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 microM each, DeltapI=0.2) at an Rs value of 2.29. The determined concentration of angiotensin II (0.1-5 microM) well correlated (R=0.999) with that obtained by the conventional RP-HPLC method. The limit of detection was 0.22 microM, which was about 10 times lower than that by UV detection (2 microM). 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; beta-lactoglobulin B) with acceptable accuracy (<1.9%) and repeatability ( approximately 10% at 1g/L).
Kuroda
Yukihiro
Y
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606 8501, Japan. y.kuroda@scas.co.jp
Yukinaga
Hideo
H
Kitano
Masahiro
M
Noguchi
Taeko
T
Nemati
Mahboob
M
Shibukawa
Akimasa
A
Nakagawa
Terumichi
T
Matsuzaki
Katsumi
K
D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D009862:Online Systems; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D012756:Sheep
D010455:Peptides; D011506:Proteins