Elsevier Inc. All rights reserved.In high-throughput biochemistry, mass spectrometry is the method of choice for the fast and reliable identification of proteins in large-scale surveys of physiological or pathological processes [1][2][3]. This makes protein mass spectrometry an integral part of biological network analysis [4] and the discovery of novel disease biomarker signatures [5]. Disorder-specific protein markers play a central diagnostic, prognostic, and therapeutic role in skeletal muscle pathology and the systematic application of proteomics has greatly expanded the range of biomarkers for neuromuscular disorders [6]. Proteome-wide studies combine protein separation methods, such as high-resolution two-dimensional gel electrophoresis [7-9] and liquid chromatography [10], with sophisticated mass spectrometric techniques to determine potential changes in protein concentration, isoform expression patterns, protein-protein interactions and posttranslational modifications [11][12][13].However, proteomic findings from comparative studies focusing on total protein extracts from highly complex and dynamic types of tissues, such as skeletal muscle fibers, are often limited to mostly soluble and relatively abundant proteins [14][15][16], missing especially the classes of very low abundance proteins and hydrophobic proteins. Thus, to cover all of the assessable constituents in a heterogeneous assembly of proteins with a greatly differing abundance and physicochemical properties, as are found in contr...

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Section: Introductionmentioning

confidence: 99%

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle

Carberry

Zweyer

Swandulla

et al. 2014

Analytical Biochemistry

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“…Hay que contar con la limitación intrínseca que supone un estudio in vitro, que no refleja las distintas interacciones que tendrán lugar en el conjunto del organismo. Asimismo, la identificación de las proteínas mediante la doble electroforesis no es exacta, y puede subestimar la presencia de elementos poco abundantes, las proteínas integradas en la membrana o componentes con peso molecular muy elevado (158)(159)(160)(161). Además, aquellas proteínas con valores de pI extremos no suelen ser bien detectadas en sistemas con un amplio rango de pI.…”

Section: Contenido De Lactato Y Piruvatounclassified

Efecto del alprostadil en los cultivos de músculo esquelético isquémico. Estudio proteómico comparativo frente a músculo sano

