Capillary Isoelectric Focusing of Proteins and Peptides Using an In-Line cIEF-ESI Interface with Improved MS Characteristics

Intact protein analysis using mass spectrometry (MS) is an important technique to characterize and provide a comprehensive overview of protein complexity. It is also the basis of “top-down” approaches in proteomics to describe the proteoforms of single protein’s post-translational modifications (PTMs). MS-based analysis of intact proteins benefits from high-resolution separations prior to electrospray ionization. Capillary isoelectric focusing (cIEF) is a high-resolution separation for proteins and peptides which is capable of separating proteoforms. MS detection coupled to cIEF can separate, detect, and characterize proteoforms at the molecular level. However, cIEF with MS detection is a compromised process. The concentration of ampholytes required for cIEF is mutually exclusive with mass spectrometer contamination. We have improved an online cIEF-ESI-MS interface to reduce (desalt) amino acid ampholytes in-line after cIEF and prior to electrospray ionization. In proof of principle experiments, >90% increase in area under the curve of the electropherograms was observed with the interface compared to without the interface. Protein standards including proteoforms of cytochrome C, myoglobin, and α-casein were separated and resolved with high reproducibility. The interface did not compromise the linearity of the cIEF pH gradient separations, achieving a high linearity with a R² of 0.99. In addition, a tryptic digest of BSA demonstrates baseline resolution of peptides with as little as 0.02 pI unit difference and a full width at half-maximum average of 7.1 s.