Internal Peptide Sequence of Proteins Digested In-Gel after One- or Two- Dimensional Gel Electrophoresis

Ferrara, Pascual; Rosenfeld, Jorge; Guillemot, Jean Claude; Capdevielle, Joël

doi:10.1016/b978-0-12-058757-5.50046-9

Cited by 15 publications

(7 citation statements)

References 14 publications

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“…Considering that MALDI‐MS has a low limit of detection of <500 fmol per peptide fragment, we conclude from this primary analysis that the mutant protein does not share extensive stretches of identity with RBP. To investigate further the primary sequence of the 28 kDa protein, we also carried out an analysis of the N‐terminal sequence of the 28 kDa mutant protein (Ferrara et al ., 1993). This analysis revealed, for the first 15 N‐terminal amino acids of the 28 kDa protein, no similarity in the primary sequences for mouse RBP and the 28 kDa protein.…”

Section: Resultsmentioning

confidence: 99%

Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein

1999

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Retinol-binding protein (RBP) is the sole specific transport protein for retinol (vitamin A) in the circulation, and its single known function is to deliver retinol to tissues. Within tissues, retinol is activated to retinoic acid, which binds to nuclear receptors to regulate transcription of >300 diverse target genes. In the eye, retinol is also activated to 11-cis-retinal, the visual chromophore. We generated RBP knockout mice (RBP -/-) by gene targeting. These mice have several phenotypes. Although viable and fertile, they have reduced blood retinol levels and markedly impaired retinal function during the first months of life. The impairment is not due to developmental retinal defect. Given a vitamin A-sufficient diet, the RBP -/-mice acquire normal vision by 5 months of age even though blood retinol levels remain low. Deprived of dietary vitamin A, vision remains abnormal and blood retinol declines to undetectable levels. Another striking phenotype of the mutant mice is their abnormal retinol metabolism. The RBP -/-mice can acquire hepatic retinol stores, but these cannot be mobilized. Thus, their vitamin A status is extremely tenuous and dependent on a regular vitamin A intake. Unlike wild-type mice, serum retinol levels in adult RBP -/-animals become undetectable after only a week on a vitamin A-deficient diet and their retinal function rapidly deteriorates. Thus RBP is needed for normal vision in young animals and for retinol mobilization in times of insufficient dietary intake, but is otherwise dispensable for the delivery of retinol to tissues.

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Section: Resultsmentioning

confidence: 99%

Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein

1999

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“…In-gel digestion of proteins was carried out as described by Ferrara et al (1993). Briefly, spots from Coomassie bluestained gels were cut with clean blades and placed in microcentrifuge tubes that had been cleaned by predigestion with trypsin.…”

Section: In-gel Trypsin Digestion Of Proteins and Maldi-tof Ms Peptidmentioning

confidence: 99%

Sleep deprivation‐induced protein changes in basal forebrain: Implications for synaptic plasticity

Basheer

Brown

Ramesh

et al. 2005

J of Neuroscience Research

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The need to sleep is universal and lack of sleep often results in decreases in alertness and cognitive function. Data suggest that sleep-related mechanisms and deficits resulting from loss of sleep are associated anatomically with the basal forebrain. Long-term effects of sleep deprivation, those lasting a day or more, likely require transcriptional changes leading to changes in protein synthesis, whereas short-term effects may be mediated by rapid changes in the functional status of proteins. To understand sleep deprivation-induced changes in proteins in the wake-promoting area of the basal forebrain in rat, proteomic analysis was carried out by a combination of 2D gel electrophoresis to separate and visualize proteins and matrix-assisted laser desorption/ionization time-of flight mass spectrometry for protein identification. Among 969 protein spots that were compared, 89 spots showed more than a twofold difference between 6-hr sleep-deprived rats and undisturbed sleeping controls. We have identified 11 of these proteins to be either cytoskeletal or associated with synapses. The changes in four of these proteins were analyzed further by Western blots of 1D and 2D. Two proteins associated with the cytoskeleton, tubulin and GAP43, show posttranslational modifications. Increased tyrosination of alpha tubulin isoforms and increased phosphorylation of GAP43 was observed after 6-hr sleep deprivation when compared to that in sleeping controls. The synaptic protein synaptosomal-associated protein-25 (SNAP25) is decreased whereas amphiphysin is phosphorylated after sleep deprivation. These changes in proteins in the basal forebrain during short-term sleep deprivation are suggestive of changes in the substrate for neuronal transmission and plasticity.

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“…Peptides pl, p4, and p6 were obtained from S. purpuratus REJ by running lanes of 80 Ixg WGA eluate on 5% SDS-PAGE gels, visualizing REJ using the CuC1 z staining kit (Bio Rad Laboratories, Richmond, CA) and excising the center of the REJ band from the gel. The cut-outs were exposed to trypsin, and the tryptic peptides were separated by reverse-phase HPLC (Ferrara et al, 1993). The peak fractions were gas-phase microsequenced by the University of California, San Diego Protein Sequencing Laboratory.…”

Section: Lane Bmentioning

confidence: 99%

The sea urchin sperm receptor for egg jelly is a modular protein with extensive homology to the human polycystic kidney disease protein, PKD1.

Moy

Mendoza

Schulz

et al. 1996

The Journal of Cell Biology

237

184

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Abstract. During fertilization, the sea urchin sperm acrosome reaction (AR), an ion channel-regulated event, is triggered by glycoproteins in egg jelly (E J). A 210-kD sperm membrane glycoprotein is the receptor for EJ (RE J). This conclusion is based on the following data: purified REJ binds species specifically to EJ dotted onto nitrocellulose, an mAb to REJ induces the sperm AR, antibody induction is blocked by purified RE J, and purified REJ absorbs the AR-inducing activity of EJ. Overlapping fragments of REJ cDNA were cloned (total length, 5,596 bp). The sequence was confirmed by microsequencing six peptides of mature REJ and by Western blotting with antibody to a synthetic peptide designed from the sequence. Complete deglycosylation of REJ followed by Western blotting yielded a size estimate in agreement with that of the mature amino acid sequence. REJ is modular in design; it contains one EGF module and two C-type lectin carbohydrate-recognition modules. Most importantly, it contains a novel module, herein named the REJ module (700 residues), which shares extensive homology with the human polycystic kidney disease protein (PKD1). Mutations in PKD1 cause autosomal dominant polycystic kidney disease, one of the most frequent genetic diseases of humans. The lesion in cellular physiology resuiting from mutations in the PKD1 protein remains unknown. The homology between REJ modules of the sea urchin REJ and human PKD1 suggests that PKD1 could be involved in ionic regulation. SEA urchin eggs possess an extracellular matrix termed egg jelly (E J) t. Glycoprotein ligands in EJ induce the sperm acrosome reaction (AR) (Keller and Vacquier, 1994a;Suzuki, 1995). The AR is required for fertilization; it consists of the exocytosis of the acrosome granule and the polymerization of acrosomal actin to form the bindin-coated acrosomal process used by the sperm to attach to and fuse with the egg (Vacquier et al., 1995). Underlying the E J-induced AR, increased sperm respiration,

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Internal Peptide Sequence of Proteins Digested In-Gel after One- or Two- Dimensional Gel Electrophoresis

Cited by 15 publications

References 14 publications

Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein

Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein

Sleep deprivation‐induced protein changes in basal forebrain: Implications for synaptic plasticity

The sea urchin sperm receptor for egg jelly is a modular protein with extensive homology to the human polycystic kidney disease protein, PKD1.

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