Evaluation of two-beam fluorescence cross correlation spectroscopy for electrophoretic analysis of protein digests

Abstract

In this report, we evaluate experimentally and through the use of simulations and calculations the possibility of using 2-beam fluorescence cross correlation spectroscopy (2BFCCS) for the multicomponent electrophoretic analysis of peptides. The concept described has potential as a high throughput, extraordinarily sensitive means of identifying proteins and accelerating proteome studies. We present Monte Carlo simulation methods and results using them that help in understanding the capabilities and limitations of 2BFCCS for protein identification. We have calculated the expected pH dependent mobility and resultant 2BFCCS fingerprint spectra for a randomly selected subset of the peptides present upon digestion of horse myoglobin by trypsin. We demonstrate experimentally the multicomponent analysis of a mixture containing a fluorescently labeled peptide and a free fluorophore. We also demonstrate experimentally the ability to measure migration rates of dilute, single-fluorophore species over more than 2 orders of magnitude in linear velocity (between 4.2 mm s⁻¹ and 640 mm s⁻¹).