Sample preparation project for the subcellular proteome of mouse liver

Song, Yanping; Hao, Yunwei; Sun, Aihua; Li, Tao; Li, Wenrui; Guo, Lihai; Yan, Yujuan; Geng, Chao; Chen, Ning; Zhong, Fan; Huang, Wei; Jiang, Ying; He, Fuchu

doi:10.1002/pmic.200500893

Cited by 38 publications

(39 citation statements)

References 34 publications

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“…The resulting mitochondrial crude extract was dissolved in 12 ml 25% Nycodenz (Axis-Shield Co.) for full suspension. Density gradient centrifugation was performed as previously described (19); the total time was not more than 1 h. Finally, mitochondria were fully suspended in the isolation buffer.…”

Section: Methodsmentioning

confidence: 99%

Magnetic nanoparticles: An improved method for mitochondrial isolation

et al. 2012

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Abstract. The ultrastructure, function, and physical and chemical properties of mitochondria have become important issues in scientific research. Current mitochondrial isolation methods mainly rely on the physical and chemical properties of mitochondria. Our team presents a fast and reliable new isolation method based on magnetic nanoparticles binding with monoamine oxidase-A (MAO-A) expressed in the mitochondria. MAO-A is expressed in the outer membrane of human mitochondria and is localized on the cytoplasmic side of the membrane, which makes it possible to isolate the mitochondria effectively, using a magnetic field. As shown in the present study, in comparison with differential centrifugation and density gradient centrifugation, the yield of mitochondria isolated by magnetic nanoparticle binding is higher, with greater mitochondrial purity and activity. The entire process, from cell harvesting to final isolation of the mitochondria, takes approximately 1 h. Magnetic nanoparticles provide a simple, practical approach for mitochondrial isolation.

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Section: Methodsmentioning

confidence: 99%

Magnetic nanoparticles: An improved method for mitochondrial isolation

et al. 2012

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“…The complete workflow for our experiments is shown in Figure 2. Suborganelle preparation by sucrose density gradient centrifugation was conducted as previously described in the paper of Song et al [18]. The integrity and purity of suborganelle which prepared by this method were assessed by electron microscopy analysis and Western blotting.…”

Section: Identification Of N-glycoproteins From Mouse Livermentioning

confidence: 99%

“…The procedure for ER preparation has been described previously [18]. All procedures were conducted at 4 °C except where noted.…”

Section: Preparation Of Mouse Liver Er and Er Proteinmentioning

confidence: 99%

N-glycosylation proteome of endoplasmic reticulum in mouse liver by ConA affinity chromatography coupled with LTQ-FT mass spectrometry

et al. 2010

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Glycosylation is the most versatile and one of the most significant protein post-translational modifications. It is generally classified into three categories according to the amino acid to which the glycan is attached: N-glycosylation, O-glycosylation and C-glycosylation. Synthesis of N-glycoproteins occurs in the rough endoplasmic reticulum (rER), and all N-glycoproteins synthesized in rER have uniform glycan endings with mannose (Man) and glucose (Glc). A systematic strategy was developed to comprehensively profile N-glycoproteins in complex biological samples. The lectin Concanvalin A (ConA), which has a high affinity for glycans ending with Man, was used to extract the N-glycoproteins synthesized or located in the rER, and identified the N-glycoproteins and their glycosylation sites by LTQ-FT MS. The analysis was repeated three times at both the biological sample and mass spectrometry levels. In total, 323 glycosylation sites on 212 N-glycoproteins were identified in the mouse liver. Of these, 131 were known N-glycoproteins in the Swissprot database. The identified N-glycoproteins were classified according to their cell location by the Swissprot database and GO software. The identified N-glycoproteins in this study would serve as a good complement to the N-glycoprotein database for the mouse liver. glycosylation, rER, lectin affinity chromatography, ConA, LTQ-FT MS

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“…12 Additionally, proteins or peptides within a sample can be separated by gel electrophoresis or liquid chromatography-based multi-dimensional protein identification technology (MudPIT).…”

Section: Sample Fractionation Techniquesmentioning

confidence: 99%

The application of proteomics technology to thrombosis research: the identification of potential therapeutic targets in cardiovascular disease

Howes

Keen

Findlay

et al. 2008

Diabetes and Vascular Disease Research

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T hrombus formation underpins the development of cardiovascular diseases, including acute coronary syndromes and ischaemic stroke. A number of wellcharacterised cardiovascular risk factors which contribute to the development of the majority of cardiovascular events have been identified, including dyslipidaemia, hypertension and diabetes. Individuals with type 2 diabetes mellitus (T2DM) have a 3-to 5-fold increased risk for development of cardiovascular disease (CVD). They may have a cluster of haemostatic abnormalities, including elevated levels of plasminogen activator inhibitor-1 (PAI-1) and fibrinogen, which contribute to acute thrombotic events. It is clear that additional unidentified risk factors contribute to the pathogenesis of cardiovascular events, and so the search for novel biomarkers and effectors, particularly in individuals with T2DM, remains a major challenge of cardiovascular medicine.Plasma and cellular proteins which contribute to thrombus formation have the potential to confer a prothrombotic state and represent a link between genotype, environment and disease phenotype. The comprehensive analysis of these proteins is now increasingly facilitated through the continued development of proteomic technologies which provide multifaceted approaches to the identification of novel biomarkers and/or effectors of thrombus formation and on which future anticoagulant and thrombolytic therapies may be based.This review provides an overview of current proteomic technologies. It focuses on the recent studies in which these technologies have been applied in the search for novel proteins that may confer increased risk of acute cardiovascular diseases and therefore that may influence disease progression and therapy.

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Sample preparation project for the subcellular proteome of mouse liver

Cited by 38 publications

References 34 publications

Magnetic nanoparticles: An improved method for mitochondrial isolation

Magnetic nanoparticles: An improved method for mitochondrial isolation

N-glycosylation proteome of endoplasmic reticulum in mouse liver by ConA affinity chromatography coupled with LTQ-FT mass spectrometry

The application of proteomics technology to thrombosis research: the identification of potential therapeutic targets in cardiovascular disease

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