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Calculating Known Peptide or Oligonucleotide Masses

ProMass can automatically calculate the theoretical molecular masses of peptide, protein, and oligonucleotide sequences and display them in the web-based results report.  ProMass will automatically evaluate the mass of the BioSequence and determine if the target mass was found in the data set. This is very useful if you want to confirm the masses of known components.  ProMass will calculate masses based on a sequence string entered directly in the Xcalibur sample list or can read in a text file containing a sequence string.  Here's how it's done:

Specifying a BioSequence string in the sample list:

ProMass can calculate the mass of a biomolecule by entering the sequence string directly in the Xcalibur sample list.  You can use this method if your sequence is less than 255 characters or less in length.  All you need to do is paste your sequence in the BioSequence field in the Xcalibur Sequence Setup window.  If the BioSequence and Target Info fields are not present in your Xcalibur Sequence Setup window, read this first. Copy your complete sequence (in single letter codes) to the clipboard.  Double-click on the BioSequence field and right click your mouse to be able to paste the sequence into the Sequence Setup window.

Specifying a BioSequence file in the sample list:

ProMass can also read a text file containing a BioSequence string.  This is particularly useful for sequences exceeding 255 characters in length.  Enter the filename including the path in the BioSequence field of the Xcalibur sample list to have ProMass automatically read it in.  The file must be a plain text file and have either the .pep, .txt, or .fasta file extension.  The file should contain only a single protein or oligonucleotide sequence.

Specifying sequence type:

Regardless of the method you use to enter a BioSequence, you also need to instruct ProMass what type of sequence is represented.  To do this, you specify, one of the two options shown below in the Target Info field in the Xcalibur Sequence Setup program.  You must enter one of the sequence types as shown below or ProMass will not calculate masses. 

sequence = peptide (or protein)

sequence = nucleotide (or dna, or oligo)

sequence = rna

Specifying termini:

You may also specify termini in the Target Info field.  Normally, ProMass assumes that H, and OH must be added to the sequence to calculate a mass of either a polypeptide or oligonucleotide.  Therefore, you do not have to specify termini if for example your peptide contains a free amino N-terminus and a free acid at the C-terminus, or if your oligonucleotide contains hydroxyl groups at the 5' and 3' ends. Therefore, entering no termini options is the same as entering:

termini = H, OH

However, if you are analyzing oligonucleotides and you have a phosphate at the 3' end you could enter:

termini = H, POH

(The POH group has been pre-defined to be equal to H2PO4).

ProMass uses a text file to store the masses of the amino acid, nucleotide, termini, and custom groups.  The text file is called  znova_masses.ini and it can be found in your ProMassXcali\ZNova install directory.  You can create your own amino acid or nucleotide groups by editing the znova_masses.ini file.  You may use single letters (upper or lower case) or numbers to represent amino acids or nucleotides.  Termini groups can contain more than one letter or number.  Before specifying termini, make sure they have been defined in the znova_masses.ini file.

As an example, we'll use the myoglobin LC/MS file from the Getting Started section.

• Set up the Xcalibur sequence as described previously in the Getting Started section with the myolcmsdata.raw test data file.  The amino acid sequence of horse myoglobin is shown below:

GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKG
HHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG

• Paste this entire sequence in the BioSequence field in the Xcalibur Sequence Setup program.  (Note: The Xcalibur Setup will terminate input of any field at a carriage return, so you may want to check that the whole sequence gets pasted in.  If not, copy the above sequence to a notepad, remove the line-ending character, then copy/paste into the Xcalibur Sequence Setup).  Also enter, sequence = peptide in the Target Info field.  Entry of the termini = H, OH in the Target Info field is optional.  When you're done your Xcalibur Sequence Setup should look something like this:

• Select Row 1 with the mouse, and hit the batch reprocess button ( ). Make sure the following options are checked in the batch reprocess dialogue box: Qual - Peak detection and integration, Programs, and Replace Sample Info.
• When the file is finished processing, display the resulting index.html file in your browser.  It should look something like the web page shown below.  Note how the myoglobin sequence appears in the report along with the calculated average and monoisotopic masses.

Related topics:

• How to setup target masses
• The top-level summary report