Proteomics of rat liver Golgi complex: Minor proteins are identified through sequential fractionation

Taylor, Randall S.; Wu, Christine C.; Hays, Lara G.; Eng, Jimmy K.; Yates, John R.; Howell, Kathryn E.

doi:10.1002/1522-2683(20001001)21:16<3441::aid-elps3441>3.0.co;2-g

Cited by 95 publications

(67 citation statements)

References 68 publications

(44 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this vein, Fountoulakis and colleagues (38) performed a 2D PAGE analysis of crude human brain homogenates; many spots identified in the mouse myelin proteome are consistent (with respect to pI and molecular mass) with their findings, making crossover identifications possible. A second approach recognizes that a cornerstone of proteomic research is prior fractionation (39). The high abundances of two myelin proteins, proteolipid protein (Ϸ50%) and isoforms of myelin basic protein (Ϸ30%), suggest a requirement for further enrichment of the low-abundance proteins.…”

Section: Discussionmentioning

confidence: 99%

Proteomic mapping provides powerful insights into functional myelin biology

Taylor

Marta

Claycomb

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

108

107

View full text Add to dashboard Cite

Myelin is a dynamic, functionally active membrane necessary for rapid action potential conduction, axon survival, and cytoarchitecture. The number of debilitating neurological disorders that occur when myelin is disrupted emphasizes its importance. Using highresolution 2D gel electrophoresis, mass spectrometry, and immunoblotting, we have developed an extensive proteomic map of proteins present in myelin, identifying 98 proteins corresponding to at least 130 of the Ϸ200 spots on the map. This proteomic map has been applied to analyses of the localization and function of selected proteins, providing a powerful tool to investigate the diverse functions of myelin.M yelin is a dynamic, functionally active membrane (1), the loss or damage of which results in serious neurological disorders including leukodystrophies, central and peripheral neuropathies, and inflammatory demyelinating diseases such as multiple sclerosis (2-4). Rapid and efficient action potential conduction in the nervous system depends on myelin, which traditionally has been viewed as a passive contributor to conduction by increasing internodal membrane resistance and decreasing membrane capacitance (5). However, recent work has revealed additional active roles for myelin in nervous system development and function. For example, myelin regulates axon diameter and the formation of axon microtubular networks, and it is a key player in ion channel clustering at nodes of Ranvier (6-11). In return, the axon regulates myelin gene expression (12) and oligodendrocyte survival (13). The functional coupling of myelin and axons is further illustrated by the transfer of phospholipids (14) and N-acetylaspartate (15) from the axon to the myelin sheath.A major impediment to understanding the active biological functions of myelin is the relative lack of myelin proteins that have been described and characterized. Thus, we have undertaken an extensive proteomic analysis of central nervous system myelin, developed a 2D PAGE map of myelin proteins, identified 98 of these proteins, and illustrated the power and utility of this approach through specific applications of comparative proteomics. For example, a comparison of CNS and peripheral nervous system (PNS) myelin proteins has revealed a previously undescribed component of Schwann cell microvilli, a proteomic matching of myelin from normal and genetically modified mice lacking key myelin lipids has demonstrated a dramatic reduction of two newly recognized myelin protein kinases, and an application of the map to a neuroimmunological analysis of demyelinating disease has identified a signaling mechanism (16).

show abstract

Section: Discussionmentioning

confidence: 99%

Proteomic mapping provides powerful insights into functional myelin biology

Taylor

Marta

Claycomb

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

108

107

View full text Add to dashboard Cite

show abstract

“…Using methods, such as differential centrifugation, electrophoresis, or affinity fractionation, cell or tissue lysates are preseparated into fractions containing particular organelles [8,12,14]. After this prefractionation, the "organelle proteome" can be analyzed by use of established proteomics techniques, such as 1-and 2-DE and chromatographic methods followed by MS [8,[12][13][14][15][25][26][27][28].…”

Section: Introductionmentioning

confidence: 99%

“…After isolation of organelles or subcellular structures, and before electrophoretic separation and final identification by MS, fractionation by selective extraction with different agents and chromatographic separation with different resins can further facilitate proteome analysis [4,12,[23][24][25][26][27][28][29][30].…”

Section: Introductionmentioning

confidence: 99%

“…Less hydrophobic, mostly membrane-associated proteins are separated into the water-rich phase, away from more hydrophobic, integral membrane proteins in the Triton X-114 phase. Recently, this method was successfully used for prefractionation of proteins from Golgi membranes and the enrichment of less-abundant proteins from this organelle [27,28]. Methods for selective capture of plasma membrane proteins from the cell surface following biotinylation of whole cells have also been developed.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

et al. 2005

View full text Add to dashboard Cite

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteinsA model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion-and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.

show abstract

“…The separation of the protein mixture into organelles or other multiprotein complex fractions prior to a proteomics analysis is usually the first step to increase the probability of detecting low-copynumber proteins (1)(2)(3)(4)(5). Subcellular fractionation and purification of organelles provide attractive additions to protein separation techniques commonly used in proteomic analysis.…”

mentioning

confidence: 99%

A High-throughput Approach for Subcellular Proteome

Jiang

Zhou

Zhang

et al. 2004

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Four fractions from rat liver (a crude mitochondria (CM) and cytosol (C) fraction obtained with differential centrifugation, a purified mitochondrial (PM) fraction obtained with nycodenz density gradient centrifugation, and a total liver (TL) fraction) were analyzed with two-dimensional liquid chromatography tandem mass spectrometry analysis. A total of 564 rat proteins were identified and were bioinformatically annotated according to their physicochemical characteristics and functions. While most extreme alkaline ribosomal proteins were identified in the TL fraction, the C fraction mainly included neutral enzymes and the PM fraction enriched alkaline proteins and proteins with electron transfer activity or oxygen binding activity. Such characteristics were more apparent in proteins identified only in the TL, C, or PM fraction. The Swiss-Prot annotation and the bioinformatic prediction results proved that the C and PM fractions had enriched cytoplasmic or mitochondrial proteins, respectively. Combination usage of subcellular fractionation with twodimensional liquid chromatography tandem mass spectrometry was proved to be a high-throughput, sensitive, and effective analytical approach for subcellular proteomics research. Using such a strategy, we have constructed the largest proteome database to date for rat liver (564 rat proteins) and its cytosol (222 rat proteins) and mitochondrial fractions (227 rat proteins). Moreover, the 352 proteins with Swiss-Prot subcellular location annotation in the 564 identified proteins were used as an actual subcellular proteome dataset to evaluate the widely used bioinformatics tools such as PSORT, TargetP, TMHMM, and GRAVY.

show abstract

Proteomics of rat liver Golgi complex: Minor proteins are identified through sequential fractionation

Cited by 95 publications

References 68 publications

Proteomic mapping provides powerful insights into functional myelin biology

Proteomic mapping provides powerful insights into functional myelin biology

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

A High-throughput Approach for Subcellular Proteome

Contact Info

Product

Resources

About