1. Getting Help:
In all modules: press the “h” key to display the help screen listing all key combinations for zooming, scaling, labeling and shortcut procedures. |
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2. How to plot exact Mass Chromatograms:
From the Browser first zoom in on the mass peak on interest. Select from the menu Task > Exact Mass Chromatogram and zoom in on the requested mass range. Confirm the m/z ranges to extract the EIC. Exact mass chromatogram plotting is much faster if you zoom in on the retention time range of interest. To overlay different exact mass chromatograms, select Overlay (“o”) and repeat the above sequence.
From MPeaks and IPeaks: convert the result table to exact mass values, then select: Menu > Options > Plot exact Mass Chromatograms. You will be asked to enter an m/z margin to extract the EIC e.g. +/- 0.035 Dalton.
To switch from nominal to exact mass spectra plotting in the Browser press the “m” key.
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3. Mass Spectrum Labeling Styles:
In all modules: for centroided mass spectra press “r”, for profile mass spectra press “q”. |
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4. Measure percentage peakheight for any peak:
The mass chromatogram title will display the percentage peakheight value for the largest peak in the mass chromatogram. To measure the peakheight for smaller peaks in the same mass chromatogram, zoom in around this peak and press the Normalize button. The title will now show the height for the zoomed peak.
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5. Measure the width of a peak:
All modules contain a peak width measuring tool which can be accessed from the toolbar. Click on the left and right of a peak to get the peakwidth information. |
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6. MPeaks Peak Detection – Try a baseline correction:
Sometimes it will not be easy to know if a baseline correction will be necessary. In general, for heavily varying baselines or when having many peaks in the same mass chromatograms, try a baseline correction to detect more peaks.
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7. Copying peaks from MPeaks and IPeaks to the Browser:
In MPeaks and IPeaks press the “b” key to copy the current mass chromatogram to the Browser to be able to explore it in more detail. |
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8. Copy a Data File from MS Compare to the Browser:
To load a data file from a selected sample to the Browser, press the Browser button in MS compare. After the file has been read, the Browser will become the active window. |
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9. Finding peaks for which no MSMS spectra have been acquired:
Run MPeaks, convert to exact mass values and select from the Menu: Tasks > Link MS2 Scans and enter the search criteria. Peaks for which MS2 spectra are available will be commented with retention time and the m/z value of the precursor ion.
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10. Impurity Profiling – Overlay MS and UV signals:
From the Browser select as external signal (top right corner list box) the UV you want to overlay. Scaling will be based on maximum values of both signals. To zoom in and rescale both traces press “r” to rescale the TIC trace, followed by “e” to rescale the external signal. To detect very small impurities that are difficult to identify use a combination of smoothing or try to extract the average baseline corrected mass spectrum over the retention time range of interest. See Chapter |
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11. Impurity Profiling: from peaks to components:
To obtain a clean list of component instead of all peaks: 1. run MPeaks, 2. delete all 13C isotopes, apply clustering and keep the largest peak from each cluster only. |
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12. Impurity Profiling: Peak Purity Analysis:
Run MPeaks using the time range of interest (parent compound). You may also run a clustering to remove co-eluting peaks. Plotting all detected peaks in overlay after normalization is a very good way to detect co-eluting or nearly co-eluting peaks. Peak Purity Analysis can also be done using the MPeaks Viewer and selecting the Dot Plot to graphically detect co-eluting peaks.
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13. Run IPeaks Isotope Pattern Recognition for charged (1+, 2+, 3+, etc.) peaks
The general procedure to run IPeaks in which the Isotope pattern is dependent on charge state is outline below. The algorithms to be used are MPeaks Re-Search and MIDAR. See also Chapter
A GUI automating the individual tasks will be implemented in the next version of MsXelerator. |
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14. MS Compare: Use Combinations of Alignment
Alignment of chromatographic data can be a very difficult issue. The Alignment algorithms in MS compare use the TIC, BPC, or a synthetic EIC to correct for shift problems over the full chromatographic region. Indeed many peaks can be properly aligned using a overall correction procedure, but it is often seen that the shift in the same region is different for different components.
Sometimes it can be easier to apply a combination of corrections to your data. E.g. start with a simple offset correction on a common peak in all the samples. This might already give a large improvement. Secondly try to use Reference peak Warping and finally apply COW on the selection of peaks.
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15. MS Compare: Preprocess and Save before running Biomarker Surface Maps:
When pre-processing is used during the creation of Biomarker Surface Maps (full 2D LC/MS Search) the computations can take a long time to complete due to the pre-processing used (smoothing). As an alternative you may select the pre-processing option from the menu and subsequently pre-process and save your data. In general this will be much faster. When you want to undo the pre-processing you should read in the original files again. This can be done using: Options > Conversion > Convert MAT files to Compare Format. Select individual files or use the list to read in the files again. |
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