In this study, we have investigated the structure of the native myelin proteolipid protein (PLP), DM-20 protein and several low molecular mass proteolipids by mass spectrometry. The various proteolipid species were isolated from bovine spinal cord by size-exclusion and ion-exchange chromatography in organic solvents. Matrix-assisted laser desorption ionizationtime of flight-mass spectrometry (MALDI-TOF-MS) of PLP and DM-20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester-bound fatty acids. Electrospray ionization-MS analysis of the deacylated proteins in organic solvents produced the predicted molecular masses of the apoproteins (29.9 and 26.1 kDa), demonstrating that palmitoylation is the major post-translational modification of PLP, and that the majority of PLP and DM-20 molecules in the CNS are fully acylated. A series of myelin-associated, palmitoylated proteolipids with molecular masses raging between 12 kDa and 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N-and C-terminal sequencing, tryptic digestion and peptide mapping by MALDI-TOF-MS. The results clearly showed that these polypeptides correspond to the N-terminal region (residues 1-105/112) and C-terminal region (residues 113/131-276) of the major PLP, and they appear to be produced by natural proteolytic cleavage within the 60 amino acid-long cytoplasmic domain. These proteolipids are not postmortem artifacts of PLP and DM-20, and are differentially distributed across the CNS. Keywords: mass spectrometry, myelin, palmitoylation, proteolipid protein, proteolysis. (Macklin et al. 1987;Nave et al. 1987). In addition to these species, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of brain proteolipids reveals the presence of minor low molecular weight forms (LMWPs, 12-18 kDa) (Lees and Bizzozero 1992). Two LMWPs have been isolated from bovine brain and appear to correspond to the first 113 amino acid of PLP plus an unknown N-terminal sequence, and to the last 160 residues with a blocked N-terminus (Lepage et al. 1986 Abbreviations used: DM-20, minor myelin proteolipid protein; ESI, electrospray-ionization; GLC, gas-liquid chromatography; IEC, ionexchange chromatography; LMWPs, low molecular weight (12-18 kDa) proteolipids; MALDI-TOF, matrix-assisted laser desorption ionizationtime of flight; MS, mass spectrometry; m/z, mass to charge ratio; PAGE, polyacrylamide gel electrophoresis; PLP, major myelin proteolipid protein; SDS, sodium dodecyl sulfate; SEC, size-exclusion chromatography.

We have investigated the structure of the native PLP, DM‐20 and several low molecular weight proteolipids (LMWPs) by mass spectrometry (MS). The various proteolipid species were isolated from bovine spinal cord by size‐exclusion and ion‐exchange chromatography. Matrix‐assisted laser desorption ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) of PLP and DM‐20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester‐bound fatty acids. Electrospray ionization‐MS analysis of the deacylated proteins produced the predicted molecular masses of the apoproteins, demonstrating that palmitoylation is the major modification of PLP, and that the majority of PLP and DM‐20 molecules in the CNS are fully acylated. A series of proteolipids with molecular masses from 12 to 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N‐ and C‐terminal sequencing, tryptic digestion and peptide mapping by MALDI‐TOF‐MS. The results showed that these polypeptides correspond to the N‐terminal region (residues 1‐105/112) and C‐terminal region (residues 113/131‐276) of PLP, and they appear to be generated by natural proteolytic cleavage within the 50 amino acid‐long cytoplasmic domain. These LMWPs are derived from myelin, and are not generated during their isolation or during the postmortem period. Moreover, they are present in other species and are unevenly distributed across the CNS. It is tempting to speculate that these minor proteolipids may play a role in myelin formation. Acknowledgements:  Supported by PHHS grant NS 38325.