Generally electrospray ion intensities do not reflect directly the quantity of a molecule in solution. For obtaining ion concentrations or quantities normalisation procedures have to be applied.
Arcadiate allows two level of normalisations, the ion or peptide level and the protein level.

Ion level normalisation:
The ion intensities in a chromatogram can be normalized by two means:
  • using isotopically labelled standards
  • using a set of molecules whose concentration is known or always the same - a set of internal standard molecules

a) Isotopically labelled standards
When using isotopically labelled standards the ion intensity of a molecule is compared to the ion intensity of a structurally mostly identical molecule that carries some heavy isotopes. Isotope standards can be defined by defining Molecular References in a Molecular References View. The measurement expresses the quantity of the molecule in relation to the quantity of the isotopic standard - as a fold change.

b) A set of standard molecules, an internal standard
In cases when isotopic labels cannot be used the ion intensities of a chromatogram can be normalised to the ion intensities of a set of molecules whose concentration is known or is always the same. It is preferable that the retention time distribution of these ions covers a wide range of the analytically useful separation width. This set of ions can be defined using a Normalization Ion Group View. Ions can be transferred from an Ion Group to a Normalization Ion Group using copy - paste. Every ion in the Normalization Ion Group should have a Normalized Quantity. By dropping the link ion onto a chromatogram the ions actual quantities in this chromatogram are measured. With the actual and the normalized quantities available a set of normalisation factors is calculated that brings the measured quantities onto the level of the normalized ion quantities. These factors are at the origin of the normalisation function that covers the chromatogram. The function’s parameter are displayed in the lower part of the Normalization Ion Group View. The normalisation factors and the normalisation function can be displayed graphically by dropping the Normalization Views link icon onto an empty Data Graph View. The Data Graph View allows the editing of individual normalisation function values.

The normalisation function is not automatically applied to the chromatogram. To apply it its respective column in the lower part of the Normalization Ion Group View has to be selected and the has to be pressed. After activating the function all ion quantities determined in this chromatogram will be multiplied with the retention time depending normalisation factor of the normalisation function. The exceptions are ion quantities displayed in Normalization Ion Groups. They always represent ion quantities as they have been measured by the mass spectrometer.

When employing internal standards it is possible to use the total ion count as an additional normalisation. The total ion count in a proteomic experiment can be a good estimate for the total amount of sample applied to the chromatogram. Normalising it to a given value can compensate for slight variations in the total material used to run different mass spectrometric investigations.

Protein level normalisation:
All protein quantifications can be seen in relation to the level of a chosen protein. This protein should be a member of a Database Search Result View. Its line in the protein table should be dropped onto a chromatogram map or chromatogram spectrum to set the norm-protein of the chromatogram. Whether protein level normalisations are used is controlled by a preference setting: Arcadiate > Preferences… > Quantification > Display Normalized Protein Quantities.