High-performance liquid chromatography of amino acids, peptides and proteins. LVIII. Application of reversed-phase high-performance liquid chromatography to the separation of tyrosine-specific phosphorylated polypeptides related to human growth hormone
Procedures for the purification of native and phosphorylated human growth hormone (hGH), S-carboxymethylated hGH, and hGH tryptic peptides, based exclusively on reversed-phase chromatography have been developed. Combinations of several volatile ion-pairing systems with small- and large-pore n-alkylsilicas were exploited in a general strategy, which allowed high recoveries of various hGH-related polypeptides from enzymatic incubations, as well as rapid desalting of samples following chemical modifications of the native protein, such as reductive alkylation in 6 M guanidine hydrochloride. The influence of the elution conditions on retention behaviour of phosphorylated hGH and its tryptic peptides in reversed-phase high-performance liquid chromatography is discussed.