High‐efficiency peptide analysis on monolithic multimode capillary columns: Pressure‐assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization‐mass spectrometry

Abstract

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.

Capillary electrophoresis‐mass spectrometry: Recent advances to the analysis of small achiral and chiral solutes

“…The applications of CEC-MS for achiral analysis continued to increase in the last 2-3 years as a convenient approach for small molecules, such as peptides [89,90], carbohydrates [91,92], pharmaceutical samples [93,94], and other small organic compounds [95]. Readers are also referred to a recent review from Barrachina and co-workers [96] entitled "State-of-the-art of the hyphenation of capillary electrochromatography with mass spectrometry".…”

“…Fast separation of peptides with high efficiency was obtained using a methacrylate-based monolithic column [89]. The peptide mass fingerprinting of bovine serum albumin was possible by applying a gradient of 5-30% acetonitrile in 0.1% acetic acid more than 10 min with a flow rate in the range of 180-185 nL/min.…”

Recent advances in polymeric monolithic stationary phases for electrochromatography in capillaries and chips

“…High-efficiency peptide analysis using multimode pressure-assisted CEC/CE with both UV and ESI-MS/MS was presented by Ivanov et al [15]. The authors used monolithic columns prepared by the copolymerization of EDMA, methyl methacrylate, and glycidyl methacrylate in the presence of a porogen consisting of formamide and n-propanol at different composition.…”

Recent advances in capillary separations for proteomics

“…Ivanov and co-workers [49] have recently modified the chromatographic surface of the monolith from neutral to cationic and applied the modified column to perform high-efficiency peptide analysis using multimode CEC. Under acidic conditions, the peptide separation was mainly controlled by electrophoretic migration along with limited chromatographic retention.…”