A method of protein quantitative analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is described. To decouple the quantitative approach from the extreme dependence of absolute ion signal on the experimental parameters of the matrix-assisted technique, the method relies on the normalization of analyte molecular ion signals to internal reference molecular ion signals. Further, as protein molecular ion signal responses are dependent on the nature of the solutions from which they are analyzed, experiments were carried out using a quantitative approach of standard addition. Linear relationships were observed, over at least 1 order of magnitude, between the normalized analyte signal and analyte concentration, with average relative errors on the order of ∼ 15%. For molecular ion signals which were well resolved from quasi- molecular adduct ion signals, little difference in average relative error was observed when the analyte concentration was plotted versus either the normalized intensity or the normalized integral. The average relative error was observed to be lower when the normalized intensity of nonresolved ion signals was used. In model solutions containing more than just analyte and reference proteins, little suppression of tertiary protein component signals was observed due to increasing the analyte concentration. In natural solutions, however, analyte signal was observed to be suppressed due to the complexity of the biological fluid analyzed. Through the use of simple purification techniques, this suppression was overcome to an extent which allowed the quantitative analysis of a clinically significant protein present in a natural biological fluid. General considerations, with respect to limitations and application of the quantitative and purification approaches used, are given.
ASJC Scopus subject areas
- Analytical Chemistry