User Manual for KVL Aminosequencer
KVL Aminosequencer supports manual de novo sequencing of proteins by
interpretation of tandem mass spectrometry data (MS/MS).
This page provides preliminary documentation of the user
interface. Especially the description of the automatic sequence
proposal mechanisms is incomplete.
Starting the program
First you must download and unpack the
program. Your computer must have a Java runtime version 1.3
installed, see http://java.sun.com/j2se/1.3/jre/.
Then start the program by executing, at the Unix or MS Windows
java -jar aminoseq.jar
Opening a spectrum file
File | Open to open and read a spectrum file. The
spectrum file must be in Sequest DTA or Mascot format.
The user interface
Here is a screen dump of the user
interface. The user interface has the following parts:
- Message window
- The spectrum window
- The current mouse position and a list of observed ions that
support this peak
- The current interpretation of the spectrum as an amino acid
sequence; an uninterpreted bit is indicated by a mass in brackets,
such as [19.0]
- Facilities for automatic sequence proposals
The spectrum window
The spectrum window shows the current spectrum with mass (in amu) on
the x-axis and relative abundance on the y-axis.
An amino acid sequence can be found manually by marking peaks in
the spectrum. At any point during this process, the spectrum will be
decorated with zero or more subsequences, each consisting of:
with green marks on either side of the red mark, and blue marks only
on the left and right fringes. The accepted (green) amino acids of
two subsequences may meet but cannot overlap. The potential (blue)
amino acids belonging to one subsequence may overlap with potential
(blue) amino acids of another sequence. If one peak is decorated with
potential amino acids from two different sequences, then clicking on
it will accept both, and make the sequences meet in that peak.
- a red mark: the starting point
- zero or more green marks: accepted amino acids
- zero or more blue marks: potential amino acids
Building a sequence
- Click on some peak to select it as the starting point; it will be
shown in red.
Every neighbouring peak whose distance from the selected one
corresponds to the mass of an amino acid is a potential new peak to
select, and will be labelled in blue.
- Clicking on a potential (blue) amino acid will color it green,
remove all the blue ones relating to the same previously accepted
(green) amino acid, and add new potential (blue) amino acids
corresponding to the recently accepted (green) one. The best-matching
potential amino acid is highlighted with a red ring.
- Clicking on an accepted (green) amino acid will backtrack to the
time just before is was accepted. More precisely, all blue and green
marks further out will be removed, and blue marks will be added
corresponding to the mark most recently accepted before this one.
- Clicking on the red mark will remove all markings altogether, on
both sides, and will allow you to select a new starting point.
- The program can generate sequence proposals automatically, see Automatic sequence proposals below.
|z ||Zoom in around the cursor
|o ||Zoom out around the cursor
|c ||Recenter around the cursor
|r ||Reset to full view of the spectrum
|arrows ||Step left or right
|m ||Show masses of 10 more peaks; may be repeated
|n ||Hide masses of 10 more peaks; may be repeated
||ctrl-O||Open file and read a new spectrum
||ctrl-S||Save log to file
||ctrl-P||Print the spectrum window
||ctrl-Q||Quit the program
||ctrl-Z||Undo change to sequence
||ctrl-Y||Redo change to sequence
|Add mirrored spectrum
||ctrl-E||Show spectrum backwards also
|Show sequence backwards
||ctrl-B||Show amino acid sequence backwards
|Show local levels
||ctrl-L||Show average local abundance (peak height)
|Use log scale
||ctrl-W||Use log scale for abundance (peak height)
|Show backwards spectrum
||ctrl-R||Show spectrum backwards also
|Show reinterpreted spectrum
||Shift spectrum by an amino acid modification
|Allow K and R
||ctrl-A||Allow K and R amino acids inside sequence
||ctrl-D||Decrease macth tolerance for potential amino acids
||ctrl-I||Increase match tolerance for potential amino acids
||Set parameters for automatic proposals
||ctrl-F1||Show brief help (outdated)
||Show brief program information
||Show monoisotopic amino acid masses
||Show composition of amino acid modifications
||Show information about the current spectrum
Automatic sequence proposals
(This section yet to be written).
The program can generate sequence proposals automatically. If you
press the Compute button in the Computed proposals field, purple marks
will appear on a sequence of peaks that correspond well to a series of
peptide fragments. Repeatedly pressing the Compute button will cycle
through some proposals. Right-clicking on a purple-marked peak, you
can select Follow proposal right to obtain a sequence corresponding to
all the purple peaks to the right. This is the same as left-clicking
on the all those peaks, from left to right.
The program uses the monoisotopic masses, found here and here;
some differ by 0.00001 Dalton from those of AminoCalc from Protana.
Authors and credits
KVL Aminosequencer was developed by Niels Jørgen Østergaard Kokholm
and Peter Sestoft (email@example.com).
Natascha Helena Beyer (firstname.lastname@example.org) provided background
in proteomics and comprehensive MS/MS data sets, and tested the
Gerhard Saalbach (email@example.com) provided
the initial inspiration for this work.
Peter Sestoft (firstname.lastname@example.org) 2002-01-04,