Producing a Sequence Ladder Summary

A sequence ladder is a list of all partial sequences that contain either terminus of a biomolecule sequence.  For example, a 5' sequence ladder for a 20-mer oligonucleotide with the sequence TAATACGACTCACTATAGGG would look something like this:

Position  Mass     Sequence

T1-G20   6125.1  TAATACGACTCACTATAGGG
A2-G20   5820.9  AATACGACTCACTATAGGG
A3-G20   5507.7  ATACGACTCACTATAGGG
T4-G20   5194.5  TACGACTCACTATAGGG
A5-G20   4890.3  ACGACTCACTATAGGG
C6-G20   4577.0  CGACTCACTATAGGG
G7-G20   4287.9  GACTCACTATAGGG
A8-G20   3958.7  ACTCACTATAGGG
C9-G20   3645.4  CTCACTATAGGG
T10-G20  3356.3  TCACTATAGGG
C11-G20  3052.1  CACTATAGGG
A12-G20  2762.9  ACTATAGGG
C13-G20  2449.7  CTATAGGG
T14-G20  2160.5  TATAGGG
A15-G20  1856.3  ATAGGG
T16-G20  1543.1  TAGGG
A17-G20  1238.9  AGGG
G18-G20   925.7  GGG
G19-G20   596.5  GG
G20-G20   267.2  G

Conversely, a 3' oligo sequence ladder would contain the 5' end and would produce a list of sequences with successive removal of nucleotides from the 3' end.

ProMass can calculate sequence ladders for either proteins or oligonucleotides and indicate experimental masses which matched these calculated masses in the deconvoluted data. This feature is particularly useful for identifying failure sequences in LC/MS data of synthetic oligonucleotides.  The feature can also be used to confirm masses resulting from exonuclease digestion of oligonucleotides.  For proteins, the feature can be used to look for components resulting from either N- or C-terminal enzymatic processing.  A sequence ladder summary cannot be used to look for all possible internal fragments from a given biomolecule sequence.  This feature may become available in a future version of ProMass.

In order for ProMass to produce a sequence ladder summary you need to do the following:

1. Setup the Xcalibur sample list to process your data file
2. Enter the nucleotide or amino acid sequence in the BioSequence field of the sample list for the data file you want to process
3. Enter information in the Target Info field of the Xcalibur Sequence Setup that indicates what type of sequence and ladder will be used.  In the Target Info field, you enter the type of sequence using one of the following:

sequence = nucleotide

or

sequence = protein

If you want a 5' (oligonucleotide) or N-terminal (protein) sequence ladder summary enter one of the following in the Target Info field to indicate removal of residues from the left terminus:

ladder = left

or

ladder = 5'

If you want a 3' (oligonucleotide) or C-terminal (protein) sequence ladder enter the following in the Target Info field to indicate removal of residues from the right terminus:

ladder = right

or

ladder = 3'

If you want to look for masses that contain either termini, enter the following in the Target Info field:

ladder = both

The screenshot below shows an Xcalibur sample list properly configured to produce a 5' sequence ladder summary.

The sequence ladder summary appears in the chromatogram-level report after the target mass summary section.  An example is shown below for the data file processed in the Xcalibur sample list shown above.  In the example below, the 5' sequence ladder summary is used to identify oligonucleotide sequence failures from the synthesis of an 80-mer oligonucleotide.