We have MS evidence that the endogenous protein is phosphorylated, but phosphatase treatment doesn't resolve the two bands. Neither does treatment with EGTA, switching out BME for DTT, or altering the timing of protein harvest.
We sent the doublet from the recombinant protein and get the same coverage at the N and C termini of the protein. Both bands are missing coverage for the 6xHIS tag at the N-terminus of the protein, but I would guess that is due to the peptide not flying well, since the protein was purified twice over a Nickel column. The only difference between the two bands is the presence of two acetylated lysines in the higher molecular weight band (a few amino acids away from each other, on the same peptide).
Both the endogenous protein and the tagged protein are approximately 20 kDa and the two bands of the doublet are 2-3 kDa apart.
Acetylation of lysine removes the positive charge on the epsilon amino group. This might cause the protein to migrate at a different rate in SDS-PAGE, since the rate of migration depends upon the charge on the protein.
Isoforms in proteins are quite common and can be due to post-transnational modification that, as suggested by Adam, can alter the interaction of the protein with its environment including your separation system. I like Adam's hypothesis as loss of the + charge could lead to less SDS-protein interaction, resulting in slower migration towards the anode. It is not uncommon for aberrant migration of proteins under SDS-PAGE conditions due to charge anomalies.
I agree with Adam's comments and would like to add a couple more:
You did not mention about your particular phosphorylated protein (is it cytoplasmic or lysosomal protein ?). Did you try to use each of the phosphatase - acid phosphatase and neutral one ?
If the higher MW bands have two acetylated Lys residues, that might explain why those bands had higher MW. The SDS has a negative charge, therefore it binds mostly to positively charged amino acids. When the positively charged amino acids such as Lys change their charge because of acetylation, the amount of the SDS bound to the protein molecule is getting less. And the number of SDS molecule bound to any protein molecule is always the determining factor of the mobility of your protein in SDS-PAGE.
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