A peptide preconcentration approach for nano‐high‐performance liquid chromatography to diminish memory effects

Schaefer, Heike; Chervet, Jean-Pierre; Bunse, Christian; Joppich, Cornelia; Meyer, Helmut E.; Marcus, Katrin

doi:10.1002/pmic.200300801

Cited by 53 publications

(28 citation statements)

References 8 publications

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“…Spots were manually picked and in-gel trypsin digested [22,23]. Tryptic peptides were extracted twice with 10 μl acetonitrile/5% formamide (50/50 [vol./vol.]).…”

Section: D-dige and Image Analysismentioning

confidence: 99%

The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes

et al. 2012

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Aims/hypothesis The molecular mechanisms underlying insulin resistance in skeletal muscle are incompletely understood. Here, we aimed to obtain a global picture of changes in protein abundance in skeletal muscle in obesity and type 2 diabetes, and those associated with whole-body measures of insulin action. Methods Skeletal muscle biopsies were obtained from ten healthy lean (LE), 11 obese non-diabetic (OB), and ten obese type 2 diabetic participants before and after hyperinsulinaemic-euglycaemic clamps. Quantitative proteome analysis was performed by two-dimensional differential-gel electrophoresis and tandem-mass-spectrometry-based protein identification.Results Forty-four protein spots displayed significant (p< 0.05) changes in abundance by at least a factor of 1.5 between groups. Several proteins were identified in multiple spots, suggesting post-translational modifications. Multiple spots containing glycolytic and fast-muscle proteins showed increased abundance, whereas spots with mitochondrial and slow-muscle proteins were downregulated in the OB and obese type 2 diabetic groups compared with the LE group. No differences in basal levels of myosin heavy chains were observed. The abundance of multiple spots representing glycolytic and fast-muscle proteins correlated negatively with insulin action on glucose disposal, glucose oxidation and lipid oxidation, while several spots with proteins J. Giebelstein, G. Poschmann and K. Højlund contributed equally to this work. involved in oxidative metabolism and mitochondrial function correlated positively with these whole-body measures of insulin action. Conclusions/interpretation Our data suggest that increased glycolytic and decreased mitochondrial protein abundance together with a shift in muscle properties towards a fasttwitch pattern in the absence of marked changes in fibretype distribution contribute to insulin resistance in obesity with and without type 2 diabetes. The roles of several differentially expressed or post-translationally modified proteins remain to be elucidated.

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“…Spots were manually picked and in-gel trypsin digested [22,23]. Tryptic peptides were extracted twice with 10 μl acetonitrile/5% formamide (50/50 [vol./vol.]).…”

Section: D-dige and Image Analysismentioning

confidence: 99%

The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes

et al. 2012

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“…Nano-HPLC/ESI-MS 2 -On-line reversed-phase capillary HPLC separations were performed using the Dionex LC Packings HPLC systems (Dionex LC Packings, Idstein, Germany) as described previously (25). ESI-MS 2 was performed on a Bruker Daltonics HCT plus ion trap instrument (Bremen, Germany) equipped with a nanoelectrospray ion source (Bruker Daltonics) and distal coated SilicaTips (FS360-20-10-D; New Objective, Woburn, MA).…”

Section: Purification Of Peroxisomes From Mouse Kidney-peroxisomesmentioning

confidence: 99%

Proteomics Characterization of Mouse Kidney Peroxisomes by Tandem Mass Spectrometry and Protein Correlation Profiling

Wiese

Gronemeyer

Ofman

et al. 2007

Molecular & Cellular Proteomics

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The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by ␣-and ␤-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate ϳ50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

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“…after gel scanning the spots of interest were manually isolated from the preparative gel and in-gel digested with trypsin (Promega, Mannheim, Germany) in 10 mM ammonium bicarbonate buffer (pH 7.8) at 37°C overnight (16,17).…”

Section: In-gel Tryptic Digestion and Protein Identification Using Mamentioning

confidence: 99%

“…The resulting extracts were combined before the sample volume was reduced in vacuo, and samples were acidified by addition of 5% formic acid to a final volume of 20 l. Online reversed-phase capillary HPLC separations were performed using HPLC systems (Dionex LC Packings, Idstein, Germany) as described previously (16). ESI-MS/MS analyses were performed on a Bruker Daltonics HCT plus ion trap instrument equipped with a nanoelectrospray ion source (Bruker Daltonics) and distal coated SilicaTips (FS360-20-10-D; New Objective, Woburn, MA).…”

Section: In-gel Tryptic Digestion and Protein Identification Using Mamentioning

confidence: 99%

Identification of Proteomic Differences between Squamous Cell Carcinoma of the Lung and Bronchial Epithelium

Poschmann

Sitek

Sipos

et al. 2009

Molecular & Cellular Proteomics

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Proteins that exhibit different expression levels in normal and malignant lung cells are good candidate biomarkers to improve early diagnosis and intervention. We used a quantitative approach and compared the proteome of microdissected cells from normal human bronchial epithelium and squamous cell carcinoma tumors of histopathological grades G2 and G3. DIGE analysis and subsequent MS-based protein identification revealed that 32 non-redundant proteins were differentially regulated between the respective tissue types. These proteins are mainly involved in energy pathways, cell growth or maintenance mechanisms, protein metabolism, and the regulation of DNA and RNA metabolism. The expression of some of these proteins was analyzed by immunohistochemistry using tissue microarrays containing tissue specimen of 55 patients, including normal bronchial epithelium, squamous cell carcinomas, adenocarcinomas, and large cell carcinomas. The results of the immunohistochemical studies correlated with the proteome study data and revealed that particularly HSP47 and a group of cytokeratins (i.e. cytokeratins 6a, 16, and 17) are significantly co-regulated in squamous cell carcinoma. Furthermore cytokeratin 17 showed significantly higher abundance in G2 grade compared with G3 grade squamous cell carcinomas in both the gel-based and the immunohistochemical analysis. Therefore this protein might be used as a marker for stratification between different tumor grades. Molecular & Cellular

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A peptide preconcentration approach for nano‐high‐performance liquid chromatography to diminish memory effects

Cited by 53 publications

References 8 publications

The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes

The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes

Proteomics Characterization of Mouse Kidney Peroxisomes by Tandem Mass Spectrometry and Protein Correlation Profiling

Identification of Proteomic Differences between Squamous Cell Carcinoma of the Lung and Bronchial Epithelium

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