scholarly journals Membrane Protein Identification in Rodent Brain Tissue Samples and Acute Brain Slices

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 423 ◽  
Author(s):  
Sarah Joost ◽  
Stefan Mikkat ◽  
Michael Wille ◽  
Antje Schümann ◽  
Oliver Schmitt
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Sample Size ◽  
Protein Identification ◽  
Disease Modeling ◽  
Brain Slices ◽  
Small Sample ◽  
Mass Spectrometric ◽  
Plasma Membrane Proteins

Acute brain slices are a sample format for electrophysiology, disease modeling, and organotypic cultures. Proteome analyses based on mass spectrometric measurements are seldom used on acute slices, although they offer high-content protein analyses and explorative approaches. In neuroscience, membrane proteins are of special interest for proteome-based analysis as they are necessary for metabolic, electrical, and signaling functions, including myelin maintenance and regeneration. A previously published protocol for the enrichment of plasma membrane proteins based on aqueous two-phase polymer systems followed by mass spectrometric protein identification was adjusted to the small sample size of single acute murine slices from newborn animals and the reproducibility of the results was analyzed. For this, plasma membrane proteins of 12 acute slice samples from six animals were enriched and analyzed by liquid chromatography-mass spectrometry. A total of 1161 proteins were identified, of which 369 were assigned to membranes. Protein abundances showed high reproducibility between samples. The plasma membrane protein separation protocol can be applied to single acute slices despite the low sample size and offers a high yield of identifiable proteins. This is not only the prerequisite for proteome analysis of organotypic slice cultures but also allows for the analysis of small-sized isolated brain regions at the proteome level.

Expression and compartmentalization of integral plasma membrane proteins by hepatocytes and their progenitors in the rat pancreas

1991 ◽  
Vol 98 (1) ◽  
pp. 45-54
Author(s):  
J.R. Bartles ◽  
M.S. Rao ◽  
L.Q. Zhang ◽  
B.E. Fayos ◽  
C.L. Nehme ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Apical Plasma Membrane ◽  
Specific Marker ◽  
Plasma Membrane Protein ◽  
Plasma Membrane Proteins ◽  
Dipeptidylpeptidase Iv ◽  
Copper Depletion ◽  
Pancreatic Hepatocytes

A combination of Western blotting, Northern blotting and immunofluorescence was used to examine the expression and compartmentalization of plasma membrane proteins by those hepatocyte-like cells that arise in the pancreases of rats subjected to sequential dietary copper depletion and repletion. The pancreatic hepatocytes were found to: (1) express several integral membrane proteins known to be concentrated within the apical, lateral or basolateral domains of the plasma membranes of hepatocytes in liver; and (2) compartmentalize the membrane proteins to equivalent plasma membrane domains, despite the organization of these cells into clusters instead of highly vascularized plates. The apical plasma membrane proteins dipeptidylpeptidase IV and HA 4 were found to line bile canaliculus-like openings between adjacent pancreatic hepatocytes; these openings were shown to be continuous with the pancreatic exocrine duct by India ink infusion. In contrast, the basolateral plasma membrane protein rat hepatic lectin-1 and lateral plasma membrane protein HA 321 were detected elsewhere about the surfaces of the pancreatic hepatocytes: by analogy to their respective localizations on hepatocytes in liver, rat hepatic lectin-1 was concentrated on those surfaces exposed to the pancreatic matrix at the periphery of the hepatocyte clusters (the basal surface equivalent), whereas HA 321 was concentrated on those surfaces exposed to adjacent hepatocytes within the clusters. The hepatocyte plasma membrane proteins were found to be expressed in the pancreas at different times during the copper depletion/repletion protocol: for example, rat hepatic lectin-1 and the bulk of the HA 4 were expressed relatively late in the protocol, only after large numbers of pancreatic hepatocytes had appeared; whereas dipeptidylpeptidase IV was induced greater than 10-fold early in the protocol and proved to be an apical-specific marker for those ductular epithelial cells that are believed to be the progenitors of the pancreatic hepatocytes.

scholarly journals Characterization of Plasma Membrane Proteins from Ovarian Cancer Cells Using Mass Spectrometry

2004 ◽  
Vol 19 (4-5) ◽  
pp. 219-228 ◽  
Author(s):  
David L. Springer ◽  
Deanna L. Auberry ◽  
Mamoun Ahram ◽  
Joshua N. Adkins ◽  
Jane M. Feldhaus ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cancer Cells ◽  
Protein Identification ◽  
Ovarian Cancer Cells ◽  
Plasma Membrane Proteins ◽  
Targeted Interventions ◽  
Crude Homogenate

