Mass spectrometry of native rat amelogenins: primary transcripts, secretory isoforms, and C-terminal degradation.
Cloning technologies have established unambiguously that amelogenins always seem larger in molecular weight (Mr) by gel electrophoresis (SDS-PAGE) than by mass spectrometry (MS). This has caused many problems relating cloned versions of amelogenin to proteins actually secreted by ameloblasts in vivo. In this study, discrete protein fractions at 31-20 kDa (Mr(SDS)) were prepared from freeze-dried rat incisor enamel by techniques optimized for preserving protein integrity. N-terminal sequence and amino acid compositional analyses indicated that the major protein forming these fractions was amelogenin. As expected, the molecular weights estimated by matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) MS were significantly less than their apparent molecular weights estimated by SDS-PAGE. Plots of Mr(SDS) vs. Mr(MS) for all fractions showed high linear correlation (r = 0.992). Analysis of MS data further indicated that the major protein in the 27-kDa fraction corresponded to the R180 secretory isoform of rat amelogenin, whereas some minor proteins in the 23-kDa fraction likely corresponded to a R156 secretory isoform. This was in contrast to major proteins forming the 25-, 24-, and 23-kDa fractions (Mr(SDS)), which seemed to represent proteolytic fragments of R180 progressively altered at the P169-A170, P164-L165, and F151-S152 C-terminal cleavage sites, respectively. Proteins in the 20-kDa fraction (Mr(SDS)) most closely matched by ESI-MS fragments of the R156 secretory isoform that were C-terminally-modified at the equivalent P164-L165 site.
Chen
W Y
WY
Division of Oral Biology, Faculty of Dentistry, McGill University, Montreal, QC, Canada.
Bell
A W
AW
Simmer
J P
JP
Smith
C E
CE
D053523:Amelogenin; D000595:Amino Acid Sequence; D000818:Animals; D003746:Dental Enamel Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D016014:Linear Models; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020033:Protein Isoforms; D051381:Rats; D017208:Rats, Wistar; D012635:Secretory Component / Q000737:chemistry / Q000302:isolation & purification
D053523:Amelogenin; C507201:Amelx protein, rat; D003746:Dental Enamel Proteins; D020033:Protein Isoforms; D012635:Secretory Component