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Protein Statistics

Irene Saxby
  • Updated

This article describes the various protein statistics generated by Geneious Biologics, and how to turn on this functionality.

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How to Calculate Protein Statistics

To turn on Protein Statistic calculations, select the appropriate option under Analysis Options when running either Antibody Annotator, NGS Antibody Annotator or Single Cell Antibody Annotator:

Screenshot 2025-02-24 at 2.02.40 PM.png

The various properties are calculated according to user settings of what the target molecule is. We support calculating the protein statistics of:

  • mAb
    • This will calculate all the properties for a molecule consisting of two heavy and two light chains. We only calculate IgG statistics (the C gene must be from the IgG family)
    • Note: This is currently only available when using the 2025 Human and/or Mouse Ig Reference Databases
  • fAb
    • This will calculate all the properties for a molecule consisting of a Heavy variable region (in addition to the CH1) and a Light chain. The identified C gene must be from the IgG family.
    • Note: This is currently only available when using the 2025 Human and/or Mouse Ig Reference Databases
  • VDJ or VJ
    • This will calculate all the properties for the VDJ and VJ regions separately (if both chains are present) 
  • VDJC or VJC
    • This will calculate all the properties for the entire V(D)JC region, separately for each Heavy and Light chain.
  • Two chains with linker
    • This option is available if you have selected either the scFv option or the Two heavy chains in single sequence with linker option (Found under Main Options). This will calculate all the properties across the two chains as one protein product, including the linker.

Why can't I select some of the options?

This will be for one of two reasons:

  1. You are not using a supported reference database.
    • Our new 2025 databases have updated C full genes, these are necessary for calculating the correct values for the full mAb
  2. You are using a different Selected sequences are: option under the Main Options.
    • Depending on the format of your sequences, some options might not be available. For example, to determine mAb or fAb properties, heavy and light chains must be either found on one read together, or as associated heavy-light pairs.

Why are my fAb/mAb statistics not being calculated?

This is likely due to either of the following reasons:

  • Your sequences were not of IgG antibodies (we do not calculate mAb/fAb statistics for IgM/IgA/etc. antibodies)
  • The sequenced region did not contain a full V(D)J region, along with a partial match to a C gene.
    • For example, your sequences might be missing the FR1 region

Please reach out to us if you would like to see different behaviour here (for example, calculating statistics for the IgM complex). You can contact us here.

Calculated Properties

Molecular Weight (kDa)

The molecular weight is given as Kilodaltons. The following weights (in Daltons) are used for each amino acid:

A=71.0788 R=156.1875 N=114.1038 D=115.0886 C=103.1388 E=129.1155 Q=128.1307 G=57.0519 H=137.1411 I=113.1594 L=113.1594 K=128.1741 M=131.1926 F=147.1766 P=97.1167 S=87.0782 T=101.1051 W=186.2132 Y=163.1760 V=99.1326 U=150.0388 O=237.3018

 

Isoelectric Point (pI)

Geneious Biologics calculates the isoelectric point of a protein using the bisection method described at isoelectric.org.

Amino acid pKa values were taken from:
Lide, D (2009). CRC Handbook of Chemistry and Physics. CRC Press. https://doi.org/10.1021/ja906434c

General pKa values for terminal amino and carboxy groups taken from:
Berg, J., Tymoczko, J., Gatto, G., Strye, L (2015). Biochemistry (8th Edn). W.H.Freeman and Co.

Charge at pH 7

Geneious Biologics estimates the overall charge of a protein at pH 7.0 using methods described at isoelectric.org.

Amino acid pKa values were taken from the CRC Handbook of Chemistry and Physics 90th Edition, with general pKa values for terminal amino and carboxy groups taken from:
Lide, D (2009). CRC Handbook of Chemistry and Physics. CRC Press. https://doi.org/10.1021/ja906434c

Extinction Coefficient

Geneious Biologics calculates the extinction coefficient of a protein as per:
Gill, S. C., & von Hippel, P. H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry, 182(2), 319–326. https://doi.org/10.1016/0003-2697(89)90602-7

The following values for the amino acids are used: C=62.5 (only counting up to an even number) W=5500 Y=1490. All cysteines are assumed to be paired in a disulfide bridge (making cystine).

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