RESUMEN

Polysorbate (PS) degradation in monoclonal antibody (mAb) formulations poses a significant challenge in the biopharmaceutical industry. PS maintains protein stability during drug product's shelf life but is vulnerable to breakdown by low-abundance residual host cell proteins (HCPs), particularly hydrolytic enzymes such as lipases and esterases. In this study, we used activity-based protein profiling (ABPP) coupled with mass spectrometry to identify acyl-protein thioesterase-1 (APT-1) as a polysorbate-degrading HCP in one case of mAb formulation with stability problems. We validated the role of APT1 by matching the polysorbate degradation fingerprint in the mAb formulation with that of a recombinant APT1 protein. Furthermore, we found an agreement between APT1 levels and PS degradation rates in the mAb formulation, and we successfully halted PS degradation using APT1-specific inhibitors ML348 and ML211. APT1 was found to co-purify with a specific mAb via hitchhiking mechanism. Our work provides a streamlined approach to identifying critical HCPs in PS degradation, supporting quality-by-design principles in pharmaceutical development.

Asunto(s)

Anticuerpos Monoclonales , Polisorbatos , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/química , Polisorbatos/química , Animales , Tioléster Hidrolasas/metabolismo , Humanos , Cricetulus , Proteínas Recombinantes/metabolismo , Células CHO , Espectrometría de Masas/métodos , Estabilidad Proteica

RESUMEN

Manufacturing of biopharmaceuticals involves recombinant protein expression in host cells followed by extensive purification of the target protein. Yet, host cell proteins (HCPs) may persist in the final drug product, potentially reducing its quality with respect to safety and efficacy. Consequently, residual HCPs are closely monitored during downstream processing by techniques such as enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS). The latter is especially attractive as it provides information with respect to protein identities. Although the applied HPLC-MS/MS methodologies are frequently optimized with respect to HCP identification, acquired data is typically analyzed using standard settings. Here, we describe an improved strategy for evaluating HPLC-MS/MS data of HCP-derived peptides, involving probabilistic protein inference and peptide detection in the absence of fragment ion spectra. This data analysis workflow was applied to data obtained for drug products of various biotherapeutics upon protein A affinity depletion. The presented data evaluation strategy enabled in-depth comparative analysis of the HCP repertoires identified in drug products of the monoclonal antibodies rituximab and bevacizumab, as well as the fusion protein etanercept. In contrast to commonly applied ELISA strategies, the here presented workflow is process-independent and may be implemented into existing HPLC-MS/MS setups for drug product characterization and process development. Graphical abstract.

Asunto(s)

Bevacizumab/química , Contaminación de Medicamentos , Etanercept/química , Rituximab/química , Productos Biológicos/química , Cromatografía Líquida de Alta Presión/métodos , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteínas Recombinantes de Fusión/química , Espectrometría de Masas en Tándem/métodos

RESUMEN

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de-etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll-binding protein complexes and development of water splitting via O2 production in plastids (etiochloroplasts) isolated during de-etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light-harvesting antenna [PSII-light-harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de-etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de-etiolation, etiochloroplasts revealed the same water-splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de-etiolation precedes assembly of the PSII-LHCII supercomplexes. Taken together, data show a rapid establishment of water-splitting activity during etioplast-to-chloroplast transition and emphasize that assembly of the functional water-splitting site of PSII is not the rate-limiting step in the formation of photoactive thylakoid membranes.

Asunto(s)

Etiolado , Hordeum/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Agua/metabolismo , Clorofila/metabolismo , Biogénesis de Organelos

RESUMEN

The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.).

Asunto(s)

Clorofila/metabolismo , Hordeum/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Clorofila A , Cloroplastos/metabolismo , Luz , Oxidorreductasas/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Fitol/metabolismo , Unión Proteica/fisiología

RESUMEN

Host cell proteins (HCPs) are process-related impurities present in biopharmaceuticals and are generally considered to be critical quality attributes. Changes in a biopharmaceutical production process may result in qualitative shifts in the HCP population. These shifts are not necessarily detectable when overall HCP levels are measured with traditional approaches such as enzyme-linked immunosorbent assays (ELISAs). Thus, the development of techniques that complement the ELISA's functionality is desirable. Here, a mass spectrometry (MS)-based approach for the analysis of HCP populations in biopharmaceuticals is presented. It consists of (i) the generation of exclusion lists that represent the masses of the active pharmaceutical ingredient (API), (ii) the compilation of inclusion lists based on an HCP catalog derived from the analysis of protein A-purified samples, and (iii) the analysis of purified biopharmaceuticals using the generated exclusion and inclusion lists. With this approach, it was possible to increase sensitivity for HCP detection compared with a standard liquid chromatography tandem MS (LC-MS/MS) run. The workflow was successfully implemented in a comparability exercise assessing HCP populations in drug substance samples before and after a process change. Furthermore, the results suggest that size can be an important factor in the copurification of HCPs and API.

