Effects of reductive alkylation on peptide retention times.
Reductive alkylation of primary amino groups is used to introduce nuclear magnetic resonance or radioactive probes into proteins. Because of electrostatic and conformational effects, reductive alkylation is not always complete. We describe herein a rapid high-performance liquid chromatographic method for separating and quantitating mixtures of native and reductively alkylated peptides. Small synthetic peptides were chosen to illustrate the effects of methylation and isopropylation of primary amino groups on chromatographic retention times. Mixtures of unmodified and reductively methylated or isopropylated peptides (Gly-Leu-Tyr, Gly-Gly-Lys-Arg, Arg-Lys-Asp-Val-Tyr and Pro-Gly-Lys-Ala-Arg) could be separated. Chromatography was on a 5-micron, 25 cm x 0.4 I.D., C18 reversed-phase column with 0.1% trifluoroacetic acid in a 10 to 80% linear gradient of acetonitrile in water, a system appropriate for protein digests. The relative concentrations of native, and singly and doubly alkylated peptide were determined as well as the effective retention coefficients for dimethyl and isopropyl groups. The method shows promise for the peptide mapping of partially alkylated proteins.
Brown
E M
EM
U.S. Department of Agriculture, Eastern Regional Research Center, Philadelphia, PA 19118.
Dower
H J
HJ
Greenberg
R
R
D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D010084:Oxidation-Reduction; D010455:Peptides / Q000302:isolation & purification*; D011487:Protein Conformation; D012997:Solvents
D000596:Amino Acids; D010455:Peptides; D012997:Solvents