Spectrum

Show All Peaks

As a default only the peaks currently annotated by a fragment ion is shown in the foreground. The other peaks are shown in grey in the background. Only (red) peaks in the foreground can be selected. To move all peaks to the foreground click the Show All Peaks option in the menu below the spectrum.


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Annotation

PeptideShaker automatically annotates the a spectrum with the most likely ions given the spectrum and the peptide sequence identified. This includes the automatic selection of neutral losses.

However, the user can override the default annotation by selecting different ion types in the menu below the spectrum. To change the neutral losses one first has to uncheck the Adapt option on the Loss menu.

To make sure that the same ion types are used for all spectra, the user can also turn of the automatic annotation completely in the Settings menu.


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Annotation Accuracy

In addition to deciding which ion types to use when annotating the spectrum, the user can also change the desired accuracy for the annotations. This is done by scrolling the mouse wheel while holding down the Ctrl key. Scrolling up or down (when over the Spectrum panel) will increase or decrease the accuracy required to say that a given peak should be annotated. The maximum value is set to the fragment ion accuracy set in the Search Parameters dialog (found on the Edit menu).

Scrolling without holding down any keys lets the user decide how many of the peaks PeptideShaker should try to annotate. The level is set relative to the most intense peak, e.g., 80% means that the tool only considers peaks that have an intensity that is at least 80% of the most intense peak. As the value is reduced more and more of the low intensity peaks will be annotated.


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Zooming

Zooming is achieved by clicking and holding the left mouse button where you want to start the zoom, and then drag in the direction you want to zoom, marking the area to be zoomed. Right clicking in the plot undos the zoom.


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De Novo Sequencing

In PeptideShaker there are two ways of doing de novo sequencing: manual or automatic.

Manual: Click on one peak. The distance and amino acids matching this distance (if any) will then be shown. To add the sequence, click on the second peak. Repeat the process to sequence more peaks.

An added sequence is removed by holding down the Ctrl button when clicking on the sequence. To "store" a sequence, hold down the Alt button and click on the sequence, the sequence turns red. To remove such "stored" sequences, hold down Ctrl and Alt and click in the sequence.

Automatic: Go to the De Novo option in the menu below the spectrum and select which ion type you would like to annotate. Note that this annotation only tries to find matches against the sequence identified by the PSM, and as such only gives an overview over the match between the sequence and the spectrum for the selected ion types. For a complete de novo sequencing showing all options for each distance use the manual option.


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Sequence Fragmentation

The plot in the upper left shows the how the peptide sequence is fragmented using the singly charged b- and y-ions. In this plot the blue peaks represent b-ions and red peaks y-ions. Hovering over a given peak will display the fragment ion type and number, e.g., y5.


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Intensity Histogram

The plot in the middle above the spectrum represents the intensity distribution of the annotated peaks (in green) and the non-annotated peaks (in grey), with intensity bins on the x-axis and frequency on the y-axis. In a well-annotated spectrum most of the high intensity peaks ought to be annotated, while most of the non-annotated peaks ought to have low intensities.

Zomming is available by the standard left click and drag approach. Additional options are available via the right click popup menu.


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Mass Error Plot

The plot in the upper right displays the standard mass error plot with m/z on the x-axis and mass error (in Da) on the y-axis.

Zomming is available by the standard left click and drag approach. Additional options are available via the right click popup menu.


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