New zwitterionic detergents improve the analysis of membrane proteins by two‐dimensional electrophoresis

Chevallet, Mireille; Santoni, Véronique; Poinas, Alexandra; Rouquié, David; Fuchs, Alexandra; Kieffer, Sylvie; Lunardi, Joël; Garin, Jérôme; Rabilloud, Thierry

doi:10.1002/elps.1150191108

Cited by 322 publications

(228 citation statements)

References 24 publications

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“…Overall, the effectiveness of each detergent can be easily observed by the number and abundance of spots revealed between the isoelectric points 4.5 to 9.5, and the molecular weight values within 17.9 and 88.1 kDa. Other studies have also shown the effectiveness of these sulfobetaines detergents for 2-DE analysis (4,6,12,15).…”

Section: Resultsmentioning

confidence: 94%

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Assessment of four different detergents used to extract membrane proteins from Xylella fastidiosa by two-dimensional electrophoresis

Ciero¹,

Bellato²,

Meinhardt³

et al. 2004

Braz. J. Microbiol.

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Four different detergents, ASB 14, SB 3-10, CHAPS and Triton X100, were utilized to determine the optimal detergent for the solubilization of membrane proteins from the phytopathogenic bacterium Xylella fastidiosa. These proteins were differentially solubilized in distinct buffers containing the detergent and subjected to bidimensional electrophoresis within the non-linear pH range of 3-10. The detergents ASB 14 and SB 3-10 were the most effective revealing 221 and 157 spots, respectively. CHAPS and Triton X100 were less effective and revealed only 72 and 43 spots, respectively. MALDI-TOF tryptic peptide mass fingerprinting of 18 excised proteins from the ASB 14 treatment revealed that 83% were membrane proteins and that the theoretical efficiency of solubilization for ASB 14 was estimated to be 87%. This study demonstrates the effectiveness of the detergent ASB 14 for the solubilization of membrane proteins from the bacterium X. fastidiosa.

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Section: Resultsmentioning

confidence: 94%

“…The solubility problems have been addressed, with varying success, by combining chaotropic agents, such as thiourea, urea, and guanidine hydrochloride, with detergents (sodium dodecyl sulfate, lithium dodecyl sulfate, Triton X-100, Nonidet P-40 and CHAPS) (4).…”

Section: Introductionmentioning

confidence: 99%

Assessment of four different detergents used to extract membrane proteins from Xylella fastidiosa by two-dimensional electrophoresis

Ciero¹,

Bellato²,

Meinhardt³

et al. 2004

Braz. J. Microbiol.

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“…A GRAVY score is a single-value indication of the overall hydropathicity of a protein sequence based on the Kyte and Doolittle algorithms; the more positive the score, the more hydrophobic the overall sequence (Kyte and Doolittle, 1982). The addition of thiourea as a chaotropic agent, the advent of new nonionic sulfobetaine detergents, and the use of the non-charged reductant, tributylphosphine, have all been credited with allowing more hydrophobic proteins to enter twodimensional gels for separation and analysis (Chevallet et al, 1998;Rabilloud, 1998). To date, proteins with GRAVY scores of approximately ϩ0.25 to ϩ0.30 are considered to represent the upper limit of these two-dimensional gel solubility improvements (Herbert, 1999).…”

Section: More Hydrophobic Large and Basic Proteins Were Identified mentioning

confidence: 99%

“…In particular, very small (Ͻ10 kD) and very large (Ͼ100 kD) proteins, basic proteins (pI Ͼ 10), and hydrophobic proteins (grand average of hydropathicity [GRAVY] Ͼ 0.2) are poorly resolved by this method (Chevallet et al, 1998;Herbert et al, 1998;Santoni et al, 1999Santoni et al, , 2000. Incremental improvements in chaotropic agents and nonionic detergents have improved the resolving power of classical denaturing twodimensional electrophoresis (Chevallet et al, 1998;Herbert et al, 1998). However, a substantial protein set will probably always be excluded due to the physical limitations of this approach.…”

