TY - JOUR
T1 - Capillary electrophoresis for simultaneous quantification of human proinsulin, insulin and intermediate forms
AU - Arcelloni, C.
AU - Falqui, L.
AU - Martinenghi, S.
AU - Pontiroli, A. E.
AU - Paroni, R.
PY - 1998
Y1 - 1998
N2 - Capillary electrophoresis (CE) for the simultaneous and precise quantification of human insulin (hI), proinsulin (hPI) and intermediate forms (des 31, 32; split 65-66 and des 64, 65), released in culture media by engineered cells, is described. Analytical conditions for standard proteins were optimized using a bare silica capillary (20 cm x 50 μm internal diameter). Proteins were monitored at 200 nm and separated at constant voltage. Culture supernatants (12-24 mL) were purified on Sep-Pak Vac C18 cartridges, recovered in 1 mL of acetonitrile:trifluoracetic acid mixture (60:40, v:v), concentrated, ultrafiltered and injected into CE. Protein recovery was 85 ± 14% (n = 5, mean ± standard deviation) with a sensitivity limit of 0.5 nmol/L in the culture media, corresponding to 2 fmol injected in 22 nL. Using the CE method, it was possible to detect and quantify, with precision and accuracy, the release of hPI, hI and intermediate forms directly in the cell culture media, and to compare the proteic pattern released from engineered cells transduced with different hPI gene constructs.
AB - Capillary electrophoresis (CE) for the simultaneous and precise quantification of human insulin (hI), proinsulin (hPI) and intermediate forms (des 31, 32; split 65-66 and des 64, 65), released in culture media by engineered cells, is described. Analytical conditions for standard proteins were optimized using a bare silica capillary (20 cm x 50 μm internal diameter). Proteins were monitored at 200 nm and separated at constant voltage. Culture supernatants (12-24 mL) were purified on Sep-Pak Vac C18 cartridges, recovered in 1 mL of acetonitrile:trifluoracetic acid mixture (60:40, v:v), concentrated, ultrafiltered and injected into CE. Protein recovery was 85 ± 14% (n = 5, mean ± standard deviation) with a sensitivity limit of 0.5 nmol/L in the culture media, corresponding to 2 fmol injected in 22 nL. Using the CE method, it was possible to detect and quantify, with precision and accuracy, the release of hPI, hI and intermediate forms directly in the cell culture media, and to compare the proteic pattern released from engineered cells transduced with different hPI gene constructs.
KW - Capillary electrophoresis
KW - Culture media purification
KW - Intermediate forms
KW - Proinsulin conversion
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U2 - 10.1002/elps.1150190843
DO - 10.1002/elps.1150190843
M3 - Article
C2 - 9694298
AN - SCOPUS:0031806238
VL - 19
SP - 1475
EP - 1477
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 8-9
ER -