Enrichment of low abundance proteins ofEscherichia coli by hydroxyapatite chromatography

Fountoulakis, Michael; Takács, Marie-Françoise; Berndt, Peter; Langen, Hanno; Takács, B.

doi:10.1002/(sici)1522-2683(19990801)20:11<2181::aid-elps2181>3.0.co;2-q

Cited by 99 publications

(7 citation statements)

References 20 publications

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“…For MS analysis, the gels were stained by colloidal Coomassie Blue G-250 [69]. The gel was fixed in 40% methanol/5% orthophosphoric acid for 12 h and incubated with colloidal Coomassie Blue (17% ammonium sulphate, 34% methanol, 3% orthophosphoric acid and 0.1% Coomassie G-250) for 48 h. After staining, the gels were kept in 1% acetic acid at 4˚C.…”

Section: Methodsmentioning

confidence: 99%

Proteomic analysis of post-nuclear supernatant fraction and percoll-purified membranes prepared from brain cortex of rats exposed to increasing doses of morphine

et al. 2014

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BackgroundProteomic analysis was performed in post-nuclear supernatant (PNS) and Percoll-purified membranes (PM) prepared from fore brain cortex of rats exposed to increasing doses of morphine (10–50 mg/kg) for 10 days.ResultsIn PNS, the 10 up (↑)- or down (↓)-regulated proteins exhibiting the largest morphine-induced change were selected, excised manually from the gel and identified by MALDI-TOF MS/MS: 1-(gi|148747414, Guanine deaminase), ↑2.5×; 2-(gi|17105370, Vacuolar-type proton ATP subunit B, brain isoform), ↑2.6×; 3-(gi|1352384, Protein disulfide-isomerase A3), ↑3.4×; 4-(gi|40254595, Dihydropyrimidinase-related protein 2), ↑3.6×; 5-(gi|149054470, N-ethylmaleimide sensitive fusion protein, isoform CRAa), ↑2.0×; 6-(gi|42476181, Malate dehydrogenase, mitochondrial precursor), ↑1.4×; 7-(gi|62653546, Glyceraldehyde-3-phosphate dehydrogenase), ↑1.6×; 8-(gi|202837, Aldolase A), ↑1.3×; 9-(gi|31542401, Creatine kinase B-type), ↓0.86×; 10-(gi|40538860, Aconitate hydratase, mitochondrial precursor), ↑1.3×. The identified proteins were of cytoplasmic (1, 4, 5, 7, 9), cell membrane (2), endoplasmic reticulum (3) and mitochondrial (6, 8, 10) origin and 9 of them were significantly increased, 1.3-3.6×. The 4 out of 9 up-regulated proteins (4, 6, 7, 10) were described as functionally related to oxidative stress; the 2 proteins participate in genesis of apoptotic cell death.In PM, the 18 up (↑)- or down (↓)-regulated proteins were identified by LC-MS/MS and were of plasma membrane [Brain acid soluble protein, ↓2.1×; trimeric Gβ subunit, ↓2.0x], myelin membrane [MBP, ↓2.5×], cytoplasmic [Internexin, ↑5.2×; DPYL2, ↑4.9×; Ubiquitin hydrolase, ↓2.0×; 60S ribosomal protein, ↑2.7×; KCRB, ↓2.6×; Sirtuin-2, ↑2.5×; Peroxiredoxin-2, ↑2.2×; Septin-11, ↑2.2×; TERA, ↑2.1×; SYUA, ↑2.0×; Coronin-1A, ↓5.4×] and mitochondrial [Glutamate dehydrogenase 1, ↑2.7×; SCOT1, ↑2.2×; Prohibitin, ↑2.2×; Aspartate aminotransferase, ↓2.2×] origin. Surprisingly, the immunoblot analysis of the same PM resolved by 2D-ELFO indicated that the “active”, morphine-induced pool of Gβ subunits represented just a minor fraction of the total signal of Gβ which was decreased 1.2x only. The dominant signal of Gβ was unchanged.ConclusionBrain cortex of rats exposed to increasing doses of morphine is far from being adapted. Significant up-regulation of proteins functionally related to oxidative stress and apoptosis suggests a major change of energy metabolism resulting in the state of severe brain cell “discomfort” or even death.

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Section: Methodsmentioning

confidence: 99%

Proteomic analysis of post-nuclear supernatant fraction and percoll-purified membranes prepared from brain cortex of rats exposed to increasing doses of morphine

et al. 2014

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“…Selective enrichment prior to 2-D gels uses hydrophobic interaction [23], heparin [24±26], and hydroxyapatite [27] chromatography. The method is tolerant, to a certain extent, towards the presence of small molecules, e.g., salts and detergents, and sample preparation is straightforward.…”

Section: Practical Considerationsmentioning

confidence: 99%

Mass spectrometry: A tool for the identification of proteins separated by gels

2000

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“…Low copy number proteins can be enriched by chromatography like was shown for Haemophilus by fractionating the sample with hydroxyapatite columns [18]. Since large amounts of worms can be grown in liquid cultures it should not be a problem to produce enough starting material for additional purification steps.…”

Section: Discussionmentioning

confidence: 99%

A two-dimensional protein map ofCaenorhabditis elegans

et al. 2001

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A protein map of Caenorhabditis elegans was constructed by using two-dimensional gel electrophoresis followed by peptide mass fingerprinting. A whole worm extract of a mixed population was separated on immobilized pH gradient strips 4-7 L, 3-10 NL, 6-11 L and 12% sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) gels. Gels were stained with colloidal Coomassie blue and 286 spots representing 152 proteins were subsequently identified by matrix-assisted laser desorption/ionization-mass spectrometry after in-gel digestion with trypsin. Most of the identified proteins with known cellular function were enzymes related to carbohydrate and lipid metabolism, or structural proteins with subcellular locations in the cytoplasm, mitochondria or cytoskeleton.

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Enrichment of low abundance proteins ofEscherichia coli by hydroxyapatite chromatography

Cited by 99 publications

References 20 publications

Proteomic analysis of post-nuclear supernatant fraction and percoll-purified membranes prepared from brain cortex of rats exposed to increasing doses of morphine

Proteomic analysis of post-nuclear supernatant fraction and percoll-purified membranes prepared from brain cortex of rats exposed to increasing doses of morphine

Mass spectrometry: A tool for the identification of proteins separated by gels

A two-dimensional protein map ofCaenorhabditis elegans

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