## 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 (TA_{1}) can be described as:

TA_{F1} = A_{1,F1} + A_{1,F2} + A_{1,F3} + ... A_{1,Fn}

Where:

- A_{1} is the normalised abundance of A in sample 1 in a given fraction F_{x}

- A_{1} 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, TH_{1}, across all fractions:

TH_{1} = H_{1,F1} + H_{1,F2} + H_{1,F3} + ... H_{1,Fn}

Where:

- H_{1} is the normalised abundance of H in sample 1 in a given fraction F_{x}

- A_{1} 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) / (TA_{1} abundance)] * TH_{1} 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] / 10^{6}

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

### See also

- How does absolute quantitation by Hi-N for HCP analysis work?
- How is absolute quantitation by Hi-N for HCP analysis calculated?
- How does Hi-N work?
- How does Hi-N work in the fractionation workflow?
- When using absolute quantification by Hi-N, why are the calculated amounts of my calibrant protein not equal to the value I enter?
- Normalisation in fractionated experiments
- What happens to protein measurements when the calibrant protein can't be found?