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Artefacts in the Mass Spectra Output from MALDI-TOF and MALDI-TOF/TOF Machines
Werner Van Belle1* - firstname.lastname@example.org, email@example.com
Abstract : MALDI-TOF mass spectrometry is a well known and widely used technique to fingerprint and sequence proteins. A carefull investigation of the mass spectra output from unnamed machines shows a number of artefacts produced by the machines themselves. Because these artefacts complicate a number of procedures we present a number of preliminary techniques we developed to get rid of most of the artefacts.
matrix assisten laser desorption ionisation MALDI time of flight TOF artefacts noise
Tones in Reflection Mode
Decaying tones in Lift
Mass Spectrum Denoising
Data Enhancing in High Noise Lineair Mode
Automatic Detection of important peaks
In MALDI-TOF (Matrix assisted laser desorption ionization) a sample is mixed with a matrix. When this mixture dries it forms crystals. When such a crystallized mixture is targeted with a high energy laser beam with the correct wavelength, the matrix itself will suddenly absorb the incoming energy and heat up. This rapid heating causes sublimation of the matrix and subsequent expansion of the molecules co-crystallized within the matrix. The ions are then accelerated using a strong electrical field and thus separated based on their ratio. The ions can then be detected at the end of the tube, or reflected and then be detected. This (optional) reflection phase increases the accuracy of the technique substantially.
In a typical proteomics setup a mass spectrogram is taken, the peaks are selected and then used to fingerprint proteins. Some machines offer the possibility to use an advanced lift system which makes it possible to measure the mass of the (poly)peptides within a larger fragment of a specific weight. This makes sequencing of proteins possible.
We performed a number of measurements on different mass spectrometers. Surprisingly, the output from these machines contains a number of artifacts, which were also present on machines located at other sites, such as the Flemish Biotechnology Center and freely published online spectra.
We believe that these artifacts complicate a number of possible uses of those machines
|Artefacts in a typical mass spectrometry using the reflection mode.|
The first experiment concerns the typical fingerprinting of a protein. In this experiment the reflection mode was turned on. The mass spectrum output consist of 158548 samples between 100.003 and 4019.170 Da. The window size of the SFFT is 2048 samples, which forms a good compromise between frequency-accuracy and position accuracy. In all the figures we present, both the m/z axis and the energy axis have been normalized. The frequency analysis has also been normalized and is shown in dB.
|Noise of the Lift Spectrum|
In a second experiment we measured the lift of a peak using a MALDI-TOF/TOF machine. The mixture contained a protein-fragment which was to be sequenced. The output from the machine ranges from 20.067 till 1264.626, in 67873 samples. Again, the m/z, energy and frequency content are all three normalized. The frequency analysis (figure ) shows
|Noise in lineair mode|
|Noise in lineair mode|
In a third experiment we measured the pure noise output of a MALDI machine in linear mode. The output shown in figures and covers 110296 samples between 40 kDa and 80kDa. During the experiment, the laser was switched off, as such we measure only the noise generated by the machine. The artifacts we now observed were even more interesting then the previous ones.
To investigate the feasibility to obtain more data out of the spectra, we created a number of denoising and enhancing techniques which we briefly present below.
In order to denoise the data we first tried the creation of a number of digital notch filters. Because we don't want to shift the peaks back or forth in time, such a filter was required to have a zero-phase response over its entire spectrum. Also the impulse response of the filter needed to be as small as possible because we did not want to broaden the peaks, nor introduce unwelcome echos. A number of small experiments indicates that the results of such a filter would not be so very good. It became also clear that the chirp could not easily be removed by such a time independent filter. Therefore we created another technique of which you see the result in figure . A local closeup of the denoised data (figure ) shows how the peaks are located at the same places, but now allow for fully automatic detection (certainly if you look at the SFFT of the data), which makes its very attractive in high throughput proteomics.
|Up: zoomed in sample output from the machine. Bottom: the same data denoised|
The accuracy of the algorithm we created is extremely high. It will retain position information exact. However the resolution of lower peaks will be a little bit less than the higher peaks. This however should not form a problem because these peaks are still well differentiated. As can be seen in the previous pictures, accuracies far below 0.1 dalton can be achieved for smaller peaks.
Another experiment we performed was data enhancement of a linear mode mass spectrum. The mass spectrum we present is the output from a sample containing the cell-lysaat of Hela-cells. Clearly it is a relatively bad sample to put into MALDI heavy mass linear mode. Not only are these heavy masses difficult to get suspended, but also because the noise level might suffocate what we actually want to measure. Figure shows how data enhancing helps in filtering out the noise.
The result of the algorithm on a standard protein mixture is shown in figure . Important here is that certain peaks which would normally not be selected if we simply look at the highest value now show up. Whether some of these new peaks are important might be interesting to investigate.
|Upper figure is the SFFT of a measurement of a Hela cell substrate. The figure below is the SFFT of the same substrate after data enhancing (but without removal of the pulse-train).|
|Lineair mode data enhancing of the output of a ProtMix II. Bottom is the actual output. Top is the enhanced output.|
|Correlation Measure to further select peaks|
|Technique to detect important peaks in enhanced lineair mode signal|
A phenomenon often used to detect important peaks is the fact that isotopes will weigh different. For every ionized similar fragment we will sometimes measure x Dalton, sometimes we might measure x+1 Dalton (if there is one neutron more), and so on. This knowledge can be used to automatically detect important peaks as shown in figure . The visualized graph is the autocorrelation graph which mainly measures whether a peak has 'echos'. If it has echos, then it probably is a series of peaks of the same fragment.
In a similar way, if we measure the autocorrelation of the enhanced linear mode experiment, then we clearly see vertical bands. Very likely the content of every band will allow us to detect which bands are important. However, this is merely an educated guess.
We have presented a number of artifacts we have encountered in MALDI TOF and MALDI TOF/TOF machines. These are