Simultaneous Quantification of Human Cardiac α- and β-Myosin Heavy Chain Proteins by MALDI-TOF Mass Spectrometry

We have developed a novel method for quantifying protein isoforms, in both relative and absolute terms, based on MALDI-TOF mass spectrometry. The utility of the approach is demonstrated by quantifying the α and β protein isoforms of myosin heavy chain (MyHC) in human atrial tissue. α-MyHC (726−741) and β-MyHC (724−739) were identified as isoform-specific tryptic peptides. A calibration curve was constructed by plotting ion current ratios against molar ratios of the two peptides prepared synthetically. MyHC was digested by trypsin and the ion current ratio determined for the two tryptic peptides. The ion current ratio was converted to the peptide ratio and hence the isoform ratio by reference to the standard curve. The accuracy of the method was confirmed by a comparison between these results and those determined by an established method of MyHC isoform ratio determination. So that the molar ratio could be converted to absolute values, a third peptide, an analogue of the two peptides being measured, was sy...