Profiling of myelin proteins by 2D-gel electrophoresis and multidimensional liquid chromatography coupled to MALDI TOF-TOF mass spectrometry.
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.
Vanrobaeys
Frank
F
Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, Belgium.
Van Coster
Rudy
R
Dhondt
Goedele
G
Devreese
Bart
B
Van Beeumen
Jozef
J
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002490:Central Nervous System; D002845:Chromatography; D002853:Chromatography, Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008055:Lipids; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009185:Myelin Proteins / Q000737:chemistry*; D009186:Myelin Sheath / Q000737:chemistry*; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013395:Sucrose / Q000737:chemistry; D014466:Ultraviolet Rays
D008055:Lipids; D009185:Myelin Proteins; D010455:Peptides; D011506:Proteins; D020543:Proteome; D013395:Sucrose