1. 1 Brukin2d software presentation Tsagkrasoulis A. Dimosthenis Email: dimostsag@gmail.com dimostsag@gmail.com

2. 2 Functionality 2d representation of LC-MS data, acquired by the Bruker ‘DataAnalysis’ program 2d representation of LC-MS data, acquired by the Bruker ‘DataAnalysis’ program Mass Spectrum comparison between different chromatograph runs Mass Spectrum comparison between different chromatograph runs

3. 3 Chromatograph details Y-axis: intensity(in arbitrary units) Y-axis: intensity(in arbitrary units) X-axis: Time X-axis: Time Chromatograph Compound

4. 4 Mass Spectrum Details Y-axis: intensity(in arbitrary units) Y-axis: intensity(in arbitrary units) X-axis: mass to charge ratio(m/z) X-axis: mass to charge ratio(m/z) Every chromatograph compound has one (mean) mass spectrum, showing the mass peaks(peptides) that exist in that compound. The mass spectrum can be deconvoluted, in order to include only single- charged main isotopes. This is done with the Bruker ‘DataAnalysis’.

5. 5 2d representation (1) Y-axis: time(according to the chromatograph) Y-axis: time(according to the chromatograph) X-axis: mass to charge ratio X-axis: mass to charge ratio

6. 6 2d representation (2) Y-axis: time(according to the chromatograph) Y-axis: time(according to the chromatograph) X-axis: mass to charge ratio X-axis: mass to charge ratio One spot corresponds to one peak in the mass spectrum One spot corresponds to one peak in the mass spectrum Spot Height: the duration of the chromatograph compound in which the peak belongs to Spot Height: the duration of the chromatograph compound in which the peak belongs to Spot Width: According to the peak’s FWHM parameter in the mass spectrum Spot Width: According to the peak’s FWHM parameter in the mass spectrum Spot Intensity: According to the peak’s intensity in the mass spectrum Spot Intensity: According to the peak’s intensity in the mass spectrum

7. 7 Contour details The contour consists of patches that are drawn in specific intensity levels. The higher the intensity level, the darker the patch. The colormap is used in order to provide the desirable gradation and correspondence between intensity values and colors.

8. 8 Brukin2d - Main Window Parse XML Panel Export image Panel Chromatograph Window Mass Spectrum window Open dual view window Show quantization table Contour parameters panel Contour plot Window

9. 9 Main Window - Parse XML Panel Parse XML file and plot contour –XML file includes compound data and is exported from ‘DataAnalysis’ –Brukin2d uses only profile data –The chromatograph must be exported in a separate mzXML file(with the same name as the XML file) from ‘DataAnalysis’, due to the fact that the chromatograph plot data is not included in the XML file

10. 10 Main Window - Contour Parameters Panel How many levels will the contour include and at which intensity values How many levels will the contour include and at which intensity values Correspondence between the colormap and the intensity values Correspondence between the colormap and the intensity values Smoothing the spots outline Smoothing the spots outline Horizontal stretching of the spots(m/z axis) Horizontal stretching of the spots(m/z axis) Mass intensity limits Mass intensity limits Deconvoluted or undeconvoluted data Deconvoluted or undeconvoluted data Include peak labels Include peak labels

11. 11 Main Window - Export Panel Export contour plots as images –Jpeg or Jiff format –Complete plot or split into pieces –The width and height parameters determine the size of the images

12. 12 Main Window - Quantization Table Shows the quantization data for all the mass peaks of a mix. The data is taken from the xml file. Some fields may be empty due to deconvolution factors.

13. 13 Viewing the compound mass spectrum and zooming in a peak or a compound The mass spectrum of a compound can be seen by clicking in the area of that compound in the chromatograph window. The squares in the first column of the quantization table are used in order to zoom in spots at the contour plot window.

14. 14 Brukin2d - Dual View Window Shift vectors panel Peak matching panel Contour plot window Chromatograph windows Contour matching panel Zoom options Contour color correspondence Shift vectors

15. 15 Dual View Window - Shift Vectors panel The shift vectors are used in order to eliminate time differences originating from the two chromatographs, which may appear during the lc-ms procedure.

16. 16 Dual View Window - Contour Matching Panel This panel is used in order to match the two contour plots according to the shift vectors that were entered. The shift diagram shows the resulting shift values for all the compounds.

17. 17 Dual View Window - Peak Matching Panel Comparison of the two contour plots –m/z leniency: Maximum distance allowed between two matched peaks in the x-axis. –Time leniency: Maximum distance allowed between two matched peaks in the y-axis. –Intensity leniency: Maximum intensity difference allowed between two matched peaks.

18. 18 Viewing Peak Matching Results (1) Matching Log and Quantization Tables The quantization table has a primary and a secondary mix. The first column group includes all the peaks of the primary, while the others show the matched peaks, if they exist.

19. 19 Viewing Peak Matching Results (2) Compound Stats Green bars: number of matched peaks per compound in the two mixes Green bars: number of matched peaks per compound in the two mixes Purple bars: Percentage of matched peaks to the total number of peaks per compound Purple bars: Percentage of matched peaks to the total number of peaks per compound Black bars: Compounds without peaks(due to intensity cutoff) Black bars: Compounds without peaks(due to intensity cutoff) The compound correspondence lists show the compound matching, as deduced by the peak matching results.

20. 20 Applications Used Bruker ‘DataAnalysis’ 2.0 Bruker ‘DataAnalysis’ 2.0 Matlab 7.4(r2007a) Matlab 7.4(r2007a) Thanks to… Be continued Be continued

21. 21 FIN