Asunto(s)

Técnicas de Química Analítica/métodos , Proteínas/análisis , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Peso Molecular , Preparaciones Farmacéuticas/análisis , Proteínas/metabolismo , Proteínas S100/análisis , Proteínas S100/metabolismo , Tripsina/metabolismo

RESUMEN

Chloroplasts are descendants of cyanobacteria and divide by binary fission. The number of chloroplasts is regulated in a cell type-specific manner to ensure that specialized cell types can perform their functions optimally. Several protein components of the chloroplast division apparatus have been identified in the past several years, but how this process is regulated in response to developmental status, environmental signals and stress is still unknown. To begin to address this we undertook a proteomic analysis of three accumulation and replication of chloroplasts mutants that show a spectrum of plastid division perturbations. We show that defects in the chloroplast division process results in changes in the abundance of proteins when compared to wild type, but that the profile of the native stromal and membrane complexes remains unchanged. Furthermore, by combining BN-PAGE with protein interaction assays we show that AtFtsZ2-1 and AtFtsZ2-2 assemble together with rpl12A and EF-Tu into a novel chloroplast membrane complex.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Proteoma , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/fisiología , Cloroplastos/fisiología , Cromatografía Líquida de Alta Presión , Regulación de la Expresión Génica de las Plantas , Mutación , Factor Tu de Elongación Peptídica/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Plantas Modificadas Genéticamente , Proteómica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Plantones/genética , Plantones/metabolismo , Plantones/fisiología , Espectrometría de Masas en Tándem , Nicotiana/citología , Nicotiana/genética , Nicotiana/metabolismo , Electroforesis Bidimensional Diferencial en Gel , Técnicas del Sistema de Dos Híbridos

RESUMEN

CyDye labeling and DIGE have not only been proven to work for soluble proteins but also at the level of whole membrane protein complexes. After complex solubilization and CyDye labeling, proteins can be separated by native PAGE which is often combined with SDS PAGE in a subsequent step. By this combination, sizes of complexes as well as their subunit composition can be compared after mixing samples from different physiological states. Plants interact specifically with light via protein-bound pigments. This can be used in combination with CyDye technology to extend the "classical" approach in plant research. As an example, chlorophyll can be excited for fluorescent scanning at the Cy5 excitation wavelength. This property can be used to identify pigment-binding plant complexes and complex subunits isolated from plastid membranes. In this protocol, we present a combination of the conventional CyDye labeling technique with 2D native/SDS PAGE and parallel scanning for CyDyes and fluorescence from endogenous bound chlorophyll for identification of pigment-binding complexes and complex subunits.

Asunto(s)

Colorantes Fluorescentes/química , Proteínas de Plantas/análisis , Proteínas de Plantas/química , Electroforesis Bidimensional Diferencial en Gel/métodos , Métodos Analíticos de la Preparación de la Muestra , Proteínas de Plantas/aislamiento & purificación

RESUMEN

Angiosperms grown in darkness develop etioplasts during skotomorphogenesis. It is well known that etioplasts accumulate large quantities of protochlorophyllideoxidoreductase, are devoid of chlorophyll and are the site to assemble the photosynthetic machinery during photomorphogenesis. Proteomic investigation of the membrane protein complexes by Native PAGE, in combination with CyDye labelling and mass spectrometric analysis revealed that etioplasts and chloroplasts share a number of membrane protein complexes characteristic for electron transport, chlorophyll and protein synthesis as well as fatty acid biosynthesis. The complex regulatory function in both developmental states is discussed.

Asunto(s)

Proteínas de Cloroplastos/química , Cloroplastos/química , Secuencia de Aminoácidos , Clorofila/metabolismo , Cloroplastos/genética , Complejo de Citocromo b6f/metabolismo , Oscuridad , Hordeum/crecimiento & desarrollo , Hordeum/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Proteómica

RESUMEN

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b(559), with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and ß-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q(Y) region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.

