Thursday, January 9, 2014

De Novo Sequencing using Spectral Pairs or Triplets

PEAKS supports de novo sequencing using spectral pairs or triplets that are generated using different fragmentations. This is a feature introduced in PEAKS 7. Here is the poster that briefly described the method.

To use this function, simply specify the fragmentation mode as 'Mixed' during project creation, PEAKS will then automatically detect spectral pairs or triplets when doing de novo sequencing.

There are a few things to watch out though. When the fragmentation mode is set to 'Mixed', PEAKS will try to get this information from the data for each individual spectra. From the many datasets we have tested, this is generally not a problem for Thermo raw files. Other formats, especially some in-house generated XML files(e.g. mzXML, mzML) may be problematic as they either do not include the fragmentation mode information at all or PEAKS won't be able to retrieve the information for various reasons. In this case, PEAKS will ignore those spectrum, thus generating empty de novo sequencing results.

When the fragmentation mode information is available, the spectral pairs or triplets will be formed if the spectrum are from the same precursor ion in the same MS scan. The de novo sequencing result from a pair or triplet will be presented in a single row in the peptide table. The spectrum annotations for different fragmentation mode will be stacked vertically on the screen.

If you click the 'All candidates' button, all the calculated de novo sequences will be displayed in a pop up window. You can then click on those sequences to examine the spectrum annotation from individual fragmentation mode.

In the above example, although PEAKS has figured out the correct sequence using any one of the fragmentation mode, the confidence of the sequence from the triplet is much better.