Research methods and applications rapid separation and characterization of protein and peptide mixtures using 1.5 μm diameter non-porous silica in packed capillary liquid chromatography/mass spectrometry

Abstract

Octadecyl-modified 1.5 μm diameter non-porous silica particles were packed in 150 μm i.d. (360 μm o.d.) capillaries with lengths of 20 cm which were used to separate proteins and peptides generated from enzymatic digests of proteins. Gradients were produced using an exponential dilution method at pressures of 520 Bar (7500 psi) and electrospray ionization mass spectrometry was used for detection. This system was similar to packed capillary perfusion chromatography with respect to chromatographic resolution and analysis time and had a limit of detection comparable to traditional packed capillaries which use 5 μm diameter porous particles. The analyses required as little as 250 femtomol of protein or 500 femtomol of peptide on-column in approximately 30 min. This technique was then applied to verify the existence of an overexpressed protein in an E. coli cell lysate and to confirm the presence of four glycoforms of a peptide generated hi the proteolytic digest of an antibody.