Asunto(s)

Complejos de Proteína Captadores de Luz/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Multimerización de Proteína , Synechocystis/metabolismo , Immunoblotting , Complejos de Proteína Captadores de Luz/aislamiento & purificación , Espectrometría de Masas , Métodos , Complejo de Proteína del Fotosistema II/aislamiento & purificación , Subunidades de Proteína/aislamiento & purificación

RESUMEN

The phototrophic consortium 'Chlorochromatium aggregatum' is a highly structured association of green sulfur bacterial epibionts surrounding a central, motile bacterium and is the most specific symbiosis currently known between two phylogenetically distinct bacterial species. Genes and gene products potentially involved in the symbiotic interaction were identified on the genomic, transcriptomic and proteomic level. As compared with the 11 available genomes of free-living relatives, only 186 open reading frames were found to be unique to the epibiont genome. 2-D differential gel electrophoresis (2-D DIGE) of the soluble proteomes recovered 1612 protein spots of which 54 were detected exclusively in consortia but not in pure epibiont cultures. Using mass spectrometry analyses, the 13 most intense of the 54 spots could be attributed to the epibiont. Analyses of the membrane proteins of consortia, of consortia treated with cross-linkers and of pure cultures indicated that a branched chain amino acid ABC-transporter binding protein is only expressed in the symbiotic state of the epibiont. Furthermore, analyses of chlorosomes revealed that an uncharacterized 11 kDa epibiont protein is only expressed during symbiosis. This protein may be involved in the intracellular sorting of chlorosomes. Application of a novel prokaryotic cDNA suppression subtractive hybridization technique led to identification of 14 differentially regulated genes, and comparison of the transcriptomes of symbiotic and free-living epibionts indicated that 328 genes were differentially transcribed. The three approaches were mostly complementary and thereby yielded a first inventory of 352 genes that are likely to be involved in the bacterial interaction in 'C. aggregatum'. Notably, most of the regulated genes encoded components of central metabolic pathways whereas only very few (7.5%) of the unique 'symbiosis genes' turned out to be regulated under the experimental conditions tested. This pronounced regulation of central metabolic pathways may serve to fine-tune the symbiotic interaction in 'C. aggregatum' in response to environmental conditions.

Asunto(s)

Chlorobi/genética , Consorcios Microbianos/genética , Simbiosis/genética , Chlorobi/fisiología , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Genoma Bacteriano , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteoma , Análisis de Secuencia de ADN , Transcriptoma

RESUMEN

One major problem in proteomics is the biochemical complexity of living cells. Therefore, strategies are needed to reduce the number of proteins to a manageable amount, enabling researchers to make a statement concerning protein functions. One possibility is the isolation of organelles, which reduces the protein complexity, e.g., for the chloroplast to an estimated number of 2,700 different proteins. For further limitation of the protein number, proteins can be divided into membrane and soluble proteins, which can be analyzed separately in a subsequent step. For membrane proteins, blue native polyacrylamide gel electrophoresis (BN-PAGE) in combination with enzymatic in-gel assays (e.g. detection of NADPH dehydrogenases) is a suitable method for a fast and easy visualization and identification of only one class of membrane proteins.

Asunto(s)

Cloroplastos/química , Electroforesis en Gel de Poliacrilamida/métodos , Orgánulos/química , Proteínas de Plantas/análisis , Proteoma/análisis , Proteómica/métodos

RESUMEN

The proteome of the cell is at the frontier of being too complex for proteomic analysis. Organelles provide a step up. Organelles compartmentalize the cell enabling a proteome, physiology and metabolism analysis in time and in space. Protein complexes separated by electrophoresis have been identified as the next natural level to characterize the organelles' compartmentalized membrane and soluble proteomes by mass spectrometry. Work on mitochondria and chloroplasts has shown where we are in the characterization of complex proteomes to understand the network of endogenous and extrinsic factors which regulate growth and development, adaptation and evolution.

