On-line capillary isoelectric focusing-mass spectrometry for quantitative analysis of peptides and proteins

On-line capillary isoelectric focusing-mass spectrometry (cIEF-MS) was applied to determine concentrations of peptides and proteins using angiotensin II and human tetrasialo-transferrin as the model samples. The concentration of the carrier ampholyte was optimized for both resolution and ion intensity. cIEF-MS employing 1% Pharmalyte 3-10 and a sheath liquid containing water/methanol/acetic acid (50/49/1) resolved angiotensin I and II (5 μM each, ΔpI = 0.2) at an Rs value of 2.29. The determined concentration of angiotensin II (0.1-5 μM) well correlated (R = 0.999) with that obtained by the conventional RP-HPLC method. The limit of detection was 0.22 μM, which was about 10 times lower than that by UV detection (2 μM). The repeatability and accuracy were <15 and <11%, respectively. cIEF-MS was also applied to determine human tetrasialo-transferrin concentration. The good linearity (R2 = 0.998) was also observed between the transferrin concentration (0.5-1.2 g/L) and peak area ratio (IS; β-lactoglobulin B) with acceptable accuracy (<1.9%) and repeatability (∼10% at 1 g/L).