To determine how the repertoire of plasma membrane proteins change with disease state, specifically related to cancer, several methods for preparation of plasma membrane proteins were evaluated. Cultured cells derived from stage IV ovarian tumors were grown to 90% confluence and harvested in buffer containing CHAPS detergent. This preparation was centrifuged at low speed to remove insoluble cellular debris resulting in a crude homogenate. Glycosylated proteins in the crude homogenate were selectively enriched using lectin affinity chromatography. The crude homogenate and the lectin purified sample were prepared for mass spectrometric evaluation. The general procedure for protein identification began with trypsin digestion of protein fractions followed by separation by reversed phase liquid chromatography that was coupled directly to a conventional tandem mass spectrometer (i.e. LCQ ion trap). Mass and fragmentation data for the peptides were searched against a human proteome data base using the informatics program SEQUEST. Using this procedure 398 proteins were identified with high confidence, including receptors, membrane-associated ligands, proteases, phosphatases, as well as structural and adhesion proteins. Results indicate that lectin chromatography provides a select subset of proteins and that the number and quality of the identifications improve as does the confidence of the protein identifications for this subset. These results represent the first step in development of methods to separate and successfully identify plasma membrane proteins from advanced ovarian cancer cells. Further characterization of plasma membrane proteins will contribute to our understanding of the mechanisms underlying progression of this deadly disease and may lead to new targeted interventions as well as new biomarkers for diagnosis.

scholarly journals Ubiquitination, quality control and degradation of membrane proteins – chance for therapies?

2018 ◽  
Vol 72 ◽  
pp. 512-525
Author(s):  
Donata Wawrzycka ◽  
Katarzyna Mizio
Keyword(s):  
Quality Control ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Membrane Integrity ◽  
Adaptor Proteins ◽  
Control Mechanisms ◽  
Plasma Membrane Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Ubiquitination

Plasma membrane integrity maintenance is crucial for cell survival. Plasma membrane proteins are under tight regulation and under certain conditions are actively removed from the membrane allowing cells to adapt to changing environment. Proteins blocked in the cell membrane may interact with other molecules and form toxic aggregates. Ubiquitin is one of the most important modifiers targeting proteins for degradation and/or regulating protein functions. Several quality control mechanisms have been identified in eukaryotic cells: chaperone- dependent system that recognizes and ubiquitinates misfolded or redundant membrane proteins; protein-intrinsic LID-degron system, based on recognition of degron and ARTs-Rsp5 network that controls quality of membrane transporters. Rsp5, a Nedd4-family E3 ubiquitin ligase, is crucial for plasma membrane proteins ubiquitination. Rsp5-dependent ubiquitin action acts as a sorting signal for internalization of a membrane protein via endocytosis, recognition by the ESCRT system and vacuolar degradation. Rsp5 builds poliUb-chains on K63 and recognizes substrates through various adaptor proteins. Most of the identified Rsp5 adaptors belongs to the α-arrestin family. Plasma membrane protein ubiquitination and degradation disorders may cause neurodegenerative and cardiovascular diseases. The yeast Saccharomyces cerevisiae is one of the best models for studying trafficking pathways of membrane proteins and ubiquitination systems.

Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

2009 ◽  
Vol 18 (6) ◽  
pp. 527-535 ◽  
Author(s):  
Andreas Lange ◽  
Claudia Kistler ◽  
Tanja B. Jutzi ◽  
Alexandr V. Bazhin ◽  
Claus Detlev Klemke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membrane Proteins ◽  
Subtractive Suppression Hybridization

scholarly journals pH-dependent Cargo Sorting from the Golgi

2011 ◽  
Vol 286 (12) ◽  
pp. 10058-10065 ◽  
Author(s):  
Chunjuan Huang ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Atpase Activity ◽  
Secretory Pathway ◽  
Ph Dependence ◽  
Glucose Deprivation ◽  
Ph Homeostasis ◽  
Plasma Membrane Proteins ◽  
Cytosolic Ph ◽  
Cargo Sorting

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.

scholarly journals 5-Iodonaphthyl-1-azide labeling of plasma membrane proteins adjacent to specific sites via energy transfer

1997 ◽  
Vol 1324 (2) ◽  
pp. 320-332 ◽  
Author(s):  
Bruce I Meiklejohn ◽  
Noorulhuda A Rahman ◽  
Deborah A Roess ◽  
B.George Barisas
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Membrane Proteins ◽  
Plasma Membrane Proteins
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