Document Detail

Use of Pico-Tag methodology in the chemical analysis of peptides with carboxyl-terminal amides.
MedLine Citation:
PMID:  3733929     Owner:  NLM     Status:  MEDLINE    
A chemical method has been established for the detection of carboxyl-terminally amidated peptides in tissue extracts. Tissue was homogenized in an acidic medium designed to solubilize peptides while precipitating high-molecular-weight protein. The homogenate supernatant was in turn subjected to reversed-phase extraction with C18 Sep-Pak cartridges. The eluates were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). Individual fractions were exhaustively digested with thermolysin, derivatized with phenylisothiocyanate (PITC), and then subjected to ethyl acetate extraction under basic conditions. The phenylthiocarbamyl (PTC)-amino acid amide derivatives were selectively taken up into the organic phase, while the other digestion products remained in the aqueous phase. The organic phase was analyzed by RP-HPLC on a Pico-Tag amino acid analysis column, monitoring eluates at 254 nm. PTC-amino acid amides were identified and quantitated by comparing their elution positions and peak areas, respectively, with those of standards. Their identities were confirmed by amino acid analysis, following hydrolysis with hydriodic acid. The technique was applied to extracts of bovine posterior pituitaries and a human medullary thyroid carcinoma. Vasopressin (-Leu-Gly-amide), oxytocin (-Gly-amide), Lys1 gamma 1-melanotropin (-Phe-amide), and various acetylated and non-acetylated forms of alpha-melanotropin (-Val-amide) were identified in the posterior pituitary extract. Various forms of calcitonin (-Val-Gly-Ala-Pro-amide) were detected in the tumour extract. For vasopressin and calcitonin the thermolytic digest resulted in di- and tetra-peptides, respectively, reflecting thermolytic cleavage at more favoured sites.
H P Bennett; S Solomon
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Journal of chromatography     Volume:  359     ISSN:  -     ISO Abbreviation:  J. Chromatogr.     Publication Date:  1986 May 
Date Detail:
Created Date:  1986-09-16     Completed Date:  1986-09-16     Revised Date:  2013-12-13    
Medline Journal Info:
Nlm Unique ID:  0427043     Medline TA:  J Chromatogr     Country:  NETHERLANDS    
Other Details:
Languages:  eng     Pagination:  221-30     Citation Subset:  IM    
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MeSH Terms
Amides / analysis*
Amino Acids / analysis
Chromatography, High Pressure Liquid
Indicators and Reagents
Peptides / analysis*
Pituitary Gland, Posterior / analysis
Grant Support
Reg. No./Substance:
0/Amides; 0/Amino Acids; 0/Indicators and Reagents; 0/Isothiocyanates; 0/Peptides; 0/Thiocyanates; 0D58F84LSU/phenylisothiocyanate

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