Asunto(s)

Orgánulos/química , Proteoma/análisis , Proteómica/métodos , Cloroplastos/química , Electroforesis en Gel de Poliacrilamida/instrumentación , Electroforesis en Gel de Poliacrilamida/métodos , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Mitocondrias/química , Proteómica/instrumentación

RESUMEN

In Photosystem II (PSII), a high number of plastid encoded and membrane integral low molecular weight proteins smaller than 10 kDa, the proteins PsbE, F, H, I, J, K, L, M, N, Tc, Z and the nuclear encoded PsbW, X, Y1, Y2 proteins have been described. Here we show that all low molecular weight proteins of PSII already accumulate in the etioplast membrane fraction in darkness, whereas PsaI and PsaJ of photosystem I (PSI) represent the only low molecular weight proteins that do not accumulate in darkness. We found by BN-PAGE separation of membrane protein complexes and selective MS that the accumulation of one-helix proteins from PSII is light independent and occurs in etioplasts. In contrast, in chloroplasts isolated from light-grown plants, low molecular weight proteins were found to specifically accumulate in PSI and II complexes. Our results demonstrate how plants grown in darkness prepare for the induction of chlorophyll dependent photosystem assembly upon light perception. We anticipate that our investigation will provide the essential means for the analysis of protein assembly in any membrane utilizing low molecular weight protein subunits.

Asunto(s)

Cloroplastos/metabolismo , Hordeum/metabolismo , Proteínas de la Membrana/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/metabolismo , Peso Molecular , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray

RESUMEN

The N-termini of the NADPH : protochlorophyllide oxidoreductase (POR) proteins A and B from barley and POR from pea were determined by acetylation of the proteins and selective isolation of the N-terminal peptides for mass spectrometry de novo sequence analysis. We show that the cleavage sites between the transit peptides and the three mature POR proteins are homologous. The N-terminus in PORA is V48, that in PORB is A61, and that in POR from pea is E64. For the PORB protein, two additional N-termini were identified as A62 and A63, with decreased signal intensity of the corresponding N-terminal peptides. The results show that the transit peptide of PORA is considerably shorter than previously reported and predicted by ChloroP. A pentapeptide motif that has been characterized as responsible for binding of protochlorophyllide to the transit peptide of PORA [Reinbothe C, Pollmann S, Phetsarath-Faure P, Quigley F, Weisbeek P & Reinbothe S (2008) Plant Physiol148, 694-703] is shown here to be part of the mature PORA protein.

Asunto(s)

Cloroplastos/enzimología , Hordeum/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Proteínas de Plantas/química , Acetilación , Secuencia de Aminoácidos , Sitios de Unión , Datos de Secuencia Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Proteínas de Plantas/metabolismo , Espectrometría de Masas en Tándem

RESUMEN

Blue native PAGE is an electrophoretic technique for high-resolution separation of membrane proteins. The method has been proven especially useful for investigation of native protein complexes enabling a characterization of potential protein-protein interactions in the context of functional proteomics. Blue native PAGE is easy to realise, results are reproducible and a high number of protocols are available. However, care should be taken during solubilization of protein complexes to achieve significant results in BN-PAGE analysis. Solubilization of membranes and proteins is not only influenced by detergent-lipid and detergent-protein interactions but also by lipid-lipid, lipid-protein and protein-protein interactions. Interactions have been investigated experimentally and theoretically. But, in practice, the experimental results do not always mirror the theoretical basis and therefore optimal solubilization conditions for each membrane and membrane protein complex should be investigated individually to tap the full potential of BN-PAGE analysis.

Asunto(s)

Electroforesis en Gel de Poliacrilamida/métodos , Proteómica/métodos , Animales , Detergentes/química , Detergentes/farmacología , Proteínas Fúngicas/química , Iones , Focalización Isoeléctrica/métodos , Lípidos/química , Proteínas de la Membrana/química , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Mapeo de Interacción de Proteínas , Proteoma

RESUMEN

Dark-grown angiosperm seedlings are etiolated and devoid of chlorophyll. Deetiolation is triggered by light leading to chlorophyll dependent accumulation of the photosynthetic machinery. The transfer of chlorophyll to the chlorophyll-binding proteins is still unclear. We demonstrate here that upon illumination of dark-grown barley seedlings, two new pigment-binding protein complexes are de novo accumulated. Pigments bound to both complexes are identified as chlorophyll a and protochlorophyll a. By auto-fluorescence tracking and mass spectrometry, we show that exclusively Lil3 is the pigment-binding complex subunit in both complexes.