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Towards an Analysis of the Rice Mitochondrial Proteome

et al. 2003

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Purified rice (Oryza sativa) mitochondrial proteins have been arrayed by isoelectric focusing/polyacrylamide gel electrophoresis (PAGE), by blue-native (BN) PAGE, and by reverse-phase high-performance liquid chromatography (LC) separation (LC-mass spectrometry [MS]). From these protein arrays, we have identified a range of rice mitochondrial proteins, including hydrophilic/hydrophobic proteins (grand average of hydropathicity ϭ Ϫ1.27 to ϩ0.84), highly basic and acid proteins (isoelectric point ϭ 4.0-12.5), and proteins over a large molecular mass range (6.7-252 kD), using proteomic approaches. BN PAGE provided a detailed picture of electron transport chain protein complexes. A total of 232 protein spots from isoelectric focusing/PAGE and BN PAGE separations were excised, trypsin digested, and analyzed by tandem MS (MS/MS). Using this dataset, 149 of the protein spots (the products of 91 nonredundant genes) were identified by searching translated rice open reading frames from genomic sequence and six-frame translated rice expressed sequence tags. Sequence comparison allowed us to assign functions to a subset of 85 proteins, including many of the major function categories expected for this organelle. A further six spots were matched to rice sequences for which no specific function has yet been determined. Complete digestion of mitochondrial proteins with trypsin yielded a peptide mixture that was analyzed directly by reverse-phase LC via organic solvent elution from a C-18 column (LC-MS). These data yielded 170 MS/MS spectra that matched 72 sequence entries from open reading frame and expressed sequence tag databases. Forty-five of these were obtained using LC-MS alone, whereas 28 proteins were identified by both LC-MS and gel-based separations. In total, 136 nonredundant rice proteins were identified, including a new set of 23 proteins of unknown function located in plant mitochondria. We also report the first direct identification, to our knowledge, of PPR (pentatricopeptide repeat) proteins in the plant mitochondrial proteome. This dataset provides the first extensive picture, to our knowledge, of mitochondrial functions in a model monocot plant.The ATP-synthesizing organelles of eukaryotic cells, mitochondria, can trace their origins back to an event in which one prokaryotic cell was engulfed by another cell to form a new cellular lineage containing two genomes. These genomes became dependent over time, and mitochondria lost the ability to be viable outside the host cell. A significant net transfer of genetic information occurred from the mitochondrial to the nuclear genome during this time. It is predicted that mitochondria now synthesize only a few percent of the proteins required for their function, with the majority of their proteins being encoded in the nucleus, translated in the cytosol, and transferred back to the mitochondria as protein precursors by means of targeting information in the protein sequence (Gray et al., 1999). Biochemical investigations have revealed that isolated plant mitochondria shar...

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“…The 2D-PAGE has other limitations, concerning the detection of low-abundance proteins, hydrophobic proteins, and proteins with extreme size and charge values (9,13,14). In particular, the analysis of integral membrane proteins (IMPs) remains a critical challenge, although new detergents have been designed to enhance membrane protein solubility for analysis by 2D-PAGE (15,16). …”

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confidence: 99%

A High-throughput Approach for Subcellular Proteome

Jiang

Zhou

Zhang

et al. 2004

Molecular & Cellular Proteomics

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Four fractions from rat liver (a crude mitochondria (CM) and cytosol (C) fraction obtained with differential centrifugation, a purified mitochondrial (PM) fraction obtained with nycodenz density gradient centrifugation, and a total liver (TL) fraction) were analyzed with two-dimensional liquid chromatography tandem mass spectrometry analysis. A total of 564 rat proteins were identified and were bioinformatically annotated according to their physicochemical characteristics and functions. While most extreme alkaline ribosomal proteins were identified in the TL fraction, the C fraction mainly included neutral enzymes and the PM fraction enriched alkaline proteins and proteins with electron transfer activity or oxygen binding activity. Such characteristics were more apparent in proteins identified only in the TL, C, or PM fraction. The Swiss-Prot annotation and the bioinformatic prediction results proved that the C and PM fractions had enriched cytoplasmic or mitochondrial proteins, respectively. Combination usage of subcellular fractionation with twodimensional liquid chromatography tandem mass spectrometry was proved to be a high-throughput, sensitive, and effective analytical approach for subcellular proteomics research. Using such a strategy, we have constructed the largest proteome database to date for rat liver (564 rat proteins) and its cytosol (222 rat proteins) and mitochondrial fractions (227 rat proteins). Moreover, the 352 proteins with Swiss-Prot subcellular location annotation in the 564 identified proteins were used as an actual subcellular proteome dataset to evaluate the widely used bioinformatics tools such as PSORT, TargetP, TMHMM, and GRAVY.

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New zwitterionic detergents improve the analysis of membrane proteins by two‐dimensional electrophoresis

Cited by 322 publications

References 24 publications

Assessment of four different detergents used to extract membrane proteins from Xylella fastidiosa by two-dimensional electrophoresis

Assessment of four different detergents used to extract membrane proteins from Xylella fastidiosa by two-dimensional electrophoresis

Towards an Analysis of the Rice Mitochondrial Proteome

A High-throughput Approach for Subcellular Proteome

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