Identification of iodination sites in cytochrome c by high-performance liquid chromatography and fast atom bombardment mass spectrometry.
Site-directed covalent modification of proteins is currently used to study the molecular structure of enzyme active sites. Radioactive labels together with protein sequencing by Edman degradation have been previously employed to identify the site of modification. One drawback of such a procedure is that most reagents are not commercially available in radioactive form. Moreover, the covalent bond formed upon reaction of the label with the protein may be labile under the conditions of Edman degradation. By combining reverse-phase high-performance liquid chromatography (RP-HPLC) and fast atom bombardment (FAB) mass spectrometry, we have been able to identify the modified sites of horse heart cytochrome c after reaction with iodine. By using this procedure it has been possible to ascertain that, among the four tyrosines contained in the sequence of horse heart cytochrome c, only tyrosine 74 is converted into the monoiodinated derivative. The data demonstrate also the absence of unwanted secondary reactions. The technique avoids the use of radioactive materials and prevents losses of the label since Edman degradation is not required to identify the modified peptides. These results indicate that FAB mass spectrometry along with RP-HPLC is potentially a powerful technique to study chemical modifications of proteins.
Amico
V
V
Dipartimento di Scienze Chimiche dell'Università di Catania, Italy.
Foti
S
S
Saletti
R
R
Cambria
A
A
Petrone
G
G
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000032:analysis*; D006736:Horses; D006801:Humans; D007455:Iodine / Q000032:analysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D010455:Peptides / Q000032:analysis; D011492:Protein Hydrolysates / Q000032:analysis; D014357:Trypsin; D014443:Tyrosine / Q000032:analysis
D000596:Amino Acids; D003574:Cytochrome c Group; D010455:Peptides; D011492:Protein Hydrolysates; D014443:Tyrosine; D007455:Iodine; D014357:Trypsin