Chapter 19 Isolation of Subcellular Organelles and Structures

Michelsen, Uwe; Hagen, Jörg von

doi:10.1016/s0076-6879(09)63019-6

Cited by 51 publications

(49 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, to cover all of the assessable constituents in a heterogeneous assembly of proteins with a greatly differing abundance and physicochemical properties, as are found in contractile tissues, organelle proteomics should be used to supplement the findings from whole tissue proteomics. The application of prefractionation procedures substantially reduces sample complexity and thus allows more comprehensive cataloging of complex muscle protein mixtures [17][18][19][20]. Muscle organelle proteomics has been successfully applied for studying fractions enriched in nuclei, mitochondria, surface membranes, the sarcoplasmic reticulum, the cytosol, and the contractile apparatus in normal, transforming, and pathological muscle [21].…”

Section: Introductionmentioning

confidence: 99%

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle

Carberry

Zweyer

Swandulla

et al. 2014

Analytical Biochemistry

View full text Add to dashboard Cite

a b s t r a c tIn basic and applied myology, gel-based proteomics is routinely used for studying global changes in the protein constellation of contractile fibers during myogenesis, physiological adaptations, neuromuscular degeneration, and the natural aging process. Since the main proteins of the actomyosin apparatus and its auxiliary sarcomeric components often negate weak signals from minor muscle proteins during proteomic investigations, we have here evaluated whether a simple prefractionation step can be employed to eliminate certain aspects of this analytical obstacle. To remove a large portion of highly abundant contractile proteins from skeletal muscle homogenates without the usage of major manipulative steps, differential centrifugation was used to decisively reduce the sample complexity of crude muscle tissue extracts. The resulting protein fraction was separated by two-dimensional gel electrophoresis, and 2D-landmark proteins were identified by mass spectrometry. To evaluate the suitability of the contractileprotein-depleted fraction for comparative proteomics, normal versus dystrophic muscle preparations were examined. The mass spectrometric analysis of differentially expressed proteins, as determined by fluorescence difference in-gel electrophoresis, identified 10 protein species in dystrophic mdx hindlimb muscles. Interesting new biomarker candidates included Hsp70, transferrin, and ferritin, whereby their altered concentration levels in dystrophin-deficient muscle were confirmed by immunoblotting.Ó 2013 Elsevier Inc. All rights reserved.In high-throughput biochemistry, mass spectrometry is the method of choice for the fast and reliable identification of proteins in large-scale surveys of physiological or pathological processes [1][2][3]. This makes protein mass spectrometry an integral part of biological network analysis [4] and the discovery of novel disease biomarker signatures [5]. Disorder-specific protein markers play a central diagnostic, prognostic, and therapeutic role in skeletal muscle pathology and the systematic application of proteomics has greatly expanded the range of biomarkers for neuromuscular disorders [6]. Proteome-wide studies combine protein separation methods, such as high-resolution two-dimensional gel electrophoresis [7-9] and liquid chromatography [10], with sophisticated mass spectrometric techniques to determine potential changes in protein concentration, isoform expression patterns, protein-protein interactions and posttranslational modifications [11][12][13].However, proteomic findings from comparative studies focusing on total protein extracts from highly complex and dynamic types of tissues, such as skeletal muscle fibers, are often limited to mostly soluble and relatively abundant proteins [14][15][16], missing especially the classes of very low abundance proteins and hydrophobic proteins. Thus, to cover all of the assessable constituents in a heterogeneous assembly of proteins with a greatly differing abundance and physicochemical properties, as are found in contr...

show abstract

Section: Introductionmentioning

confidence: 99%

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle

Carberry

Zweyer

Swandulla

et al. 2014

Analytical Biochemistry

View full text Add to dashboard Cite

show abstract

“…In this scheme, separation of organelles depends on their sedimentation velocity, a function of their size and density. Generally purification is improved by combining velocity sedimentation centrifugation with density gradient centrifugation (Michelsen and von Hagen, 2009;Wiederhold et al, 2010). Alternatively, higher purity is obtained by affinity purification using e.g., an antibody directed against a surface protein of the organelle, free flow electrophoresis (Islinger et al, 2010) or by modification of organelles density.…”

Section: Ms Based Proteomics Of Purified Organellementioning

confidence: 99%

Analysis of Organelle Dynamics by Quantitative Mass Spectrometry Based Proteomics

Fröhlich¹,

Walther²,

Christiano³

2012

Proteomics - Human Diseases and Protein Functions

View full text Add to dashboard Cite

“…The process of subcellular fractionation is generally made up of three parts: lysing cells, separating cytosolic organelles, and isolating the target organelle [52]. There are several ways to perform these steps, each of which must also be optimized for the type of cells being used.…”

Section: Endosome Isolation Methodsmentioning

confidence: 99%

“…Large nuclei require low speeds to pellet (~600 x g), while much smaller mitochondria and endosomes require much higher speeds to pellet (~10,000-20,000 x g), and still smaller ribosomes and endoplasmic reticulum fragments require extremely high speeds to pellet (~100,000 x g) [52].…”

Section: Endosome Isolation Methodsmentioning

confidence: 99%

“…The affinity of the antibody for its antigen, as well as the substrate to which the antibody is conjugated, are two critical components of this method [52]. Magnetic (Dynabeads), sepharose, and agarose beads are commercially available binding substrates with either Protein A or Protein G (or a mixture of both) coupled to the beads.…”

Section: Endosome Isolation Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Isolation of EGFR-containing early endosomes.

Gosney¹

View full text Add to dashboard Cite

Chapter 19 Isolation of Subcellular Organelles and Structures

Cited by 51 publications

References 26 publications

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle

Analysis of Organelle Dynamics by Quantitative Mass Spectrometry Based Proteomics

Isolation of EGFR-containing early endosomes.

Contact Info

Product

Resources

About