Absolute Myoglobin Quantitation in Serum by Combining Two-Dimensional Liquid Chromatography−Electrospray Ionization Mass Spectrometry and Novel Data Analysis Algorithms

To measure myoglobin, a marker for myocardial infarction, directly in human serum, two-dimensional liquid chromatography in combination with electrospray ionization mass spectrometry was applied as an analytical method. High-abundant serum proteins were depleted by strong anion-exchange chromatography. The myoglobin fraction was digested and injected onto a 60 mm × 0.2 mm i.d. monolithic capillary column for quantitation of selected peptides upon mass spectrometric detection. The addition of known amounts of myoglobin to the serum sample was utilized for calibration, and horse myoglobin was added as an internal standard to improve reproducibility. Calibration graphs were linear and facilitated the reproducible and accurate determination of the myoglobin amount present in serum. Manual data evaluation using integrated peak areas and an automated multistage algorithm fitting two-dimensional models of peptide elution profiles and isotope patterns to the mass spectrometric raw data were compared. When the automated method was applied, a myoglobin concentration of 460 pg/μL serum was determined with a maximum relative deviation from the theoretical value of 10.1% and a maximum relative standard deviation of 13.4%. Keywords: absolute quantitation • serum • myoglobin • high-performance liquid chromatography • electrospray ionization mass spectrometry • two-dimensional HPLC • standard addition • monoliths • computational proteomics • algorithms