Nuclear extrusion in dissociated rat heart cells

Kyte, Jack

doi:10.1007/bf00338845

Cited by 101 publications

(130 citation statements)

References 10 publications

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“…The secondary structure consensus prediction was performed using self-optimized prediction method with alignment (SOPMA) [49] and PSIPRED [24]. The composition and the hydrophobicity and hydrophilicity analysis of amino acid sequence were fulfilled using the pepstats analysis tool and Protscale program, respectively [50,51]. The physicochemical parameters that are associated with biological activity were computed using the ProtParam tool on the Expasy proteomics server (http://us.expasy.org/tools/ protparam.html).…”

Section: Secondary Structure Predictionmentioning

confidence: 99%

Structure prediction, expression, and antigenicity of c‐terminal of GRP78

Aghamollaei

Gargari

Ghanei

et al. 2016

Biotech and App Biochem

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Glucose-regulated protein 78 (GRP78) is a typical endoplasmic reticulum luminal chaperone having a main role in the activation of the unfolded protein response. Because of hypoxia and nutrient deprivation in the tumor microenvironment, expression of GRP78 in these cells becomes higher than the native cells, which makes it a suitable candidate for cancer targeting. Suppression of survival signals by antibody production against C-terminal domain of GR78 (CGRP) can induce apoptosis of cancer cells. The aim of this study was in silico analysis, recombinant production, and characterization of CGRP in Escherichia coli. Structural prediction of CGRP by bioinformatics tools was done and the construct containing optimized sequence was transferred to E. coli T7 shuffle. Expression was induced by isopropyl-β-d-thiogalactoside, and recombinant protein was purified by Ni-NTA agarose resin. The content of secondary structures was obtained by circular dichroism (CD) spectrum. CGRP immunogenicity was evaluated from the immunized mouse sera. SDS-PAGE analysis showed CGRP expression in E. coli. CD spectrum also confirmed prediction of structures by bioinformatics tools. The enzyme-linked immunosorbent assay using sera from immunized mice revealed CGRP as a good immunogen. The results obtained in this study showed that the structure of truncated CGRP is very similar to its structure in the whole protein context. This protein can be used in cancer researches.

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Section: Secondary Structure Predictionmentioning

confidence: 99%

Structure prediction, expression, and antigenicity of c‐terminal of GRP78

Aghamollaei

Gargari

Ghanei

et al. 2016

Biotech and App Biochem

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“…). The lower recovery of DynA could be due to its lower hydrophobicity as compared to End1 and Met . LODs were 40 ng/mL for DynA and 10 ng/mL for End1 and Met (Table ), which represent an improvement of up to a factor 500 with regard to the values obtained by CE‐UV (i.e.…”

Section: Resultsmentioning

confidence: 86%

Evaluation of fritless solid‐phase extraction coupled on‐line with capillary electrophoresis‐mass spectrometry for the analysis of opioid peptides in cerebrospinal fluid

et al. 2014

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Fritless SPE on-line coupled to CE with UV and MS detection (SPE-CE-UV and SPE-CE-MS) was evaluated for the analysis of opioid peptides. A microcartridge of 150 μm id was packed with a C18 sorbent (particle size > 50 μm), which was retained between a short inlet capillary and a separation capillary (50 μm id). Several experimental parameters were optimized by SPE-CE-UV using solutions of dynorphin A (DynA), endomorphin 1 (End1), and methionine-enkephaline (Met). A microcartridge length of 4 mm was selected, sample was loaded for 10 min at 930 mbar and the retained peptides were eluted with 67 nL of an acidic hydro-organic solution. Using SPE-CE-MS, peak area and migration time repeatabilities for the three opioid peptides were 12-27% and 4-5%, respectively. SPE recovery was lower for the less hydrophobic DynA (22%) than for End1 (66%) and Met (78%) and linearity was satisfactory in all cases between 5 and 60 ng/mL. The LODs varied between 0.5 and 1.0 ng/mL which represent an enhancement of two orders of magnitude when compared with CE-MS. Cerebrospinal fluid (CSF) samples spiked with the opioid peptides were analyzed to demonstrate the applicability to biological samples. Peak area and migration time repeatabilities were similar to the standard solutions and the opioid peptides could be detected down to 1.0 ng/mL.

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“…A trend in proteomics studies is to directly identify proteins (including integral membrane proteins) after in-solution digestion without the need of a previous electrophoretic separation [20]; yet in these conditions membrane proteins may evade identification as they would require a drastic a) GRAVY index [15] was calculated without considering PTMs b) Peptide only identified using the two-step elution procedure denaturation (i.e., boiling in 2-5% SDS for a few minutes) followed by SDS-PAGE for successful proteolysis [2]. ATPase subunit 9 is one such case.…”

Section: Discussionmentioning

confidence: 99%

An in-gel digestion procedure that facilitates the identification of highly hydrophobic proteins by electrospray ionization-mass spectrometry analysis

Castellanos‐Serra¹,

Ramos²,

Huerta³

2005

Proteomics

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A procedure is described for in-gel tryptic digestion of proteins that allows the direct analysis of eluted peptides in electrospray ionization (ESI) mass spectrometers without the need of a postdigestion desalting step. It is based on the following principles: (a) a thorough desalting of the protein in-gel before digestion that takes advantage of the excellent properties of acrylamide polymers for size exclusion separations, (b) exploiting the activity of trypsin in water, in the absence of inorganic buffers, and (c) a procedure for peptide extraction using solvents of proven efficacy with highly hydrophobic peptides. Quality of spectra and sequence coverage are equivalent to those obtained after digestion in ammonium bicarbonate for hydrophilic proteins detected with Coomassie blue, mass spectrometry-compatible silver or imidazole-zinc but are significantly superior for highly hydrophobic proteins, such as membrane proteins with several transmembrane domains. ATPase subunit 9 (GRAVY 1.446) is a membrane protein channel, lipid-binding protein for which both the conventional in-gel digestion protocol and in solution digestion failed. It was identified with very high sequence coverage. Sample handling after digestion is notably simplified as peptides are directly loaded into the ESI source without postdigestion processing, increasing the chances for the identification of hydrophobic peptides.

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Nuclear extrusion in dissociated rat heart cells

Cited by 101 publications

References 10 publications

Structure prediction, expression, and antigenicity of c‐terminal of GRP78

Structure prediction, expression, and antigenicity of c‐terminal of GRP78

Evaluation of fritless solid‐phase extraction coupled on‐line with capillary electrophoresis‐mass spectrometry for the analysis of opioid peptides in cerebrospinal fluid

An in-gel digestion procedure that facilitates the identification of highly hydrophobic proteins by electrospray ionization-mass spectrometry analysis

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