Profiling of Myelin Proteins by 2D-Gel Electrophoresis and Multidimensional Liquid Chromatography Coupled to MALDI TOF−TOF Mass Spectrometry

Abstract

The myelin sheath is an electrically insulating layer that consists of lipids and proteins. It plays a key role in the functioning of the nervous system by allowing fast saltatory conduction of nerve pulses. Profiling of the proteins present in myelin is an indispensable prerequisite to better understand the molecular aspects of this dynamic, functionally active membrane. Two types of protein, the myelin basic protein and the proteolipid protein, account for nearly 85% of the protein content in myelin. Identification and characterization of the other "minor" proteins is, in this respect, a real challenge. In the present work, two proteomic strategies were applied in order to study the protein composition of myelin from the murine central nervous system. First, the protein mixture was separated by 2D-gel electrophoresis and, after spot excision and in-gel digestion, samples were analyzed by mass spectrometry. Via this approach, we identified 57 protein spots, corresponding to 38 unique proteins. Alternatively, the myelin sample was digested by trypsin and the resulting peptide mixture was further analyzed by off-line 2D-liquid chromatography. After the second-dimension separation (nanoLC), the peptides were spotted "on-line" onto a MALDI target and analyzed by MALDI TOF-TOF mass spectrometry. We identified 812 peptides by MALDI MS/MS, representing 93 proteins. Membrane proteins, low abundant proteins, and highly basic proteins were all represented in this shotgun proteomic approach. By combining the results of both approaches, we can present a comprehensive proteomic map of myelin, comprising a total of 103 protein identifications, which is of utmost importance for the molecular understanding of white matter and its disorders.

Adolescent Rats Find Repeated Δ9-THC Less Aversive Than Adult Rats but Display Greater Residual Cognitive Deficits and Changes in Hippocampal Protein Expression Following Exposure

“…This may be linked to structural changes or remodeling occurring after D 9 -THC exposure in adolescents and is consistent with observations of cytoarchitectural changes occurring with cannabinoid treatment (Tagliaferro et al, 2006) and other reports of altered expression of the structural-related proteins tubulin and actin (Tahir et al, 1992;Wilson et al, 1996). MBP is a major constituent of the myelin sheath that allows fast saltatory conduction of nerve pulses (Vanrobaeys et al, 2005). Previous studies employing microarray techniques have demonstrated downregulated MBP expression immediately after chronic D 9 -THC administration (Kittler et al, 2000;Grigorenko et al, 2002).…”

Development of an integrated approach for evaluation of 2‐D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2‐D gel/MALDI workflow

“…Among the traditional mass spectrometric methods available for identification of proteins separated by 2-D gel, PMF by MALDI-TOF MS [13] and MALDI-TOF/TOF MS [14], and ESI-MS/MS [15] have been used almost exclusively for this application. Due to its inherent simplicity and relatively high sample throughput, MALDI-TOF is commonly used as a screening method for direct analysis of gel-extracted peptides.…”

“…Due to its inherent simplicity and relatively high sample throughput, MALDI-TOF is commonly used as a screening method for direct analysis of gel-extracted peptides. Confidence in correct identification of the proteins by this approach can be increased through the inclusion of a tandem TOF (TOF/TOF) mass analysis [14]. For spots that fail to be identified in the initial screening process, an additional analysis using either LC-MALDI-MS/MS, or LC-ESI-MS/MS can be applied.…”

Neural MMP‐28 expression precedes myelination during development and peripheral nerve repair

“…MAG-related peptides were not detected in these experiments; however, it is unclear if this finding is due to lack of XMMP-28 degradation or difficulty in proteomic detection of this protein (Vanrobaeys et al, 2005).…”