Automated on-line ionic detergent removal from minute protein/peptide samples prior to liquid chromatography-electrospray mass spectrometry.
An automated on-line ionic detergent removal pre-column system coupled to capillary liquid chromatography-electrospray mass spectrometry is described. The system involves two micro precolumns, composed of a specific ionic detergent trapping column and a preconcentration column, respectively, and a packed 300 microns I.D. analytical column. Sample loading to the micro precolumns and regeneration of the detergent trapping column were performed at a flow-rate of 50 microliters/min, while the flow-rate through the analytical column was set at 5.0 microliters/min. Ionic detergent-containing tryptic-digested protein samples were directly applied to the micro precolumns without sample pretreatment and were analysed by UV absorption detection and electrospray mass spectrometry. The presented system allows for the fully automated removal of SDS with virtually no loss in protein/peptides. Maximum SDS load and breakthrough have been determined. Excellent protein recovery and complete removal of SDS is found. The chromatographic separation after SDS removal was completely restored and equalled the reference chromatogram. Mass spectral data confirm these findings. Finally, this technique allows for SDS removal from minute protein samples without the need for any sample handling.
Vissers
J P
JP
LC Packings, Amsterdam, Netherlands.
Hulst
W P
WP
Chervet
J P
JP
Snijders
H M
HM
Cramers
C A
CA
D001331:Automation; D002853:Chromatography, Liquid / Q000379:methods*; D003902:Detergents / Q000302:isolation & purification*; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate / Q000302:isolation & purification*
D003902:Detergents; D010455:Peptides; D011506:Proteins; D012967:Sodium Dodecyl Sulfate