Increased throughput and ultra-high mass resolution in DESI FT-ICR MS imaging through new-generation external data acquisition system and advanced data processing approaches

Desorption electrospray ionisation-mass spectrometry imaging (DESI-MSI) is a powerful imaging technique for the analysis of complex surfaces. However, the often highly complex nature of biological samples is particularly challenging for MSI approaches, as options to appropriately address mass spectral complexity are limited. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers superior mass accuracy and mass resolving power, but its moderate throughput inhibits broader application. Here we demonstrate the dramatic gains in mass resolution and/or throughput of DESI-MSI on an FT-ICR MS by developing and implementing a sophisticated data acquisition and data processing pipeline. The presented pipeline integrates, for the first time, parallel ion accumulation and detection, post-processing absorption mode Fourier transform and pixel-by-pixel internal re-calibration. To achieve that, first, we developed and coupled an external high-performance data acquisition system to an FT-ICR MS instrument to record the time-domain signals (transients) in parallel with the instrument’s built-in electronics. The recorded transients were then processed by the in-house developed computationally-efficient data processing and data analysis software. Importantly, the described pipeline is shown to be applicable even to extremely large, up to 1 TB, imaging datasets. Overall, this approach provides improved analytical figures of merits such as: (i) enhanced mass resolution at no cost in experimental time; and (ii) up to 4-fold higher throughput while maintaining a constant mass resolution. Using this approach, we not only demonstrate the record 1 million mass resolution for lipid imaging from brain tissue, but explicitly demonstrate such mass resolution is required to resolve the complexity of the lipidome

Phase relationships in two-dimensional mass spectrometry

Two-dimensional mass spectrometry (2D MS) is a data-independent tandem mass spectrometry technique in which precursor and fragment ion species can be correlated without the need for prior ion isolation. The behavior of phase in 2D Fourier transform mass spectrometry is investigated with respect to the calculation of phase-corrected absorption-mode 2D mass spectra. 2D MS datasets have a phase that is defined differently in each dimension. In both dimensions, the phase behavior of precursor and fragment ions is found to be different. The dependence of the phase for both precursor and fragment ion signals on various parameters (e.g., modulation frequency, shape of the fragmentation zone) is discussed. Experimental data confirms the theoretical calculations of the phase in each dimension. Understanding the phase relationships in a 2D mass spectrum is beneficial to the development of possible algorithms for phase correction, which may improve both the signal-to-noise ratio and the resolving power of peaks in 2D mass spectra

Phase correction of fourier transform ion cyclotron resonance mass spectra using MatLab

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