How to Calculate Mole %

Mole % (i.e., residues per 100 residues) may be used for evaluation of AAA data without knowing molecular weight. See attached software example.

  • a. Enter pmol analyzed for each amino acid.

  • b. Sum the total picomoles of amino acids in the analysis. Make sure peaks are identified and integrated accurately.

  • c. Divide the picomoles of each residue by the sum total and multiply by 100. Tabulate results.

  • d. Notes: Cys and Trp are usually not quantified, Ser and Thr are partially destroyed by hydrolysis, Met values are frequently low due to oxidation and Gly, Ala, Ser are common contaminants.

    Software Set-up Example for Calculating Mole %

    Software: Microsoft Excel Version 2.2 Data entry commands are shown e.g., =SUM(B10:B25) sum column B, rows 10-25; (B10/B27) yields Asp mole % by dividing column B line 10 by column B line 27.

    Calculation Example: Mole %

    Calculation Example: Mole %
    DATE RUN 3/31/96  
    TYPE IN ID#    
    NAME AND METHOD NBS-BSA  
    TOTAL VOL IN UL    
    VOL HYD ul 10  
    RATIO APPLIED 1.0  
         
        COMPOSITION
    AMINO ACID pmol analyzed MOLE %
    ASP (D) 1606.1 10.67%
    GLU (E) 2251.26 14.96%
    SER (S) 760.99 5.06%
    GLY (G) 545.79 3.63%
    HIS (H) 469.49 3.12%
    ARG (R) 637.16 4.23%
    THR (T) 887.22 5.89%
    ALA (A) 1316.07 8.74%
    PRO (P) 832.03 5.53%
    TYR (Y) 621.56 4.13%
    VAL (V) 967.31 6.43%
    MET (M) 112.87 0.75%
    ILE (I) 298.79 1.98%
    LEU (L) 1844.82 12.26%
    PHE (F) 799.26 5.31%
    LYS (K) 1102.34 7.32%
    TOTAL PMOL ANALYZED = 15053.06 100.00%
    % ERROR = 8.7%  

    How to Calculate Residues per Molecule and Quantify Unknown Samples

    The molecular weight must be known or an assumed value used in order to calculate residues per molecule. See attached software set-up example.

  • a. Enter approximate molecular weight.

  • b. Enter pmol analyzed for each amino acid.

  • c. Divide the molecular weight by 112 (approximate average residue weight) to estimate total residues per molecule (B30).

  • d. Sum total pmol analyzed (column B). Divide total pmol analyzed (B26) by residues per molecule (B30) to estimate pmol protein analyzed (D29).

  • e. Divide pmol of each amino acid by pmol of protein (D29) to estimate residues per molecule (column C).

  • f. To calculate the original sample concentration, be aware of the sample volume hydrolyzed, the fraction of the hydrolysate applied to the amino acid analyzer and the fraction of the applied hydrolysate actually analyzed. Know whether the data reduction method normalized the pmol values to 100% analyzed.

  • g. Column D is Mole % .

  • h. Column E is the monoisotoptic residue weight for each amino acid.

  • i. Column F is the sum of the residue weights (number residues x residue weight).

    Software Set-up Example for Calculating Residues/Molecule Unknown

    Software: Microsoft Excel Version 2.2. Data entry commands are shown. See Software instructions for more details.

    Calculation Example of Residues/Molecule-Unknowns

    Using software: Microsoft Excel Version 2.2 and the above Set-up Example.

    How to Calculate Residues per Molecule and Quantify Known Samples

    For samples of known composition and mass, determine residues per molecule based upon the amount of sample analyzed. See attached software set-up example.

    • a. Enter molecular weight.

    • b. Enter pmol analyzed for each amino acid.

    • c. Divide pmol of each residue by the known residue value (column B) to determine pmol protein (column D).

    • d. Calculate an average amount analyzed (B27) by averaging all the values in column D.

    • e. Discard pmol protein values with unacceptable deviation from the mean (e.g., greater than ± 15%). This is an arbitrary window that can be adjusted to optimize the number of relevant residues in the recalculated mean.

    • f. Recalculate the average pmol protein analyzed from the remaining relevant values (F26).

    • g. Calculate the experimental composition (column G) by dividing the amount of each amino acid by the recalculated pmol of protein.

    • h. Round off experimental values (column G) to integer values (column H).

    • i. Calculate % compositional error (column I) based on integer values (column H).

    Software Set-up Example for Calculating Residues/Molecule Known

    Please note that this is a wide spread sheet, you may wish to open this file and print in landscape direction.

    Software: Microsoft Excel Version 2.2. Data entry commands are shown. See software instructions for more details.

    Calculation Example of Residues/Molecule Known

    Using software: Microsoft Excel Version 2.2 and the above Set-up Example.

    How to Calculate Compositional Error

    % Error = 100 x |known value-measured integer value|
    known value

    = 100 x |Experimental - true residue valve|
    true

    Average % Error = S % Error for n amino acids
    n

    (usually n=16)

    Crabb, J.W, L. Ericsson, D. Atherton, A. Smith and R. Kutny (1990) Current Research in Protein Chemistry (J.J.Villafranca, ed) Academic Press, pp 49-61.

    Tarr,G.E., R.J.Paxton, Y-C Pan, L.H.Ericsson and J.W.Crabb (1991) (J.J.Villafranca, ed) Academic Press, pp 139-150.

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