Two‐dimensional electrophoresis with cationic detergents: A powerful tool for the proteomic analysis of myelin proteins. Part 2: Analytical aspects

Abstract

The ability to analyze proteins in developing and damaged myelin will be crucial to improve our understanding of the mechanisms of myelinogenesis, dysmyelination, and demyelination. Comparative two-dimensional electrophoresis (2-DE) is a powerful approach to analyze these proteins. In part 1 of this study (see accompanying paper), a method for the 2-DE analysis of myelin proteins using the cationic detergents benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide; CTAB) was described. We obtained improved separation and found that 16-BAC is the most effective agent for separation in 2-DE of myelin proteins and that CTAB is the most effective agent for solubilization of myelin proteins. Here in part 2, major myelin proteins as well as membrane proteins with multitransmembrane domains were identified by mass spectrometry after 16-BAC/SDS-PAGE and CTAB/SDS-PAGE. In addition, a high-molecular-weight protein enriched in myelin fraction was identified as unconventional myosin ID using 16-BAC/SDS-PAGE, which had previously not been detected using immobilized pH gradient isoelectric focusing (IPG)/SDS-PAGE. From these results, we concluded that combinational analysis using IPG/SDS-PAGE, 16-BAC/SDS-PAGE, and CTAB/SDS-PAGE provides a powerful technique facilitating the proteomic analysis of myelin proteins in either developmental or pathological changes.

L-MPZ, a Novel Isoform of Myelin P0, Is Produced by Stop Codon Readthrough

“…MS Analysis-PNS myelin proteins were separated with CTAB-2DE and visualized with fluorescent staining (18,21). The excised 36-kDa spot was analyzed by the Pasarow Mass Spectrometry Laboratory at the University of California at Los Angeles.…”

Hypothesis of an Energetic Function for Myelin

“…Many proteins of the OXPHOS, among which Cytochrome c oxidase (P12787), NADH-coenzyme Q reductase (Q91WD5) and Ubiquinol-cytochrome c reductase (Q9CZ13), were identified in all of the five proteomic analyses of purified myelin [24][25][26][27][28]. Taylor et al talk of ''myelin mitochondria''.…”

“…In the subsequent analyses even though all of them identified the mitochondrial OXPHOS proteins, in some cases the Authors do not mention this fact, in other cases they consider them as contaminants. In these papers control experiments were performed to exclude the possibility of mitochondrial contamination [24][25][26][27][28]. Proteomics, even though powerful, is not a functional technique, therefore, it is conceivable that most of the proteomic papers cited considered the OXPHOS proteins as contaminants.…”

A CTAB based method for the preparation of total protein extract of wine spoilage microrganisms for proteomic analysis

“…6), showing again the advantage of CTAB in terms of sharpness of resolution. According to Yamaguchi et al [21,22], CTAB was the most effective agent for the solubilisation of highly hydrophobic proteins (e.g. myelin proteins) and integral membrane proteins, thus a possible limitation of our method could lie in the loss, by entrapment in the CTAB fraction, of the more hydrophobic proteins.…”