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Batch Integration

A detailed step-by-step guide for new users on how to integrate a series of spectra automatically and write them out for analysis in a spreadsheet program. The example given is for a series of HSQC15N spectra measured for amide exchange rate determination. Once you have learned a few tricks described here, you will be able to work very efficiently. -FD

What you will need:

  1. all your spectra in one format in a single directory.
  2. an xeasy peaklist that fits your spectrum: its useful if the peak numbers equal the residue number. (See the script PeakListNumberedByResidue.lua in the Lua scripts page).

Step 1: Set up Project file and Read in your Spectrum Series:

  1. Place all the spectra from your series of measurements in the same directory
  2. For XEASY format, rename them with endings like "_001.3D.param", "_002.3D.param", "_003.3D.param" ... "_100.3D.param" etc. If you have another spectrum format do something analogous.
  3. Start CARA and load your favorite template file. (File-Open from Template) or an existing repository (File-Open).
  4. Create a new project (Project-New Project) and read in sequence if you wish (not needed for this application).
  5. Click on Spectra in the new Project tree (Main CARA window, left side)
  6. Click in the Main CARA window, right side with right mouse button.
  7. In the resulting context menu, click on Add Spectrum and select the spectrum type for your series (e.g. HSQC15N)
  8. Select the directory containing your spectra.
  9. Click on the first spectrum in the series.
  10. move the scroll bar to the right end of the window.
  11. Hold down shift key and simultaneously click on last spectrum in series. All spectra in directory should be selected. You can deselect a spectrum by holding down the Ctrl key and simultaneously clicking on the spectrum.
  12. Click on Open to import all the selected spectra in the series. They should appear in the Main-CARA window, right side in the correct order.

Step 2: Set up Peaklist and adjust peak positions.

  1. Main CARA window, right side: right-click on first spectrum in list and Open MonoScope (spectrum should appear with correct orientation X-axis=1H,Y-axis=15N)
  2. View-Show Intensity or keyboard shortcut si (toggle intensity off/on)
  3. View-Auto Contour level or keyboard shortcut sc (to turn on auto-contour mode)
  4. Peaks-Import Peaklist (to import peaklist with xeasy format)
  5. Select desired Peaklist to import (this will not be overwritten - Cara creates a local copy in the repository) click Open (crosspeak positions should appear)
  6. Peaks-Save Peaklist - enter a unique name for the peaklist (this will be stored in repository)
  7. Use Shift-Ctrl click-drag to zoom a region to inspect peak positions.
  8. To calibrate the peaklist to the spectrum: Click at the actual position for a particular peak.
  9. While holding Shift key click the crosspeak symbol "+" corresponding to the same peak.
  10. Peaks-Calibrate Peaklist (calibrate peaklist to spectrum). Now most crosspeak symbols "+" should be quite close to the actual crosspeak.
  11. View-Show List or keyboard shortcut sl (to show the peaklist in table format)
  12. Zoom into a particular peak.
  13. Double-click on the first line of the peaklist to go to the first peak in the list.
  14. Use arrow keys to adjust the cursor onto the center of the peak.
  15. Type mp or Right-click in 2D spectrum window and select Move Peak
  16. Hit Tab key 3 times (or shift-tab once) to switch focus back to peaklist.
  17. Hit Down-arrow key one time (to select next peak in list)
  18. Hit Return key to display the second peak. Adjust peak position.
  19. Continue until you reach bottom of list and all peaks are adjusted.

Step 3: Adjust Integrator Model.

  1. Double-click a peak in the peaklist to display the peak.
  2. Integrator-Show Peak Model
  3. Integrator-Tune Peak Model
  4. Drag the slider for "Width 0" to adjust the width of the model to fit the actual linewidth along X-axis (1H)
  5. Drag the slider for "Width 1" to adjust the width of the model to fit the actual linewidth along Y-axis (15N)
  6. Click on [CLOSE] to store the model parameters.
  7. Integrator-Show Base Width
  8. Integator-Tune Peak Model adjust Peak Tol. 0 to be 50% (enough to detect the maximum intensity within the range displayed by the bar above the peak model along the X-axis) adjust Peak Tol. 1 in a similar way. (Warning: It is dangerous to set tolerences to values >70% since you may find maxima from other peaks)
  9. Integrator-Integrate All (to integrate all peaks in current spectrum)
  10. Integrator-Show Backcalculation (check how well model matches data by switching back and forth)
  11. Integrator-Show Difference Spectrum (check discrepancy between model and data)
  12. Display different peaks using the peaklist as described in Step 2 and check how well the model fits different peaks.
  13. Note: You can only adjust one model for all peaks in the current version of CARA. However, it is possible to define several models by editing the repository file. You can then classify each peak to belong to a specific model. But you must edit the repository to alter the peakmodel.

Step 4: Define Batch List.

  1. Spectrum-Setup Batch List
  2. Right-Click inside the window.
  3. In the Context menu, select: Add All Spectra
  4. If you want to remove a spectrum, click on it, and Right-click Remove Spectrum
  5. When done setting up the Batch List click [OK]
  6. To display the next spectrum in the list: type "ns" or use the menu: Spectrum-Next Spectrum
  7. To display the previous spectrum type "ps" or use the menu: Spectrum-Previous Spectrum
  8. To jump to a particular spectrum type "gs" and enter the spectrum ID number and hit [RETURN]
  9. Alternatively, use the menu: Spectrum-Select Spectrum
  10. Check whether the peak positions are close in the Spectrum series. Try adjusting Peak tolerence if there are slight shifts between spectra.
  11. You can also adjust the peak positions in individual spectra. These movements in the current CARA version affect the peakposition only the displayed spectrum.
  12. To move a peak in all spectra, display the first spectrum (the one the peaklist is associated with)

Step 5: Integrate All Spectra and write out Table of Integrals

  1. Integrator-Integrate Batch List
  2. You can display the various spectra in the batch list using gs, ns and ps. The peaklist will update to reflect the integrals in the currently displayed spectrum.
  3. To find problem peaks, display the first spectrum. Click on the Amp column in the peaklist (the peaklist is now ordered by amplitude)
  4. The lowest amplitudes are at the top. Double-click and check any peaks with negative or zero Amplitude
  5. Check the plot of peak amplitude versus spectrum ID number by clicking on the peak in 2D spectrum or double-clicking in peaklist followed by: Integrator-Show Peak Curve
  6. Do not resize the Peak Curve window (otherwise the context info does not correspond to the datapoint position - see below)
  7. Systematic errors can be observed in this window quickly. If you see a spike, move the cursor on the spike to find out which spectrum is causing problems (context info). Cara will display the spectrum number in the batch list, the spectrum ID, and the spectrum name at the current cursor position. Now return to the Spectrum-Setup Batch List and remove the offending spectrum. Repeat the Integrator-Integrate Batch List and recheck the Peak Curve. The offending data point should be gone.
  8. When you are satisfied with the appearance of the Peak curves, write out the Peak Amplitudes into a text file: Peaks-Export Integration Table (one column for each peak, first row is the title, first column is the names of the spectra)
  9. Import into your favorite Plotting, Fitting program (like EXCEL) and analyse data.

Note: There are some Lua scripts available to support batch integration:

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