A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization- mass spectrometry

Yan, Jun X.; Wait, Robin; Berkelman, Tom; Harry, Rachel A.; Westbrook, Jules A.; Wheeler, Colin; Dünn, Michael J.

doi:10.1002/1522-2683(200011)21:17<3666::aid-elps3666>3.0.co;2-6

Cited by 661 publications

(295 citation statements)

References 26 publications

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“…Spot plugs were destained and proteins were digested with trypsin as described previously. 42 All samples were run on a Finnigan LTQ mass spectrometer connected to a Surveyor chromatography system incorporating an auto-sampler. Tryptic peptides were resuspended in 0.1% formic acid and were separated by means of a modular CapaLC system (Finnigan) connected directly to the source of the LTQ.…”

Section: Discussionmentioning

confidence: 99%

“…For preparative gels, the PlusOne Silver Staining kit (Amersham Biosciences, Uppsala, Sweden) was used with modifications to allow subsequent use with mass spectrometric analysis. 42 Image analysis Silver-stained gels were scanned at 100 mm resolution using a Personal SI laser densitometer (Molecular Dynamics, Sunnyvale, CA, USA). Protein spots were analysed quantitatively using the image analysis system Progenesis V2003.2 (Nonlinear Dynamics, Newcastle, UK).…”

Section: Samplesmentioning

confidence: 99%

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Prominent synaptic and metabolic abnormalities revealed by proteomic analysis of the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder

et al. 2007

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There is evidence for both similarity and distinction in the presentation and molecular characterization of schizophrenia and bipolar disorder. In this study, we characterized protein abnormalities in the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder using two-dimensional gel electrophoresis. Tissue samples were obtained from 35 individuals with schizophrenia, 35 with bipolar disorder and 35 controls. Eleven protein spots in schizophrenia and 48 in bipolar disorder were found to be differentially expressed (P < 0.01) in comparison to controls, with 7 additional spots found to be altered in both diseases. Using mass spectrometry, 15 schizophrenia-associated proteins and 51 bipolar disorder-associated proteins were identified. The functional groups most affected included synaptic proteins (7 of the 15) in schizophrenia and metabolic or mitochondrial-associated proteins (25 of the 51) in bipolar disorder. Six of seven synaptic-associated proteins abnormally expressed in bipolar disorder were isoforms of the septin family, while two septin protein spots were also significantly differentially expressed in schizophrenia. This finding represented the largest number of abnormalities from one protein family. All septin protein spots were upregulated in disease in comparison to controls. This study provides further characterization of the synaptic pathology present in schizophrenia and of the metabolic dysfunction observed in bipolar disorder. In addition, our study has provided strong evidence implicating the septin protein family of proteins in psychiatric disorders for the first time.

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Section: Discussionmentioning

confidence: 99%

Section: Samplesmentioning

confidence: 99%

Prominent synaptic and metabolic abnormalities revealed by proteomic analysis of the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder

et al. 2007

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“…The isoelectric focusing gel strips were loaded on top of 9% to 16% gradient polyacrylamide gels (200 3 250 3 1.0 mm) for a second round of SDS-PAGE using the Hoefer IsoDALT apparatus (Amersham-Pharmacia Biotech) until the tracking dye reached the anode ends of the gels. The gels were stained with silver nitrate according to the modified procedure by Yan et al (2000). Silver-stained gels were scanned using a high-resolution scanner (Bio-Rad GS-800 calibrated imaging densitometer).…”

Section: -De Analysis and Protein Identification By Maldi-tof Msmentioning

confidence: 99%

Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog

et al. 2006

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The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35STCaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35STCaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.

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“…Gels underwent silver staining using a MALDI-TOF compatible protocol, according to Yan and co-authors (14). Two-dimensional electrophoresis 2DE was performed according to Gö rg and co-authors (15).…”

Section: Laboratory Methodsmentioning

confidence: 99%

Serum albumin fragmentation in end-stage renal disease patients – a pilot study

Donadio

Pennacchio²,

Dimuccio³

et al. 2009

Clinical Chemistry and Laboratory Medicine

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Background: The goal of this study was to detect modification in the expression of plasma proteins and/or post-translational modifications of their structure in patients with end stage renal disease. Methods: Serum samples from 19 adult patients treated by maintenance hemodialysis (MHD) were analyzed in comparison to sera from six healthy controls using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2DE). Spots of interest were identified by mass spectrometry analysis. In addition, the 2DE maps were incubated with a human anti-albumin polyclonal antibody. Results: SDS-PAGE gels, 2DE maps and matrix-assisted laser desorption/ionization time of flight analysis indicated over-expression of low-molecular weight proteins (LMWP) in sera from patients. Unexpectedly, another 15 spots with estimated M r of 12.5-29 kDa from the 2DE maps of six patients were identified as fragments of albumin. 2D immunoblotting of sera from 12 other patients detected numerous albumin fragments. Conclusions:These results indicate that in addition to increased expression of LMWP, a relevant amount of albumin fragments are detectable in the serum of patients undergoing MHD. Uremia appears to facilitate the fragmentation of albumin and/or the retention of albumin fragments in blood. Clin Chem Lab Med 2009;47:1373-9.

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A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization- mass spectrometry

Cited by 661 publications

References 26 publications

Prominent synaptic and metabolic abnormalities revealed by proteomic analysis of the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder

Prominent synaptic and metabolic abnormalities revealed by proteomic analysis of the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder

Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog

Serum albumin fragmentation in end-stage renal disease patients – a pilot study

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