title: Peptide chiral purity determination: hydrolysis in deuterated acid, derivatization with Marfey's reagent and analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry.
abstract: 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).
authors.affiliation: Immunobiology Research Institute, Annandale, NJ 08801, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Goodlett
authors.forename: P A
authors.initials: PA
authors.lastname: Abuaf
authors.forename: P A
authors.initials: PA
authors.lastname: Savage
authors.forename: K A
authors.initials: KA
authors.lastname: Kowalski
authors.forename: T K
authors.initials: TK
authors.lastname: Mukherjee
authors.forename: J W
authors.initials: JW
authors.lastname: Tolan
authors.forename: N
authors.initials: N
authors.lastname: Corkum
authors.forename: G
authors.initials: G
authors.lastname: Goldstein
authors.forename: J B
authors.initials: JB
authors.lastname: Crowther
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003903:Deuterium; D004136:Dinitrobenzenes / Q000737:chemistry; D006868:Hydrolysis; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D011487:Protein Conformation
chemical_list: D000596:Amino Acids; D004136:Dinitrobenzenes; D007202:Indicators and Reagents; D010455:Peptides; C047134:Marfey's reagent; D003903:Deuterium; D000409:Alanine