Feasibility of nonvolatile buffers in capillary electrophoresis-electrospray ionization-mass spectrometry of proteins.
The combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI-MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5-10 microM (ca. 100 fmol injected). The usefulness of the CE-MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE-MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.
Eriksson
Jonas H C
JH
Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Groningen, The Netherlands.
Mol
Roelof
R
Somsen
Govert W
GW
Hinrichs
Wouter L J
WL
Frijlink
Henderik W
HW
de Jong
Gerhardus J
GJ
D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D001881:Borates; D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000592:standards; D005240:Feasibility Studies; D005561:Formates; D006801:Humans; D010710:Phosphates; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods* / Q000592:standards
D001881:Borates; D002021:Buffers; D005561:Formates; D010710:Phosphates; D011506:Proteins; C030544:formic acid; C028466:ammonium borate; D000469:Alkaline Phosphatase; C018279:sodium phosphate