Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) delivers high resolving power, mass measurement accuracy, and the capabilities for unambiguously sequencing by a top-down MS approach. Here, we report isotopic resolution of a 158 kDa protein complex - tetrameric aldolase with an average absolute deviation of 0.36 ppm and an average resolving power of ~520,000 at m/z 6033 for the 26+ charge state in magnitude mode. Phase correction further improves the resolving power and average absolute deviation by 1.3 fold. Furthermore, native top-down electron capture dissociation (ECD) enables the sequencing of 149 C-terminal amino acid (AA) residues out of 463 total AAs. Combining the data from top-down MS of native and denatured aldolase complexes, a total of 58% of the backbone cleavages efficiency is achieved. The observation of complementary product ion pairs confirms the correctness of the sequence and also the accuracy of the mass fitting of the isotopic distribution of the aldolase tetramer. Top-down MS of the native protein provides complementary sequence information to top-down ECD and CAD MS of the denatured protein. Moreover, native top-down ECD of aldolase tetramer reveals that ECD fragmentation is not limited only to the flexible regions of protein complexes and that regions located on the surface topology are prone to ECD cleavage.

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The Fourier transform spectrum can be presented in the absorption-mode (commonly used in FT-NMR), magnitude-mode (FT-ICR), and power-mode (engineering applications). As is routinely used in FT-NMR, it is well-known that the absorption-mode display gives a much narrower peak shape which greatly improves the spectrum; recently, the successful solution of the phase equation allowed broadband phase correction which makes it possible to apply the absorption-mode routinely in FT-ICR. With the empirical evidence provided herein, it has been confirmed that in addition to the improvement on resolving power, compared to the conventional magnitude-mode, the new absorption-mode improves the signal-to-noise ratio (S/N) of a spectrum by 1.4-fold and can improve the mass accuracy up to 2-fold with no extra cost in instrumentation. Therefore, it is worthwhile to apply and promote absorption-mode in routine FT-ICR experiments.

“Native” mass spectrometry (MS) has been proven increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD MS hADH dimer shows that each subunit (E and S chain) binds not only to two zinc atoms, but also the NAD+/NADH ligand, with a higher NAD+/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.

FT-ICR mass spectrometry has been limited to magnitude mode for almost 40 years due to the data processing methods used. However, it is well known that phase correction of the data can theoretically produce an absorption-mode spectrum with a mass-resolving power that is as much as twice as high as conventional magnitude mode, and that it also improves the quality of the peak shape. Temporally dispersed frequency sweep excitation followed by a time delay before detection results in a steep quadratic variation in the signal phase with frequency. Viewing this, it is possible to find the correct phase function by performing a quadratic least squares fit, modified by iterating through phase cycles until the correct quadratic function is found. Here, we present a robust manual method to rotate these signals mathematically and generate a "phased" absorption-mode spectrum. The method can, in principle, be automated. Baseline correction is also included to eliminate the accompanying baseline drift. The resulting experimental FT-ICR absorption-mode spectra exhibit a resolving power that is at least 50% higher than that of the magnitude mode.