Automated on-line ionic detergent removal from minute protein/peptide samples prior to liquid chromatography-electrospray mass spectrometry

An automated online ionic detergent removal pre-column system coupled to capillary liq. chromatog.-electrospray mass spectrometry is described. The system involves two micro precolumns, composed of a specific ionic detergent trapping column and a preconcn. column, resp., and a packed 300 micro m I.D. anal. column. Sample loading to the micro precolumns and regeneration of the detergent trapping column were preformed at a flow-rate of 5.0 micro l/min. while the flow-rate through the anal. column was set at 5.0 micro l/min. Ionic detergent-contg. tryptic-digested protein samples were directly applied to the micro precolumns without sample pretreatment and were analyzed by UV absorption detection and electrospray mass spectrometer. The presented system allows for the fully automated removal of SDS with virtually no loss in protein/peptides. Max. SDS load and breakthrough have been detd. Excellent protein recovery and complete removal of SDS is found. The chromatog. sepn. after SDS removal was completely restored and equaled the ref. 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. [on SciFinder (R)

Two-dimensional capillary liquid chromatography based on microfractionation

A two-dimensional capillary liquid chromatography (LC) method is described which is based on microfractionation, automated reinjection, and rechromatography using an automated microcolumn switching setup to separate complex peptide mixtures from different origin. Different types of separations modes, i.e., hydrophobic and charge separation mechanisms, have been applied to increase the selectivity and peak capacity and to approach orthogonal separation mechanisms. The first-dimension separations were carried out by either reversed-phase chromatography or strong anion exchange chromatography. The second-dimension separation was in all cases reversed-phase capillary LC. In case of two-dimensional reversed/reversed-phase separations, some degree of orthogonality was achieved by using either two different sets of mobile phase solvents and one reversed-phase capillary LC column or a single set of mobile phases and two capillary LC columns packed with different-reversed-phase stationary phases. Electrospray ionization mass spectrometry was conducted to confirm the orthogonality of the developed two-dimensional capillary LC technique. The analysis of tryptic digests of cytochrome C and fetuin and a major histocompatibility complex (MHC) class I peptide mixture are described

Fully automated on-line sample clean-up of minute proteinaceous samples prior to capillary LC-ESI-MS

An automated on-line ionic detergent removal precolumn 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 J.lIl1 i.d. analytical column. Sample loading to the micro precolumns - and regeneration of the detergent trapping column - was performed at a flow rate of 50 ~min while the flow rate through the analytical column was set at 5.0 ~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 proteinlpeptides. 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 of any sample handling

On-Line Electrochemical Reduction of Disulfide Bonds: Improved FTICR-CID and -ETD Coverage of Oxytocin and Hepcidin

Proteomic