Ivanov Alexander R, Horváth Csaba, Karger Barry L
Barnett Institute, Northeastern University, Boston, MA, USA.
Electrophoresis. 2003 Nov;24(21):3663-73. doi: 10.1002/elps.200305620.
High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.
本文展示了使用多模式压力辅助毛细管电色谱/毛细管电泳（pCEC/pCE）整体式聚合物柱进行高效肽分析，以及在施加电场下通过等度洗脱和梯度洗脱对模型肽混合物和蛋白质消化产物进行分离，并采用紫外和电喷雾电离质谱（ESI-MS）检测。通过在作为致孔剂的正丙醇和甲酰胺以及作为引发剂的偶氮二异丁腈存在下，对内径为50、75和100微米的硅烷化熔融石英毛细管进行热诱导原位共聚，制备了毛细管多功能柱。使用N-乙基丁胺将整体柱的色谱表面从中性改性为阳离子性。整体柱被称为多功能或多模式柱，因为它们在不同条件下显示出混合的分离机制模式。液相色谱（LC）模式下的阴离子交换分离能力可由整体柱的阳离子色谱表面决定。在酸性pH值和柱上施加高电压时，整体固定相为肽提供了主要进行毛细管电泳迁移的条件。在碱性pH值和柱上施加电场时，肽在整体毛细管柱上的色谱保留增强，使得CEC迁移机制负责分离。研究了压力、离子强度、pH值和流动相有机含量对色谱性能的影响。使用挥发性和非挥发性、酸性和碱性缓冲液时，整体柱对肽分离的效率很高（超过300000塔板/米）。对于紫外和ESI-MS检测，等度洗脱和梯度洗脱压力辅助CEC/CE分离均具有良好的重现性和稳健性。通过操纵电场和梯度条件，可以对复杂肽混合物进行高通量分析。一种简单的无鞘电喷雾发射器设计为整体多模式柱与ESI-MS提供了有效且稳健的低死体积接口。在不到5分钟的时间内，对牛血清白蛋白（BSA）胰蛋白酶消化产物进行梯度洗脱压力辅助混合模式分离CE/CEC-ESI-MS质谱指纹图谱分析和数据依赖型pCE/pCEC-ESI-MS/MS分析，获得了高序列覆盖率（73%），证明了该方法具有潜力。
