The Direct Infusion algorithm is a spectral tree construction utility rather than a component detection method. It allows the creation of one or more spectral trees from a single raw file by the reading of various MSn scans acquired in one run and constructing a tree according to their MS stage and precursor ion m/z values.

To start the spectral tree construction utility using Direct Infusion algorithm:

1. Click on the Components Detection and Spectra Deconvolution button and choose the Direct Infusion pop-up menu item

2. When the parameters setup dialog window appears, change the settings if required, and then click the Calculate button

The Direct Infusion algorithm dialog window contains various parameters that can be optimized for specific types of analysis. Please note that these parameters are interdependent, so a change of one parameter can also affect algorithms linked with other parameters.

Minimal Tree Depth: Specifies the MS stage that should only have a single precursor . The value determines the MS stage where a tree branch division will take place. If the default value 1 is used, the algorithm creates a single tree from all the scans in a run (file). If the value 2 is used and the analyzed run contains MS2 spectra with different precursor m/z values, the algorithm will create separate trees for each MS2 scan with a unique precursor ion m/z value.

Include Upper Spectra: If checked, the resulting spectral trees contain actual scans in the stage above the level set in Minimal Tree Depth. If unchecked, the spectra above the stage set in Minimal Tree Depth. contain spectra with a single peak equal to the precursor ion of the product spectra.

Average Scan: If checked, every tree node will only contain average spectra.

Calculate Envelope: If checked, the ion profile (envelope) for each tree will be calculated. This allows you to preview scans that belongs to particular tree.

A spectrum obtained from the average of scans, or from scans with a subtracted background, or from component deconvolution, can be further processed in Database Manager, Spectra Classifier, or can be directly searched for in a library from Chromatogram Processor. To transfer the extracted spectrum or tree to Database Manager or Spectra Classifier, use the copy button. After pressing the copy button in the Chromatogram Processor window, the mass spectrum can be pasted to a Database Manager or Spectra Classifier window. In addition, when you press the copy button in the Chromatogram Processor, the chromatogram graphic along with the spectrum from the Scan tab will be copied to Windows Clipboard and can be used for creating reports in any Windows application.

The Chromatogram Processor window contains a Search button for the direct searching of spectrum from the Scan tab or tree from the Tree pane in libraries. To search the extracted spectrum in libraries, just press the Search button in the Chromatogram Processor window and the Spectra Search dialog window will appear.

Mass Spectra obtained in Chromatogram Processor can be classified using Principal Component Analysis, Neural Networks, and Fuzzy Clustering methods.

To choose scans or deconvoluted spectra for classification:

• Select scans or components that you want to classify and click Add Selected Scans or Components to Spectra Classifier button.

Detected components are marked with a triangle in the original chromatogram. Because the program calculates the precise retention time (tR) of each component the component triangle can appear between scan points.

If you select a component by clicking its triangle, a deconvoluted spectrum will appear in the spectrum pane. This spectrum can be processed in the same way as any spectra in the program, e.g. searched in a library or copied to Database Manager.

If you move the mouse cursor over a component triangle, a tool tip rectangle will inform you about the component number, precise retention time and model ion m/z value that were used in the automated detection and deconvolution processes.

Chromatographic components can be edited or searched in Components Editor that can be opened using Edit Components    button.

To copy or process more than one detected component in Database Manager or in Spectra Classifier modules, you must first select them.

To select one or more components:

• Click on the Select Scans or Components button and select the desired components with the mouse cursor while holding down the LEFT mouse button

To select all detected components in a run:

1. Click anywhere in the chromatogram pane with the RIGHT mouse button and a pop-up menu will appear.

2. Click on the Select All menu item.

The program makes a strict distinction between original scans and detected components and does not mix them. When selecting a chromatographic region, components are preferred over scans. If you select a chromatographic region that contains components, only these components will be selected. If you select a region that does not contain any components, all scans in that region will be selected.

Chromatogram Processor allows you to view and process Xcalibur® data dependent experiments and product ion scanning *.raw files and extract spectral trees (MSn spectra) from chromatograms. If Xcalibur® data is opened in Chromatogram Processor, a tree view control will appear on the right side of the window. This tree view control allows you to select one or more product scans at any MSn stage. If deconvoluted components are present, they are listed at the bottom of the tree control.

If component detection and spectra deconvolution procedure is applied to data dependent chromatograms, the program generates components as spectral trees. The tree components can be processed in the same way as regular spectra. They can be edited in Components Editor, searched in spectral libraries or classified using Spectra Classifier. Spectral trees in Chromatogram Processor can only be viewed. If you wish to edit them, you must copy and paste them into Database Manager, where all editing utilities are available.

Please note that the spectrum in the bottom right of the Chromatogram Processor window is displayed for the selected tree node spectrum.

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