Eriksson Jonas H C, Mol Roelof, Somsen Govert W, Hinrichs Wouter L J, Frijlink Henderik W, de Jong Gerhardus J
Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Groningen, The Netherlands.
Electrophoresis. 2004 Jan;25(1):43-9. doi: 10.1002/elps.200305695.
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.
研究了通过三轴接口将毛细管电泳（CE）与电喷雾电离质谱（ESI-MS）相结合作为完整蛋白质表征的潜在手段。为了评估在CE中使用非挥发性电解质的可能性，通过进样实验研究了磷酸钠和硼酸铵对蛋白质胰岛素、肌红蛋白和牛血清白蛋白（BSA）的MS信号的影响，并与甲酸铵和甲酸的影响进行了比较。研究表明，使用甲酸（50 mM，pH 2.4）可获得最强的蛋白质信号，而使用磷酸钠缓冲液（5和10 mM，pH 7.5）几乎完全消除了MS响应。甲酸铵和硼酸铵（高达100 mM，pH 8.5）也会导致蛋白质离子抑制，但特别是使用硼酸盐缓冲液时仍保留有显著的MS强度。对肌红蛋白的MS分析表明，当使用酸性CE电解质时，血红素基团会丢失。使用含有25 mM硼酸铵（pH 8.5）的背景电解质，证明了可以用ESI-MS监测蛋白质测试混合物的CE分离，而不会降低MS性能，从而可以测定分离化合物的分子量。在硼酸盐存在下，检测限估计为5-10 microM（约100 fmol进样量）。对储存的BSA样品进行分析，发现了几种降解产物，这表明采用硼酸盐缓冲液的CE-MS系统是有用的。还通过CE-MS分析了胎盘碱性磷酸酶（PLAP，一种潜在的治疗剂）样品，表明存在蛋白质杂质。可能由于PLAP（一种复杂的糖蛋白）的电离不足，未观察到完整蛋白质的MS信号。
