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Efficient Disulfide Bond Determination Using Mass Spectrometry
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  1. Multi-Ions
  2. Initial Matching Threshold
  3. MS/MS Intensity/Abundance Threshold
  4. Confirmatory Matching Threshold
  5. Trimming Parameter ε
  6. Trimming Parameter δ
  7. Matching Score Threshold

1. Multi-Ions

fragmentation

The types of fragment ions observed in an MS/MS spectrum depend on many factors including primary sequence, the amount of internal energy, how the energy was introduced, charge state, the dissociation method used, etc.

Different dissociation methods generate different ion types in different abundances. MS2DB+ allows users to analyze MS/MS data considering combinations of up to twelve of the most commonly generated ions types: a, ao, a*, b, bo, b*, c, x, y, yo, y*, and z.

Some of the most common dissociation methods are: Collision Induced Dissociation (CID), Electron-Transfer Dissociation (ETD), Electron-Capture Dissociation (ECD), Electron-Detachment Dissociation (EDD) and Infrared Multiphoton Dissociation (IRMPD).

2. Initial Matching Threshold

The Initial Matching threshold is a threshold used to define a mass window centered on a experimental precursor ion mass value (from a MS/MS file) within which a correspondence between this ion mass value and a theoretical precursor ion mass value may be found. The correspondence is defined as an Initial Match (IM).

Both Initial Matching Threshold and Confirmatory Matching threshold are directly dependent on the mass spectrometer sensitivity. The more sensitive the mass spectrometer is, the smaller the mass window needs to be.

3. MS/MS Intensity/Abundance Threshold

The MS/MS Intensity/Abundance threshold is used to remove the fragment peaks (m/z data points) with low intensity in an experimental spectrum (or MS/MS file). These peaks represent noisy unreliable data, which do not contribute to the method's accuracy and efficiency. This lower bound limit allows MS2DB+ to account only for the meaningful m/z values in the entire spectra being analyzed.

4. Confirmatory Matching Threshold

The Confirmatory Match threshold is a threshold used to define a mass window centered on a experimental (product) fragment ion mass value within which a correspondence between this product ion mass value and a theoretical fragment ion mass value may be found. This correspondence is defined as an Confirmatory Match (CM).

Both Initial Matching Threshold and Confirmatory Matching threshold are directly dependent on the mass spectrometer sensitivity. The more sensitive the mass spectrometer is, the smaller the mass window needs to be.

5. Trimming Parameter ε

ε is a trimming parameter used to trim the set of mass values corresponding to all possible disulfide-bonded peptide structures that can be obtained from a digested protein (DMS). To trim the DMS set by ε means to remove as many elements from DMS as possible without losing meaningful mass values. The formula to calculate ε is:
epsilon
We modeled this functional relationship using the following independent variables: (1) the cysteine-containing peptides (CCP) mass range defined by CCPmax and CCPmin corresponding to the peptides with highest and lowest mass respectively. (2) The number of cysteine-containing peptides k. A large k implies that the average difference in the mass of any two peptide fragments is small. Conversely, a small k implies fewer fragments with putatively larger differences in their masses. (3) The cysteine-containing peptides average mass value CCPaverage. The relationship between ε and these other variables is then obtained using multiple-variable regression.

6. Trimming Parameter δ

δ is a trimming parameter used to trim the set of mass values of every disulfide-bonded fragment structure (FMS) that can be obtained from fragment ions, which can be of types a, b, bo, b*, c, x, y, yo, y* and z. To trim the FMS set by δ means to remove as many elements from FMS as possible without losing meaningful fragment ions mass values (spectrum peaks). The formula to calculate δ is:
delta
It was already expected that the functional form of δ would depend on the fragments mass range, as well as their granularity (extent to which fragments are broken down into smaller ions). In a manner similar to the case for estimating ε, we used regression to obtain the specific functional form for the dependent variable δ in terms of the variables AAmax (the largest amino acid residue mass), AAmin (the smallest amino acid residue mass), AAaverage (the average amino acid residues mass), and ||p|| (average number of amino acid residues per fragment).

7. Matching Score Threshold

A match score represents the weight or importance of a potential disulfide bond. This score represents the combined importance of each single peak match within two spectra. (one theoretical and one experimental). Each specific peak match is weighted according to its intensity. The match score is given by:
matchscore
In the equation above, the numerator corresponds to the sum of each confirmatory match for a disulfide bond multiplied by the matched MS/MS fragment normalized intensity value (In). VMi is a binary value which is set to 1 if a confirmatory match was found for fragment i. The denominator similarly contains the sum of each experimental MS/MS fragment ion from the MS/MS spectra (TMS) multiplied by In. TMSi is a binary variable which indicates the presence of a fragment i in the MS/MS spectrum.

We recommend users who did not find the expected results to lower the matching score threshold. The mass spectrometer specificity, the dissociation method used, the tuning of some of the thresholds and/or trimming parameters play an important role in scoring each disulfide bond. Maybe the default matching score threshold used by MS2DB+ turns out to be too "tight" for the data inputed.

The trade-off for lowering the matching score threshold is the possibility of finding false positive S-S bonds. However, researchers consider false negative results to have a more deleterious effect on protein characterization than false positive results, which justifies lowering the matching score threshold.