HPLC shadowing: artifacts in peptide characterization monitored by RIA.
Fischman. A J AJ; Kastin. A J AJ; Graf. M V MV
Key Findings
- HPLC-RIA can produce a "shadow" effect where peptide from a previous standard contaminates the next sample.
- The shadowing error for DSIP was as high as 10% with the regular peptide.
- Using a radiolabeled version (125I‑Tyr‑DSIP) reduced the shadowing error to about 1%.
Practical Outcomes
- When testing DSIP levels, choose assay methods that minimize carry‑over, such as using radiolabeled peptides or thorough column cleaning. Be skeptical of reported endogenous DSIP concentrations if the testing method isn’t clearly described, as they may be inflated by up to 10%.
Summary
The study shows that when measuring the peptide DSIP with a combo of HPLC and radioimmunoassay, leftover peptide from previous runs can falsely appear as if the test sample contains DSIP, creating up to a 10% error. Using a labeled version of the peptide reduces this error to about 1%. So, the measurement method can give misleading results if you’re not careful.
Abstract
High performance liquid chromatography (HPLC), a valuable tool for characterization of peptides, is frequently used in combination with sensitive radioimmunoassays (RIA). The shadow phenomenon, representing carry-over of the peptide from previous application of the standard, can appear to result in the presence of endogenous peptide in the test sample when none is actually there. With delta sleep-inducing peptide (DSIP), we found the shadowing to be as high as 10%, although it was only 1% with 125I-Tyr-DSIP. Thus, when HPLC-RIA systems are used for identification of peptides, caution must be used to avoid false positive results.
Study Information
pubmed
1984
1984-09-01T00:00:00.000Z
10.1016/0196-9781(84)90128-1
27
4