SEPARATION OF NEUROPEPTIDE-Y DIASTEREOMERS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND CAPILLARY ZONE ELECTROPHORESIS

Separation of analogues of neuropeptide Y (NPY) in which a single D-am ino acid replaced the corresponding naturally occurring residue was pe rformed by chromatographic techniques to ensure the quality of the syn thetic peptides to be used for structural and biological studies. Of t he 35 compounds, 28 were easily separated (alpha = 1.02-2.76) from nat ive NPY by standard reversed-phase high-performance chromatography (RP -HPLC) methods using a Vydac C18 column and a gradient buffer system d eveloped in our laboratory comprised of triethylammonium phosphate (TE AP) at pH 2.25 and acetonitrile at 40-degrees-C. The identical diaster eomers could be separated on the same solid support and by using 0.1 % trifluoroacetic acid (TFA) as the mobile phase modifier, however sepa ration factors were smaller and retention times were longer. Three of the remaining seven unresolved analogues were separated (alpha = 1.02- 1.96) by changing the solid-phase support to Vydac diphenyl derivatize d silica and a buffer system consisting of 0.1 % TFA and acetonitrile. Of the four remaining unresolved analogues, only two could be separat ed by capillary zone electrophoresis (CZE) in 0.1 M sodium phosphate a t pH 2.5, but all four were finally resolved by changing the electroph oretic buffer to 0.1 M TEAP buffer at pH 2.5. Migration times of the d iastereomers differed by 0.2-2.0 min from that of the natural NPY. In addition to confirming the uniqueness of each isomer, this investigati on demonstrated the expansive utility and high efficiency of the TEAP buffer system for both RP-HPLC and CZE as well as the difference in se lectivity produced by the TEAP and TFA buffers in RP-HPLC. The conditi ons described here have broad applications for the analysis and prepar ative separation of synthetic and native peptides.