High speed capillary zone electrophoresis–mass spectrometry via an electrokinetically pumped sheath flow interface for rapid analysis of amino acids and a protein digest

• We demonstrate a 2-min CZE–ESI-MS separation of an amino acid mixture.
• We demonstrate a 2-min CZE–ESI-MS separation of a BSA digest.
• We demonstrate 52% sequence coverage for BSA in the separation.
• We also demonstrate a peak capacity of ∼50.

While capillary zone electrophoresis (CZE) has been used to produce very rapid and efficient separations, coupling these high-speed separations with mass spectrometry (MS) has been challenging. Now, with much faster and sensitive mass spectrometers, it is possible to take full advantage of the CZE speed and reconstruct the fast migrating peaks. Here are three high-speed CZE–MS analyses via an electrokinetically pumped sheath-flow interface. The first separation demonstrates CZE–ESI-MS of an amino acid mixture with a 2-min separation, >50,000 theoretical plates, low micromolar concentration detection limits, and subfemtomole mass detection limits (LTQ XL mass spectrometer). The second separation with our recently improved third-generation CE–MS interface illustrates a 20 amino acid separation in ∼7 min with an average over 200,000 plate counts, and results in almost-baseline resolution of structural isomers, leucine and isoleucine. The third separation is of a BSA digest with a reproducible CZE separation and mass spectrometry detection in 2 min. CZE–MS/MS analysis of the BSA digest identified 31 peptides, produced 52% sequence coverage, and generated a peak capacity of ∼40 across the 1-min separation window (Q-Exactive mass spectrometer).