Asunto(s)

Clorofila/biosíntesis , Hordeum/efectos de la radiación , Complejos de Proteína Captadores de Luz/metabolismo , Luz , Proteínas de Plantas/metabolismo , Cloroplastos/enzimología , Cloroplastos/efectos de la radiación , Oscuridad , Hordeum/enzimología , Subunidades de Proteína/metabolismo , Semillas/enzimología , Semillas/efectos de la radiación

RESUMEN

Blue native PAGE is a discontinuous electrophoretic system that allows the separation of membrane protein complexes in a native, enzymatically active state with high resolution. Membrane protein complexes are solubilized by neutral, nondenaturing detergents like n-dodecyl-beta-D-maltoside. After addition of Coomassie G250 that binds to the surface of the proteins, separation of the negatively charged complexes according to molecular mass is possible. After electrophoresis the structure and function of the isolated protein complexes can be investigated.

Asunto(s)

Electroforesis en Gel de Poliacrilamida/métodos , Glucósidos/química , Proteínas de la Membrana/análisis , Complejos Multiproteicos/análisis , Colorantes de Rosanilina/química , Animales , Humanos , Proteínas de la Membrana/química , Peso Molecular , Complejos Multiproteicos/química

RESUMEN

The cytochrome b6f complex is a dimeric protein complex that is of central importance for photosynthesis to carry out light driven electron and proton transfer in chloroplasts. One molecule of chlorophyll a was found to associate per cytochrome b6f monomer and the structural or functional importance of this is discussed. We show that etioplasts which are devoid of chlorophyll a already contain dimeric cytochrome b6f. However, the phytylated chlorophyll precursor protochlorophyll a, and not chlorophyll a, is associated with subunit b6. The data imply that a phytylated tetrapyrrol is an essential structural requirement for assembly of cytochrome b6f.

Asunto(s)

Clorofila/análogos & derivados , Cloroplastos/enzimología , Complejo de Citocromo b6f/metabolismo , Proteínas de Plantas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Clorofila/química , Clorofila/metabolismo , Complejo de Citocromo b6f/química , Dimerización , Proteínas de Plantas/química , Ribulosa-Bifosfato Carboxilasa/química , Ribulosa-Bifosfato Carboxilasa/metabolismo

RESUMEN

Natural compartmentalization makes proteome analysis of the cell, cell organelles and organelle subfractions possible. Protein complexes are the basis for the next level of compartmentalization that can be addressed well with proteomic technology. Protein complexes organize and maintain the cellular and organelle functions on all levels of complexity in time and space. Cell development and division, transcription and translation, respiration and photosynthesis, transport and metabolism can be defined by the activity of protein complexes. Since a large part of the protein complexes of the cell body are inserted in lipid membrane phases, isolation, separation and protein subunit identification were difficult to address. Blue native polyacrylamide gel electrophoresis (BN-PAGE) provides us with the technology for high resolution separation of membrane protein complexes. Here, we show that high resolution separation of protein complexes by BN-PAGE requires the establishment of a detailed solubilisation strategy. We show that BN/SDS-PAGE provides the scientist with a high resolution array of protein subunits which allows analysis of the specific subunit stoichiometry of a protein complex as well as the assembly of protein complexes by standard protein detection methodology like DIGE, gelblot analysis and mass spectrometry. We envision BN-PAGE to precede classical 2D IEF/SDS-analysis for detailed characterization of membrane proteomes.

Asunto(s)

Electroforesis en Gel Bidimensional/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Proteínas de la Membrana/aislamiento & purificación , Complejos Multiproteicos/análisis , Compartimento Celular , Digitonina/farmacología , Glucósidos/farmacología , Focalización Isoeléctrica , Espectrometría de Masas , Micelas , Colorantes de Rosanilina , Solubilidad , Tilacoides/química

RESUMEN

Anabaena sp. PCC 7120 is a filamentous cyanobacterium that serves as a model to analyze prokaryotic cell differentiation, evolutionary development of plastids, and the regulation of nitrogen fixation. The cell wall is the cellular structure in contact with the surrounding medium. To understand the dynamics of the cell wall proteome during cell differentiation, the cell wall from Anabaena heterocysts was enriched and analyzed. In line with the recently proposed continuity of the outer membrane along the Anabaena filament, most of the proteins identified in the heterocyst cell-wall fraction are also present in the cell wall of vegetative cells, even though the lipid content of both membranes is different.

Asunto(s)

Anabaena/química , Anabaena/citología , Pared Celular/química , Proteoma/análisis , Anabaena/genética , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismo