Rapid separation and characterization of protein and peptide mixtures using 1.5 microns diameter non-porous silica in packed capillary liquid chromatography/mass spectrometry.
Octadecyl-modified 1.5 microns diameter non-porous silica particles were packed in 150 microns i.d. (360 microns 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 microns 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 in the proteolytic digest of an antibody.
MacNair
J E
JE
University of North Carolina at Chapel Hill, Department of Chemistry 27599-3290, USA.
Opiteck
G J
GJ
Jorgenson
J W
JW
Moseley
M A
MA
D002852:Chromatography, Ion Exchange; D003625:Data Collection; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012822:Silicon Dioxide; D013056:Spectrophotometry, Ultraviolet; D018909:src Homology Domains
D007202:Indicators and Reagents; D010455:Peptides; D011506:Proteins; D012822:Silicon Dioxide