I'm working with a protein which acording to Uniprot there is an isoform with a N-glycosylated asparagine in the N-terminal region. When I did the amino acid sequencing by Edman degradation, I saw that the retention time is similar to an aspartic acid residue. Can N-glycosylated Asn and Asp be confused in Edman?
Dear Gisele,
During the HPLC, at the positoin where you have Asparagine glycosylation, you will probably not detect a PTH amino acid at the established retention time. There will be a "hole" in the sequencing experiment. This is because the detection of newly generated amino acids are based on the PTH derivative of the amino acid without a modification or glycosylaton link. I am not aware that recovery and retention time for PTH derivative of glycosylated Asparagine is established.
In general, PTH Asp elutes earlier than PTH Asn. Furthermore, when you detect PTH Asn, a fraction of this amino acid is also present as deamidated form which is PTH Asp. Therefore, when you have Asn in a peptide sequence, you will always detect a small amount of Asp as the deamidated Asn. So, Asn and Asp are clear, distinct calls during Edman sequencing experiments..
Best,
Hediye.
In addition to explanation provided by Hediye, enzymatic removal of N-glycan results in deamidation of Asn to Asp. Many proteins are first N-glycosylated and then deglycosylated in cells as normal part of their processing (i.e. rhodopsin). Maybe this is a case with your protein as well.
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