Hydropathic influences on the quantification of equine heart cytochrome c using relative ion abundance measurements by electrospray ionization fourier transform ion cyclotron resonance mass spectrometry.

The number of publications documenting the utility of electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) for the analysis of biological molecules has increased in geometric proportion spanning diverse areas of research. Currently, we are investigating the capabilities of ESI-FTICR to quantify relative molecular ion abundances of biopolymers, an area which has not been explored rigorously. We present here the results of an investigation of a two-component system utilizing equine heart cytochrome c (EH) as the analyte and bovine heart cytochrome c (BH) as a constant concentration internal standard. As these compounds are relatively large ( approximately 12 kDa), they will become multiply charged during the electrospray process. Using appropriate solution and instrument conditions, the 7(+) and 8(+) charge states were enhanced for both cytochrome c species. We report that using the average of the ion abundances for the two charge states observed for each species, the linear curve (intensity ratio vs concentration ratio) had a dynamic range of 0.045-2.348 microM (1.7 orders of magnitude). Linear least-squares regression analysis (LLSRA) of these averaged ion abundances (i.e. [(EH + 7H(+))(7+)/(BH + 7H(+))(7+) + (EH + 8H(+))(8+)/(BH + 8H(+))(8+)]/2) yielded the equation y = 1.005x + 0.027. The slope of the line with its calculated precision, reported as one standard deviation, is 1.005 +/- 0.0150, which is statistically ideal (i.e. equal to unity). However, LLSRA of the ion abundances of the two individual charge states were significantly different (i.e. the slope of the (EH + 7H(+))(7+)/(BH + 7H(+))(7+) peak intensity ratio vs molar ratio data was 0.885 +/- 0.0183 and the slope of the (EH + 8H(+))(8+)/(BH + 8H(+))(8+) data was 1.125 +/- 0.0308). We attribute this difference to the variation in primary amino acid sequence for the two cytochrome c species. Both have 104 amino acids, but there are three residue substitutions between EH and BH; one of the substitutions confers an additional basic site to EH. While this extra basic residue may imply an additional charging site, the low charge states observed under the solution conditions employed indicate that most (>66%) basic sites are not protonated. However, the extra basic site also renders EH slightly more hydrophilic. These results present significant considerations when choosing internal standards for the quantification of large proteins by ESI-FTICR-MS and demonstrate that relative molecular ion signals in FTICR can be used to quantify macromolecular species in the nanomolar regime.