Fourier Transform Mass spectrometry (FT-ICR): 2 and multltidimensional acquisition and data processing for fast parallel acess to all MS/MS data from an MS spectrum – FT-ICR 2D

For the purpose of this study, all pulses were identical in amplitude and frequency range. They were built using the Apex Control pulse generator (Bruker, Bremen, Germany). Pulse amplitude attenuation was 7.0 dB (i.e. identical to the default pulse amplitude attenuation of excitation pulses). For substance P, the pulse length was 1.0 us per frequency (the default excitation pulse length is 20.0 us per frequency). For each experiment, the frequency list had a 2.0 kHz increment.
For substance P, the delay between the two first radiofrequency excitation pulses varied with a 1.0 us increment, corresponding to a Nyquist frequency of 500 kHz and an m/z 288.724 - 1500.000 mass range in the vertical dimension. For all three experiments, 2048 time transients with an incremental delay were recorded, leading to an experiment time of 71 min. The length of the time transients for each experiment was 65536 data points, which is relatively short for FT-ICR MS experiments. This choice was imposed by data processing considerations in order to have data files that were not too large to be easily handled. The modulus was applied to the result.
Data Processing. Because of the level of scintillation noise in 2D mass spectra using nanoESI ionization, an additional step using the Cadzow algorithm was added between the Fourier transform along t2 (measurement date) and the Fourier transform along t1 (incremental delay between the two first pulses). The adaptation of the Cadzow algorithm to FT-ICR MS, which has been performed in the frame of this project, allows the de-noising of 2D mass spectra in order to remove the vertical scintillation noise from the highest abundance peaks and to bring out signal peaks that are hidden in the noise.

We successfully implemented two-dimensional FT-ICR mass spectrometry on a commercial instrument and we showed that IRMPD (InfraRed Multi Photon Dissociation) and ECD (Electron Capture Dissociation) are fragmentation modes that can be used with good results in a 2D mass spectrometry experiment. We applied ECD both to peptides and glycopeptides and we obtained fragmentation patterns that are equivalent to the ones in MS/MS, with abundances that are equivalent. Processing 2D mass spectra with the Cadzow algorithm has been showed to eliminate a lot of the scintillation noise and vertical stripes that can make 2D mass spectra difficult to interpret and also increase the signal-to-noise ratio. In order to improve the sensitivity of 2D ECD FT-ICR MS, we are currently developing a data processing program that can handle large data files. By using a more efficient data format and faster algorithm, we can keep data processing of large data files manageable while improving the resolving power and the sensitivity of 2D mass spectrometry, as well as the complexity of the samples that can be analyzed by 2D FT-ICR MS. This enables us to record 2D ECD FT-ICR mass spectra both of intact proteins and tryptic digests.
By successfully recording 2D mass spectra both of peptides and glycopeptides, we have shown that 2D ECD FT-ICR mass spectrometry has the potential to be of use both in top-down and bottom-up proteomics. Because in 2D FT-ICR MS the duty cycle of the mass analyzer does not interfere with the resolving power, all ion species are fragmented instead of a few chosen ones and the signal-to-noise ratio is much higher. ECD FT-ICR mass spectrometry can become analytically useful for abundant samples like for example human plasma, petroleum and most environmental samples, since it avoids a chromatographic step which always leads to the loss of molecular families in the sample.

Several problems need to be addressed in order to obtain analytically useful 2D mass spectra. The first issue concerns the harmonics of precursor ion peaks (and, to a lesser extent, of fragment ion peaks) that appear in the vertical dimension. Fragmentation by ECD and IRMPD occurs at the center of the ICR cell, so the modulation of ionic radii resembles a Dirac comb rather than a sinusoid. As a result, harmonics in the vertical dimension are intense. In order to eliminate these problems, we are considering both pulse sequence parameter optimization and additional data processing steps, like investigating frequency correction in the vertical dimension.
The need to record large datasets in order to obtain high resolution in 2D mass spectra leads to protracted experiments and consume large amounts of sample. Fortunately, there are many partial sampling techniques that can be adapted from 2D NMR spectroscopy to 2D FT-ICR MS in order to reduce the size of the datasets without loss of resolving power. These can lead to shorter experiments and less sample consumption. We are also looking into possibilities offered by alternative pulse sequences, especially by adapting ideas like the accordion experiment from 2D NMR spectroscopy to 2D FT-ICR MS which may give access to ion life-time. We can also obtain high resolution 2D mass spectra through alternative data processing methods, like the Filter Diagonalisation Method, which shows great promise in one-dimensional FT-ICR mass spectrometry.

