PubMed · 3695966
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
U Sprandel, D J Lanz, W von Hörsten. 1987. Magnetically responsive erythrocyte ghosts.. https://doi.org/10.1016/0076-6879(87)49068-x
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
Membrane protein identification by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) requires that proteins be separated prior to MS analysis. After membrane solubilization with the nondenaturing detergent n-dodecyl-beta-D-maltoside, proteins can be separated by ion-exchange chromatography (IEC) and further resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An additional separation step by gel filtration (GF) before IEC/SDS-PAGE can be required depending on the complexity of the membrane protein mixture. Staining of final SDS-PAGE gels allows one to establish simply the protein expression pattern of a membrane fraction and to profile responses. Moreover, in-gel digestion of hydrophobic integral proteins is valuable. Finally, the resolution capacity of this separation procedure allows identification of proteins by MALDI-TOF MS. The method is illustrated by application to plant and yeast plasma membrane and to plant vacuolar membrane.
Reversible protein phosphorylation is crucially involved in all aspects of plant cell physiology. The highly challenging task of revealing and characterizing the dynamic protein phosphorylation networks in plants has only recently begun to become feasible, owing to application of dedicated proteomics and mass spectrometry techniques. The experimental methodology that identified most of the presently known proteins phosphorylated in vivo is based on protein cleavage with trypsin, following chromatographic enrichment of phosphorylated peptides and mass spectrometric fragmentation and sequencing of these phosphopeptides. This procedure is most efficient when it is limited to the tryptic digestion of proteins in distinct isolated fractions or compartments of plant cells. Immobilized metal affinity chromatography (IMAC) is most useful for phosphopeptide enrichment after methylation of the peptides in the complex protein digests. The following tandem mass spectrometry of the isolated phosphopeptides results in both identification of phosphorylated proteins and mapping of the in vivo phosphorylation sites. The relative quantitation of the extent of phosphorylation at individual protein modification sites may be accomplished by either stable isotope labeling technique or dedicated liquid chromatography-mass spectrometry measurements.
High-resolution protein separation procedures are an important prerequisite for proteome analyses. Classically, protein separations are based on 2D IEF/SDS-PAGE. Unfortunately, this technique only poorly recovers hydrophobic proteins, and it is not compatible with analyses of proteins in native state. Blue-native PAGE represents a powerful alternative. It is based on the careful integration of negative charge into proteins and protein complexes by the anionic wool dye Coomassie blue, and it allows protein analyses under native ("blue-native") conditions. Upon combination with SDS-PAGE for a second gel dimension, protein complexes separated by the blue-native dimension are dissected into their subunits, which form vertical rows of spots on the resulting 2D gels. 2D blue-native/SDS-PAGE ideally complements 2D IEF/SDS-PAGE in proteomics.