How does Hi-N for HCP analysis work in the fractionation workflow?

The non-HCP-analysis absolute quantitation method had some modifications when used in the fractionation workflow. Similarly, the process for fractionated experiments is slightly modified in this situation as well.

(1) Global normalisation

The fractions are normalised as standard for multi-fraction experiments – at the peptide ion level, across runs within a fraction, then across fractions as well. This includes the ions comprising the calibrant protein. The extra across-fraction normalisation corrects for any technical differences between fraction sets.

(2) Summation of abundance over fractions and relative protein abundance calculation

The relative abundance of every protein is then calculated after normalisation. This is the average of the integrated intensity of the N most abundant peptides for each protein, just as for the single-fraction process, but in this case the within-fraction peptide abundances are first summed over all the fractions to give a total, normalised, abundance for each peptide before the Hi-N selection of peptides and calculation is carried out.

The normalised total abundance of calibrant protein, TA, in sample 1 across the fractions (TA₁) can be described as:

TA_F1 = A_1,F1 + A_1,F2 + A_1,F3 + ... A_1,Fn

Where:
- A₁ is the normalised abundance of A in sample 1 in a given fraction F_x
- A₁ is calculated by the Hi-N approach, but selecting peptides based on their abundance over all fractions
- n is the number of fractions

The same process is also carried out for the HCP contaminants as well:

Normalised total abundance of a given HCP in sample 1, TH₁, across all fractions:

TH₁ = H_1,F1 + H_1,F2 + H_1,F3 + ... H_1,Fn

Where:
- H₁ is the normalised abundance of H in sample 1 in a given fraction F_x
- A₁ is calculated by the Hi-N approach, but selecting peptides based on their abundance over all fractions
- n is the number of fractions

(3) Calculating absolute amounts using a calibrant-derived relationship between abundance and amount

These normalised abundances are then used as for the single-fraction workflow to estimate the amount of proteins present, based on the relationship between defined calibrant amount present (X fmol) and observed calibrant abundance (TA).

To estimate the amount of a protein in fmol:

HCP protein “H” in fmol, in sample 1:

(i) [(X fmol) / (TA₁ abundance)] * TH₁ abundance

This calculates the fmol / measured signal from the calibrant within a sample across all fractions, and then applies that to the observed signal of the HCP contaminant within the same sample across all fractions.

Similarly, for amount of protein:

HCP protein “H” in ng, in sample 1:

(ii) [(fmol H in sample 1 from (i)) * mass “H” g/mol] / 10⁶

This is the fmol H multiplied by its molar mass, along with a femto-nano unit correction factor.

How does Hi-N for HCP analysis work in the fractionation workflow?

See also