Capillary electrophoresis for simultaneous quantification of human proinsulin, insulin and intermediate forms

C. Arcelloni, L. Falqui, S. Martinenghi, A. E. Pontiroli, R. Paroni

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)1475-1477
Number of pages3
JournalElectrophoresis
Volume19
Issue number8-9
DOIs
Publication statusPublished - 1998

Keywords

  • Capillary electrophoresis
  • Culture media purification
  • Intermediate forms
  • Proinsulin conversion

ASJC Scopus subject areas

  • Clinical Biochemistry

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