et al. 2014

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El presente trabajo reúne las condiciones de rigor y originalidad científica para ser presentada como tesis doctoral.Y para que conste a los efectos oportunos firmo la presente certificación en El presente trabajo reúne las condiciones de rigor y originalidad científica para ser presentada como tesis doctoral.Y para que conste a los efectos oportunos firmo la presente certificación en ABREVIATURAS• ADP: Adenosín Difosfato• AC: Actina• ATP: Adenosín Trifosfato• Arg: arginina• BCA: ácido bicinconínico• Bcl-2 :B-cell Lymphoma 2• BSA: albúmina del suero bovino• CE: Célula embrionaria• CHAPS: cholamidopropil-2 hidroxi-1 propanosulfato• CML: Célula madre linfoide• Dl: decilitros• DGºf: Variación de la energía libre de Gibbs estándar de formación• DHAP: Dihidroxiacetona-fosfato• EAP: Enfermedad arterial periférica• EDTA: ácido etilendiaminotetraacético• ESI : del inglés, ElectroSpray Ionization• FAD: Flavín adenina dinucleótido• FADD : factor associated death domain• F-6-P: Fructosa 6 fosfato• FT-MS : del inglés, Fourier Transform ion cyclotron• G3P: Gliceraldehido 3 fosfato• G6P: Gliceraldehido 6 fosfato 20• HDL: Lipoproteina de alta densidad• HEPES: ácido 4-(2-HidroxiEtil)-1-PiperacinaEtanosulfónico.• ITB: Indice tobillo brazo• IPG: del inglés Isoelectric Phocusing Gradient• LDH: lactato deshidrogenasa• LDL: Lipoproteina de baja densidad• Lys: lisina• MALDI : del ingles, Matrix-Assisted Laser Desorption/Ionization• MDH: malato deshidrogenasa• Mcl-1 : Mieloid cell leucemia-1• mg: milígramos• Mi: Miosina• ml: mililitros• mm: milímetros• mmHg: milímetros de Mercurio• mmol: milimol• MS: espectrometría de masas• NAD: Nicotinamida adenina dinucleotido• nm: nanometros• PaO2: presión parcial arterial de oxígeno• PAS: Presión arterial sistólica• PC: piruvato carboxilasa• PCR: Proteina C reactiva• PEP: fosfoenolpirúvico• PGE1: Prostaglandina E1 21• PGE2: Prostaglandina E2• pg: picogramos• PVDF :del inglés polyvinylidene difluoride• SDS-PAGE :del inglés sodium dodecyl sulfate polyacrylamide gel electrophoresis• seg: segundos• SFB: suero fetal bovino• SNC: Sistema Nervioso Central• TNF: Factor de necrosis tumoral• TOF : del inglés, time of flight• TPI: Triosafosfato isomerasa• UAD: Unidades Arbitrarias Densitométricas• μg: nanogramos• 2-DE: del inglés 2-dimensional electrophoresis Cada haz está conformado por miles de fibras, perfectamente separables porque cada una está envuelta por su propia capa de tejido conjuntivo llamada endomisio. El total de fibras que finalmente tendrá el músculo queda establecido ya a los 4 ó 5 meses de vida fetal. El grosor, en cambio, es al nacer la quinta parte de lo que tendrá de adulto. En éste es muy variable, según el músculo de donde se saque la muestra ( a través de una biopsia). En general el grosor va de 10 milimicrones a 100 milimicrones (1 micrón es una parte de mil). Su longitud suele ir a lo largo de todo el recorrido del músculo, alcanzando hasta 30 centímetros en algunos casos.En el interior de cada fibra se encuentran las miofibrillas segmentadas en una estructura histológica conocid...

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“…Despite its impressive capabilities for protein separation and identification, 2-DE may not be a good approach for the analysis of hydrophobic proteins, proteins of low abundance, or samples that contain proteins with concentrations in a large dynamic range Corthals et al, 2000;Rabilloud et al, 2010). For proteins to be resolved and appear as a single spot on a 2-D polyacrylamide gel, they should be completely soluble, denatured and reduced under the conditions employed.…”

Section: Limitations Of 2-dementioning

confidence: 99%

“…As a result, 2-DE may greatly under-represent the whole proteome. In such situations, strategies including the depletion of high abundance proteins, the use of very narrow-range IPG strips (~1 pH unit) or proteome subfractionation (see section below), can be employed to enable the detection of low abundance proteins (Greenough et al, 2004;Chevalier, 2010;Rabilloud et al, 2010).…”

Section: Limitations Of 2-dementioning

confidence: 99%

Two-dimensional gel electrophoresis in bacterial proteomics

et al. 2012

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Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples. It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques: isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension). 2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell: its proteome. Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmental and medically-relevant bacteria. Here we give a broad overview of the basic principles and historical development of gel-based proteomics, and how this powerful approach can be applied for studying bacterial biology and physiology. We highlight specific 2-DE applications that can be used to analyze when, where and how much proteins are expressed. The links between proteomics, genomics and mass spectrometry are discussed. We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by de novo peptide sequencing. The use of proteome fractionation techniques and non-gel-based proteomic approaches are also discussed. We highlight how the analysis of proteins secreted by bacterial cells (secretomes or exoproteomes) can be used to study infection processes or the immune response. This review is aimed at non-specialists who wish to gain a concise, comprehensive and contemporary overview of the nature and applications of bacterial proteomics.

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Two-dimensional gel electrophoresis in proteomics: Past, present and future

Cited by 393 publications

References 207 publications

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle

Efecto del alprostadil en los cultivos de músculo esquelético isquémico. Estudio proteómico comparativo frente a músculo sano

Two-dimensional gel electrophoresis in bacterial proteomics

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