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 (M-r) by gel electrophoresis (SDS-PAGE) th an by mass spectrometry (MS). This has caused many problems relating cloned versions of amelogenin to proteins actually secreted by ameloblasts in viv o. In this study, discrete protein fractions at 31-20 kDa (M-r(SDS)) were p repared 60m freeze-dried rat incisor enamel by techniques optimized for pre serving protein integrity. N-terminal sequence and amino acid compositional analyses indicated that the major protein forming these fractions was amel ogenin. As expected, the molecular weights estimated by matrix-assisted las er desorption ionization (MALDI) and electrospray ionization (ESI) MS were significantly less than their apparent molecular weights estimated by SDS-P ACE. Plots of M-r(SDS) vs. M-r(MS) for all fractions showed high linear cor relation (r = 0.992). Analysis of MS data further indicated that the major protein in the 27-kDa fraction corresponded to the R180 secretory isoform o f rat amelogenin, whereas some minor proteins in the 23-kDa fraction likely corresponded to a R156 secretory isoform. This was in contrast to major pr oteins forming the 25-, 24-, and 23-kDa fractions (M-r(SDS)), which seemed to represent proteolytic fragments of R180 progressively altered at the P-1 69-A(170), P-164-L-165, and F-151-S-152 C-terminal cleavage sites, respecti vely. Proteins in the 20-kDa fraction (M-r(SDS)) most closely matched by ES I-MS fragments of the R156 secretory isoform that were C-terminally-modifie d at the equivalent P-164-L-165 site.