Degree of phosphorylation of pepsinogen in gastric cancer

Phosphorylation of amino acid residues in proteins and peptides is a very i mportant modification playing a vital role in living organisms. That is a r eason why it is so important to have an efficient tool for isolation and id entification of phoshorylated peptides and proteins. The efficient technique combining IMAC (immobilized metal ion affinity chro matography) and RP-HPLC (HPLC on reversed-phase) for determination of phosp horylated peptides in proteolytic digest was verified on model proteins wit h well defined phosphorylations. Peptide fragments created by digestion of beta -casein from bovine milk with trypsin and swine pepsinogen A digested with alpha -chymotrypsin were separated in the first step by IMAC. Peptides containing phosphate group(s) were bound to ferric ions (Fe3+) immobilized on iminodiacetate (IDA)-Sepharose 6B. Phosphorylated peptides were then re leased by 0.1 M sodium phosphate (pH 5.0). Reversed phase high performance chromatography (RP-HPLC) was applied as the subsequent step for the separat ion of individual phosphopeptides from the mixture obtained by immobilized metal ion affinity chromatography. The developed method was then applied on pepsinogen A digests from stomach mucosa of patients suffering with gastric cancer, and pepsinogen A digests of patients suffering with peptic ulcer (without cancer). Significantly, hi gher amounts of phosphopeptides were found in samples obtained from patient s with gastric cancer. Thus, it seems evident that the higher level of peps inogen A phosphorylation seems to be associated with gastric cancer. The method developed represents a reliable tool for the study of phosphoryl ation degree of proteins in general. In this particular case, it could serv e, potentially, for an early diagnosis of gastric cancer.