Improvement in HPLC fractionation of thyroxine-containing thyroglobulin tryptic peptides by prior Accell ion-exchange column chromatography.
The isolation and purification of the peptides containing the hormonogenic tyrosyl residues in thyroglobulin is of great interest to the understanding of structure-function relationships in this iodoprotein. This is usually performed in reduced alkylated selectively hydrolyzed thyroglobulin by subsequent HPLC fractionation. However, the main difficulty encountered when trying to isolate these peptides is their disproportion with respect to the total number of possible peptides (14 vs a total of 508). Several HPLC runs with different mobile phases are necessary and each run is accompanied with significant losses of the peptides to be purified. In an attempt to improve the separation of the T4-containing peptides in thyroglobulin tryptic digests from the much more abundant iodotyrosine-containing ones, which are present as contaminants, we have used a very simple and fast step prior to the HPLC fractionation as it is a self-packed ion-exchange column chromatography. This preliminary step results in an improvement in the separation of these peptides and leads to a relative enrichment of the hormonogenic peptides falling in different zones of the HPLC chromatogram, which facilitates their subsequent separation and purification by HPLC.
Miguel
J
J
Unidad de Endocrinología, Universidad Autónoma de Madrid, Spain.
Asuncion
M
M
Marin
C
C
Seguido
A
A
Lamas
L
L
Mendez
E
E
D000097:Acetonitriles; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D006801:Humans; D007457:Iodine Radioisotopes; D010446:Peptide Fragments / Q000302:isolation & purification*; D013954:Thyroglobulin / Q000302:isolation & purification*; D013974:Thyroxine / Q000302:isolation & purification*; D014357:Trypsin
D000097:Acetonitriles; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D013954:Thyroglobulin; D014357:Trypsin; D013974:Thyroxine; C032159:acetonitrile