Characterization of heme ions using MALDI-TOF MS and MALDI FT-ICR MS

• Isotopic distributions of heme b and heme c ions in MALDI-MS analysis were comprehensively investigated.
• Apparent isotopic distributions of heme ions changed according to acquisition mode.
• The difference in MALDI FT-ICR MS was explained by the overlap of isobaric species.
• The difference in MALDI-TOF MS was explained by the existence of metastable ions.

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) and MALDI 15-T Fourier transform-ion cyclotron resonance (FT-ICR) MS were used to investigate the heme b ion from myoglobin and the heme c ion from tryptically digested cytochrome c. When the ion scan mode was changed from linear mode to reflectron mode in MALDI-TOF MS, the ratio of isotope distributions (intensities of M over M + 1, where M is the integer m/z value of the most abundant ion which is 616 for heme b or 617 for heme c) changed for both heme ions. Similarly, when the ion scan mode was changed from a resolution of 8000 to 800,000 in MALDI 15-T FT-ICR MS, the same phenomenon was observed. The discrepancy in the isotopic distribution of heme ions in the MALDI 15-T FT-ICR MS analysis between the two resolutions was explained by the deconvolution process of overlapping peaks, while the difference in the isotopic distribution between the linear and reflectron modes in the MALDI-TOF MS analysis was explained by the presence of metastable ions which were only detectable in the linear mode of TOF analysis.

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