Document Detail

Peptide chiral purity determination: hydrolysis in deuterated acid, derivatization with Marfey's reagent and analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry.
MedLine Citation:
PMID:  7633594     Owner:  NLM     Status:  MEDLINE    
A high-performance liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) method is presented that allows rapid and accurate determination of amino acid chiral purity in a peptide. Peptides are hydrolyzed in hydrochloric acid-d1/acetic acid-d4 and then converted to diastereomers by derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, Marfey's reagent). Mixtures of D- and L-amino acid diastereomeric pairs are resolved in one chromatographic separation using conventional reversed-phase high-performance liquid chromatography. Hydrolysis in a deuterated solvent is necessary because the original ratio of D-/L-amino acids present in a peptide changes during acid hydrolysis due to racemization. Peptide hydrolysis in deuterated acids circumvents this problem by labeling each amino acid that racemizes with one deuterium at the alpha-carbon. An increase in molecular mass of one atomic mass unit allows racemized amino acids to be distinguished from non-racemized amino acids by mass spectrometry. This procedure was used to determine the chiral purity of each amino acid in a purified, hexapeptide by-product (Arg-Lys-Lys-Asp-Val-Tyr) present in a kilogram batch of the synthetic pentapeptide, thymopentin (Arg-Lys-Asp-Val-Tyr).
D R Goodlett; P A Abuaf; P A Savage; K A Kowalski; T K Mukherjee; J W Tolan; N Corkum; G Goldstein; J B Crowther
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of chromatography. A     Volume:  707     ISSN:  0021-9673     ISO Abbreviation:  J Chromatogr A     Publication Date:  1995 Jul 
Date Detail:
Created Date:  1995-09-14     Completed Date:  1995-09-14     Revised Date:  2009-01-15    
Medline Journal Info:
Nlm Unique ID:  9318488     Medline TA:  J Chromatogr A     Country:  NETHERLANDS    
Other Details:
Languages:  eng     Pagination:  233-44     Citation Subset:  IM    
Immunobiology Research Institute, Annandale, NJ 08801, USA.
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MeSH Terms
Alanine / analogs & derivatives,  chemistry
Amino Acid Sequence
Amino Acids / chemistry
Chromatography, High Pressure Liquid / methods*
Dinitrobenzenes / chemistry
Indicators and Reagents
Mass Spectrometry / methods*
Molecular Sequence Data
Peptides / chemistry,  isolation & purification*
Protein Conformation
Reg. No./Substance:
0/Amino Acids; 0/Dinitrobenzenes; 0/Indicators and Reagents; 0/Peptides; 56-41-7/Alanine; 7782-39-0/Deuterium; 95713-52-3/Marfey's reagent

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