Continuous mode of operation for large volume dosing in analytical carrier ampholyte-free isoelectric focusing of proteins applied to off-line detection of fractions

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‐μL 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.