Online Capillary IsoElectric Focusing-ElectroSpray Ionization Mass Spectrometry (CIEF-ESI MS) in Glycerol-Water Media for the Separation and Characterization of Hydrophilic and Hydrophobic Proteins.

Capillary isoelectric focusing (CIEF) is a high-resolution technique for the separation of ampholytes, such as proteins, according to their isoelectric point. CIEF coupled online with MS is regarded as a promising alternative to 2-D PAGE for fast proteome analysis with high-resolving capabilities and enhanced structural information without the drawbacks of conventional slab-gel electrophoresis. However, online coupling has been rarely described, as it presents some difficulties. A new methodology for the online coupling of CIEF with electrospray ionization mass spectrometry (ESI-MS) has been developed in glycerol-water media. This new integrated methodology provides a mean for the characterization of a large number of hydrophilic and hydrophobic proteins.

On-line capillary isoelectric focusing hyphenated to native electrospray ionization mass spectrometry for the characterization of interferon-γ and variants.

The on-line hyphenation of Capillary IsoElectric Focusing (CIEF) with ElectroSpray Ionization Mass Spectrometry (ESI/MS) has been carried out in a non-denaturing detection mode at the CIEF-MS interface. This CIEF-MS coupling methodology relied on the use of 40% glycerol-water medium as anti-convective agent in the CE capillary and the addition of 10 mM ammonium acetate buffer, pH 5, as a volatile aqueous sheath liquid. These CIEF-MS coupling conditions allowed the characterization of the highly basic cytokine human interferon-gamma (IFN-γ) and its detection as a non-covalent homodimer (33,814.3 g mol(-1)) corresponding to the active form of this immune-regulatory protein. An experimental pI value of 9.95 was determined for the human IFN-γ homodimer in these conditions. The CIEF-MS analysis of several variants bearing punctual or deletion mutations within the two D1 and D2 basic clusters at the C-terminal end of IFN-γ revealed the different contribution of these domains to the charge properties of this heparan sulfate-binding protein.

Online CIEF-ESI-MS in glycerol-water media with a view to hydrophobic protein applications.

A new online coupling of CIEF with ESI-MS has been developed in glycerol-water media. This improved protocol provides: (i) the electric continuity during the whole analysis by a discontinuous filling of the capillary with 60:40 (cm/cm) catholyte/proteins-ampholyte mixture; (ii) the use of an anticonvective medium, i.e. 30:70 glycerol/water, v/v, compatible with MS detection and as an aid to hydrophobic protein solubilization and (iii) the use of unmodified bare fused-silica capillaries, as the glycerol/water medium strongly reduces EOF. Focusing was performed in positive polarity and cathodic mobilization was achieved by both voltage and pressure application. The setup was optimized with respect to analysis time, sensitivity and precision on pI determination. The optimized anolyte and catholyte were composed of 50 mM formic acid/1 mM glutamic acid (pH 2.35) and 100 mM NH(3)/1 mM lysine (pH 10.6), respectively. The effects of ampholyte concentration, focusing time and ESI parameters were presented for model proteins and discussed. This new integrated protocol should be an easy and effective additional tool in the field of proteome analysis, providing a means for the characterization of a large number of hydrophilic and hydrophobic proteins.

New insight into suction and dilution effects in CE coupled to MS via an ESI interface. II--dilution effect.

The hyphenation of CE with MS is nowadays accepted as a powerful analytical approach. As far as ESI, the most common interface, is concerned, one challenge is to provide the most sensitive as well as quantitative information, which is quite a difficult task, as it is linked, among other factors, to suction and dilution effects. In the coaxial ESI configuration, it has been previously demonstrated that suction effect depends on many parameters inherent to the ESI interface geometry, the prevailing ones being the CE capillary protrusion from the interface needle, the sheath liquid (SL) and the overall BGE flow rates and velocity profile. In this paper, dilution effect is studied, as the CE electrolyte is mixed with SL at the interface. Considering peak intensity and efficiency, this effect was studied as a function of the various parameters of the interface (capillary protrusion from the SL tube, nebulizing gas, SL and CE electrolyte flow rates) or of the source (skimmer and ESI voltages, drying gas flow rate and temperature). It appears that the dilution effect seems slightly lower than what can be anticipated from the proportions of the liquid flow rates. This study also indicates that suction effect has to be considered first to better understand the dilution phenomenon, as suction effect leads to an increase in peak intensity, before a dilution effect appears.

A new insight into suction and dilution effects in capillary electrophoresis coupled to mass spectrometry via an electrospray ionization interface. Part I-Suction effect.

The hyphenation of CE with MS is nowadays accepted as a powerful analytical approach. Employing ESI, the most common interface, one challenge is to provide quantitative information, which is quite a difficult task, as it is linked, among other factors, to suction and dilution effects. In the coaxial ESI configuration, the suction effect has been presented in literature as stemming from nebulizing gas (NG) flow rate and drying gas temperature. But as this interface consists in three concentric capillaries, allowing for BGE, sheath liquid (SL) and NG mixing, it is demonstrated herein that other parameters are also involved in this suction effect: the CE capillary protrusion from the interface needle, SL flow rate, and overall BGE flow rate and velocity profile. Whereas NG flow rate is the parameter affecting suction to a greater extent, separation capillary protruding length, SL, and overall BGE flow rate have a significant additional impact on this phenomenon. It is shown that SL flow rate can affect suction differently according to the NG velocity, which may be explained by modification of the Taylor cone geometry. Furthermore, it appears that suction effect is noticeably favored by a parabolic velocity profile of the BGE, again probably due to the Taylor cone shape modification. Finally, the temperature gradient created by the contact between the heated NG and the separation capillary enhances this effect.

Single-run separation of cationic, anionic, and polyanionic compounds by CE-ESI-MS.

A method for a single-run separation of cationic, anionic, and polyanionic compounds by CE hyphenated to ESI MS (CE-ESI-MS) is described. One of the main issues for coupling CE to MS with an ESI source consists in maintaining an electric contact for the electrophoretic separation. This condition is only performed if a liquid flow arising from the separation capillary is directed to the needle, making it coupling-compatible. This latter situation is incompatible with the separations of polyanionic compounds of higher electrophoretic mobility (in absolute value) than the electroosmotic mobility, performed in bare fused-silica capillaries under a negative polarity. In this study, several alternative approaches were evaluated to circumvent this difficulty, and applied to the setup of the CE-MS separation of a mixture containing both cationic and polyanionic compounds, which are synthesis intermediates of contrast agents for medical imaging. Eventually, the detection of the cationic and anionic compounds in a single run could be obtained by either using neutrally coated polymethylsiloxane (DB-1) capillaries and simultaneously applying a negative voltage polarity and a pressure allowing to compensate for the residual cathodic EOF or by dynamically modifying the inner wall of a bare fused-silica capillary with a polycationic polymer (hexadimethrine bromide) and using it afterwards under negative voltage polarity.