Efficient Separations of Intact Proteins Using Slip-Flow with Nano-Liquid Chromatography-Mass Spectrometry

A capillary with a pulled tip, densely packed with silica particles of 0.47 mu m in diameter, is shown to provide higher peak capacity and sensitivity in the separation of intact proteins by reversed-phase liquid chromatography-mass spectrometry (LC-MS). For a C18 bonded phase, slip flow gave a 10-fold flow enhancement to allow for stable nanospray with a 4-cm column length. Model proteins were studied: ribonuclease A, trypsin inhibitor, and carbonic anhydrase, where the latter had impurities of superoxide dismutase and ubiquitin. The proteins were well separated at room temperature with negligible peak tailing. The peak capacity for ubiquitin was 195 for a 10-min gradient and 315 for a 40-min gradient based on Gaussian fitting of the entire peak, rather than extrapolating the full-width at half-maximum. Separation of a cell lysate with a 60 min gradient showed extremely high peak capacities of 750 and above for a peptide and relatively homogeneous proteins. Clean, low noise mass spectra for each model protein were obtained. The physical widths of the peaks were an order of magnitude narrower than those of conventional columns, giving increased sensitivity. All proteins except ubiquitin exhibited significant heterogeneity apparently due to multiple proteoforms, as indicated by both peak shapes and mass spectra. The chromatograms exhibited excellent reproducibility in retention time, with relative standard deviations of 0.09 to 0.34%. The results indicate that submicrometer particles are promising for improving the separation dimension of LC in top-down proteomics.