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 command prompt:

   java -jar aminoseq.jar

Opening a spectrum file

Select 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:

Menu
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:

a red mark: the starting point
zero or more green marks: accepted amino acids
zero or more blue marks: potential amino acids

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.

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.

Keystroke commands

Key	Effect
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

Menu commands

Menu	Submenu	Shortcut	Effect
File	Open file	ctrl-O	Open file and read a new spectrum
	Save log	ctrl-S	Save log to file
	Print	ctrl-P	Print the spectrum window
	Quit	ctrl-Q	Quit the program
Edit	Undo	ctrl-Z	Undo change to sequence
	Redo	ctrl-Y	Redo change to sequence
	Add mirrored spectrum	ctrl-M	?
Settings	Show errors	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
	Decrease tolerance	ctrl-D	Decrease macth tolerance for potential amino acids
	Increase tolerance	ctrl-I	Increase match tolerance for potential amino acids
	Sequencing parameters		Set parameters for automatic proposals
Help	Help	ctrl-F1	Show brief help (outdated)
	About		Show brief program information
	Amino acids		Show monoisotopic amino acid masses
	Ion types		Show composition of amino acid modifications
	About		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.

Other information

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 (kokholm@dina.kvl.dk) and Peter Sestoft (sestoft@dina.kvl.dk). Natascha Helena Beyer (nhbeyer@mail.dk) provided background in proteomics and comprehensive MS/MS data sets, and tested the program. Gerhard Saalbach (g.saalbach@risoe.dk) provided the initial inspiration for this work.

Peter Sestoft (sestoft@dina.kvl.dk) 2002-01-04, 2002-01-15