RAPID SEPARATION AND CHARACTERIZATION OF PROTEIN AND PEPTIDE MIXTURESUSING 1.5 MU-M DIAMETER NONPOROUS SILICA IN PACKED CAPILLARY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY

Octadecyl-modified 1.5 mu m diameter non-porous silica particles were packed hr 150 mu m i.d. (360 mu m o.d.) capillaries with lengths of 20 cm which were used to separate proteins and peptides generated from e nzymatic digests of proteins, Gradients were produced using an exponen tial dilution method at pressures of 520 Bar (7500 psi) and electrospr ay ionization mass spectrometry was used for detection, This system wa s similar to packed capillary perfusion chromatography with respect to chromatographic resolution and analysis time and had a limit of detec tion comparable to traditional packed capillaries which use 5 mu m dia meter porous particles. The analyses required as little as 250 femtomo l of protein or 500 femtomol of peptide on-column in approximately 30 min, This technique was then applied to verify the existence of an ove rexpressed protein in an E. coli cell lysate and to confirm the presen ce of four glycoforms of a peptide generated in the proteolytic digest of an antibody. (C) 1997 by John Wiley & Sons, Ltd.