title: Continuous mode of operation for large volume dosing in analytical carrier ampholyte-free isoelectric focusing of proteins applied to off-line detection of fractions.
abstract: Mass spectrometry is being increasingly used for analysis of proteome complex samples. Sample preparation is often necessary to remove matrix interferences and to concentrate analytes prior to MS measurement. A useful method for this purpose is Carrier Ampholyte Free-Isoelectric Focusing (CAF-IEF). In this paper CAF-IEF of ampholytes was performed on a commercial apparatus EA101 (Villa Labeco, Slovakia) equipped with a specially made column for samples of large volume (up to 0.5 mL). A new continuous mode without voltage interruption or electrolyte replacement was developed. In this mode, a low molecular mass pI marker (PIM 7.4) and low concentrations of myoglobin and insulin (16 mg/L), respectively, were concentrated, and then 5-microL fractions collected for off-line analyses. The total time of focusing was 66 minutes. The concentration of PIM 7.4 in the fractions was increased up to 75 times (determined by UV-VIS spectrometry). The concentration in the fractions was increased up to 30 times for myoglobin and 10 times for insulin.
authors.affiliation: Masaryk University, Department of Analytical Chemistry, Brno, Czech Republic. buda@mail.muni.cz
authors.forename: Jana
authors.initials: J
authors.lastname: Budilová
authors.forename: Jirí
authors.initials: J
authors.lastname: Pazourek
authors.forename: Pavel
authors.initials: P
authors.lastname: Krásenský
authors.forename: Jan
authors.initials: J
authors.lastname: Pospíchal
mesh_terms: D000818:Animals; D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000032:analysis; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D009211:Myoglobin / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013056:Spectrophotometry, Ultraviolet
chemical_list: D002021:Buffers; D007328:Insulin; D009211:Myoglobin; D011506:Proteins; D020543:Proteome