DECONVOLUTION

Algorithm:
• Thrash is an algorithm used for Spectral Deconvolution.
• Thrash uses Averagine to determine monoisotopic masses.
• Most of the deconvolution tools have implemented thrash for
deconvolution.
Identifying Isotopic Clusters:
• Firstly, an isotopic cluster/spectral envelope is identified in the experimental
spectrum. To identify the cluster, the algorithm searches for a local maxima
peak in a predefined window, moving from left to right.
• The peak selected in first step is now used to identify the charge attained by
the spectral envelope. To extract the charge, we determine the peak spacing
(difference) between two peaks (call it delta), next to each other, in the same
spectral envelope. Singly charged proteins will have a delta, the difference of
m/z values, of 1 between the peaks in the isotope pattern. Doubly charged
proteins will have a delta of 0.5 between the peaks, and triply charged proteins
will have a delta of 0.33 and so on. Once the charge of protein is determined,
we multiply the mass of the peak selected in first step with the new charge.
The resultant mass is known as the average mass of the envelope.
Identifying Isotopic Clusters:
• Using
  this average mass, we determine the molecular formula using the
“Averagine” model. Averagine is a model amino acid with a molecular formula:

and an average molecular mass of 111.1254 Da obtained using statistical occurrences

of amino acids in proteins.
• We divide the average mass value by 111.1254 and the new value calculated is then
multiplied by the model amino acid given above. As a result, we obtain the number
of Carbon, Hydrogen, Oxygen, Nitrogen and Sulphur atoms. Hence, constituting a
molecular formula for the protein represented by the spectral envelope under
consideration.
Averagine Example:
•  Model amino acid molecular formula:

• For example if we find an average mass of 20000Da:

Theoretical Pattern Generation:
•  Then we generate a theoretical isotopic pattern of the molecular
formula using the “Theoretical Isotopic Pattern Generator”
• Multinomial Expansion was used to find out the intensities for a
specific number of atoms and is described below:

where
Comparison:
• The theoretical pattern is compared with the experimental isotopic
pattern, and the closeness between them is measured. Using Chi-
Squared method.
Chi-Square Test for Goodness-of-Fit
• The chi-square test for goodness-of-fit uses frequency data from a
sample to test hypotheses about the shape or proportions of a
population.

• Each individual in the sample is classified into one category on the

scale of measurement.

• The data, called observed frequencies, simply count how many

individuals from the sample are in each category.
How do we measure bias?
Processing the Spectrum:
• If the ‘chi’ value calculated is less than a user-defined threshold, the
monoisotopic mass of the protein is calculated using the same
molecular formula determined in the previous steps, and is stored in a
list. The intensities of the isotopic spectra under consideration are
subtracted from the experimental peak list.
• The window is incremented to search for a new local maximum, upon
finding it, the whole process is repeated again until the end of
experimental peak list is reached.
• At the end, a mass list is obtained that contains the monoisotopic
mass values of all the isotopic cluster identified.