Capillary zone electrophoresis of metal-binding proteins in formic acid with UV- and mass spectrometric detection using cationic transient capillary isotachophoresis for preconcentration.
A capillary zone electrophoresis (CZE) method with preceding cationic transient capillary isotachophoresis (tCITP-CZE) was developed for uncoated fused-silica capillaries to analyze metal-binding proteins (MBPs) of clinical relevance. UV detection was followed by mass spectrometry (MS). Optimization was done with model proteins of properties similar to relevant human MBPs. Using 1.0 mol x L(-1) formic acid (pH 1.78) as electrolyte resulted in up to 165000 plates m(-1) in CZE and 230000 plates m(-1) in combination with tCITP and analysis time was less than 5 min in uncoupled mode. Cationic tCITP with 125 mmol x L(-1) ammonium formate, buffered to pH 4.00, as leading electrolyte improved sample loadability considerably in comparison with sample stacking without impairing resolution. Following systematic optimization of the electrospray ionization process (ESI) the coupled system ((tCITP)-CZE-UV-ESI-MS) was tested with protein model mixtures and human MBPs. Repeatability of migration times was < 0.64% in pure CZE mode and in tCITP-CZE mode and < 0.83% in CZE-ESI-MS coupled mode. Mass accuracy was < 0.015%. Limits of detection were found to be in the range 50-160 fmol.
Stutz
Hanno
H
University of Salzburg, Institute of Chemistry and Biochemistry, Salzburg, Austria. hanno.stutz@sbg.ac.at
Bordin
Guy
G
Rodriguez
Adela Rosa
AR
D002138:Calibration; D002412:Cations; D019075:Electrophoresis, Capillary / Q000379:methods*; D005561:Formates / Q000737:chemistry*; D006801:Humans; D008670:Metals / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D002412:Cations; D005561:Formates; D008670:Metals; D011506:Proteins; C030544:formic acid