[4] van Agthoven, Maria A.; Delsuc, Marc-Andre; Bodenhausen, Geoffrey; Rolando, Christian. Towards Analytically Useful Two-Dimensional Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Analytical and Bioanalytical Chemistry 2012, 404, In revision.

[3] van Agthoven, Maria A.; Chiron, Lionel; Coutouly, Marie-Aude; Delsuc, Marc-André; Rolando, Christian. Two-Dimensional ECD FT-ICR Mass Spectrometry of Peptides and Glycopeptides. Analytical Chemistry 2012, 84, 5589-5595.
pubs.acs.org/doi/abs/10.1021/ac3004874

[2] van Agthoven, Maria A.; Delsuc, Marc-Andre; Rolando, Christian. Two-dimensional FT-ICR/MS with IRMPD as fragmentation mode. International Journal of Mass Spectrometry 2011, 306, 196-203.
www.sciencedirect.com/science/article/pii/S138738061000415X

[1] van Agthoven, Maria A.; Coutouly, Marie-Aude; Rolando, Christian; Delsuc, Marc-Andre. Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry: reduction of scintillation noise using Cadzow data processing. Rapid Communications in Mass Spectrometry 2011, 25, 1609-1616.
onlinelibrary.wiley.com/doi/10.1002/rcm.5002/abstract

The aim of this project is to develop two-dimensional mass spectrometry methods based on FT-ICR/MS (Fourier Transform Ion Cyclotron Resonance Mass Spectrometry) in order to expand the field of structural analysis of complex samples.
The first results in two-dimensional mass spectrometry using FT-ICR/MS were obtained in 1987 by the FT-ICR team of the Ecole Polytechnique of Lausanne directed by T. Gaümann in collaboration with G. Bodenhausen for NMR techniques. The mass range of these results was narrow, the amount of data was important compared to the cost of data storage and the resolution of 2D mass spectra in the second dimension was too low to study samples of real complex mixtures.
Thanks to the advances in electronics and computer science, most of the obstacles to the development of 2D FT-ICR/MS have disappeared. Its advantages are still applicable. Usual MS/MS techniques are laborious because it is necessary to measure one spectrum per ion in the MS spectrum. Furthermore, the measurement of MS/MS spectra of low-abundance ions leads to a low signal-over-noise ratio. With he acquisition of 2D FT-ICR mass spectra, MS/MS can be fully automated without information loss. Signal abundance problems can be avoided. Instead of acquiring information in a serial manner in MS/MS, it is possible to obtain it in a parallel manner and to contain it in one spectrum. As 2D NMR has simplified the interpretation of NMR data and increased the information in NMR spectra, we expect the same advantages from the development of 2D FT-ICR/MS.
Nevertheless, there are problems that need to be addressed in order to obtain readable 2D mass spectra. First, a program that analyses 2D FT-ICR/MS data needs to be developed. In preliminary studies, a program conceived for 2D NMR was used, but the files generated by 2D FT-ICR/MS experiments are much larger in size than those generated by 2D NMR. Therefore, the software needs to be adapted to mass spectrometry. The second problem is the scintillation noise caused by the fluctuation of the number of ions trapped in the ICR cell. It cannot be reduced by accumulative data acquisition. There are methods developed in 2D NMR that deal with this problem. They have to be adapted to 2D FT-ICR/MS.
A two-dimensional mass spectrometry experiment is composed of three elements: a pulse sequence, data acquisition and data processing. Therefore, our research will focus on these three axes. The development of new pulse sequences is necessary in order to minimize the acquisition time of 2D FT-ICR mass spectra without affecting resolution (superresolution, partial sampling), in order to limit data size for low density mass spectra and to develop 3D FT-ICR/MS techniques and beyond. New pulse sequences are also necessary in order to adapt 2D FT-ICR/MS to new fragmentation modes that don’t require to inject a gas in the ICR cell (IRMPD, ECD, EID). Finally, the development of data processing techniques will focus on noise reduction and the increase of resolution in the second dimension with, for instance, correlation methods. Once these multidimensional mass spectrometry techniques have been optimized, we wish to apply them to a series of biological samples of increasing complexity: apolipoprotein A1 digests, native human apolipoprotein A1, HDL samples and human blood plasma. The expected result of this project is to make multidimensional mass spectrometry a viable technique in analytical chemistry.