How are spectra of deconvoluted fragments different from those in the Fragmentation Viewer in Review? - WKB99805

ENVIRONMENT

• UNIFI
• Peptide mapping

ANSWER

The spectrum in Fragmentation Viewer is displayed in the following steps.

First, peak detection is performed using the (collision voltage) low-energy data of Function1 and the (collision voltage) high-energy data of Function2 separately.

In the three-dimension data of m/z axis, retention time axis, and intensity, if a peak with a three-dimension mountain-like shape is detected, it will be detected as a peak.

Peaks that have been detected over time, such as mobile phase impurities, are not recognized as peaks.

And, for the minimum intensity with which the peaks could be detected, the following setting in the analytical method is the threshold:

Processing Tab > Find 3D peaks > (High Energy/Low Energy) Intensity Threshold

When spectra are integrated on the Investigate screen, peaks that have been detected over time or peaks with low intensity as described above are also integrated.

 

Next, the precursor ion peak (Function1) and the product ion peak (Function2) with the same retention time are associated, and deconvolution is performed with MaxEnt3.
Here, the following settings of the analytical method are used:

Processing tab > 3D Isotope Clustering > Fraction of the chromatographic peak width to apply during cluster creation and high to low energy association

This is the setting of the acceptable value of the retention time difference when collecting the spectral peaks of the isotope ions, and the acceptable value of the retention time difference when associating the precursor ion and the product ion.

Set with the fraction against the half width of the chromatogram peak.

 

Processing tab > 3D Isotope Clustering > Fraction of the chromatographic peak width to apply during cluster creation and high to low energy association
Set the peak intensity threshold to be applied when associating precursor ions and product ions.

When the spectra of fragment ions are integrated on the Investigate screen, the spectra will be a mixture containing the fragment ions derived from precursor ions that have similar retention times.

All low-intensity peaks are also shown.

ADDITIONAL INFORMATION