Huber C G, Premstaller A
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innsbruck, Austria.
J Chromatogr A. 1999 Jul 16;849(1):161-73. doi: 10.1016/s0021-9673(99)00532-4.
Proteins ranging in molecular mass from 14,000 to 80,000 were analyzed by reversed-phase high-performance liquid chromatography-electrospray mass spectrometry (RP-HPLC-ESI-MS) using 60 x 1.0 mm I.D. microbore-columns packed with 2.3 microns highly crosslinked, octadecylated poly(styrene-divinylbenzene) particles. Proteins were eluted at temperatures of 80-90 degrees C with gradients of acetonitrile in 0.10-0.50% aqueous solutions of trifluoroacetic acid, formic acid or acetic acid. Substitution of trifluoroacetic acid, the most commonly used mobile phase additive for RP-HPLC, by formic acid resulted in a 35-160-fold improvement in analyte detectability at the cost of an only 32-104% increase in peak width at half height of eluting chromatographic peaks. The lower limits of detection for carbonic anhydrase (M(r) 29,022.7) in full scan and selected ion monitoring mode were 37 and 2.3 fmol, respectively. Measurement of protein masses by RP-HPLC-ESI-MS was accurate and highly reproducible with maximum mass deviations of 0.025% and relative standard deviations of less than 0.011%. Calibration plots of peak area versus concentration allowed the reliable quantitation of proteins in a concentration range of 0.010-1.0 mg/ml. Finally, the optimized method was applied to the separation, identification and quantification of proteins in real samples such as commercial protein preparations, monoclonal antibody fragments, allergen extracts and whey drinks.
使用内径为60×1.0 mm、填充有2.3微米高度交联的十八烷基化聚（苯乙烯 - 二乙烯基苯）颗粒的微径柱，通过反相高效液相色谱 - 电喷雾质谱（RP - HPLC - ESI - MS）分析分子量在14,000至80,000之间的蛋白质。蛋白质在80 - 90℃的温度下，用乙腈在0.10 - 0.50%的三氟乙酸、甲酸或乙酸水溶液中的梯度进行洗脱。用甲酸替代反相高效液相色谱中最常用的流动相添加剂三氟乙酸，使分析物的检测能力提高了35 - 160倍，代价是洗脱色谱峰半高宽仅增加了32 - 104%。在全扫描和选择离子监测模式下，碳酸酐酶（分子量29,022.7）的检测下限分别为37和2.3 fmol。通过RP - HPLC - ESI - MS测量蛋白质质量准确且高度可重复，最大质量偏差为0.025%，相对标准偏差小于0.011%。峰面积与浓度的校准曲线允许在0.010 - 1.0 mg/ml的浓度范围内可靠地定量蛋白质。最后，将优化后的方法应用于商业蛋白质制剂、单克隆抗体片段、过敏原提取物和乳清饮料等实际样品中蛋白质的分离、鉴定和定量。
