DEX – User’s Guide

Q & A          

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Questions and Answers

 

 

You can download a free Unix environment emulator to run DEX.  A recommended program is Cygwin (http://www.cygwin.com/), and it has a host of utilities that can be installed.  The file system is different in Cygwin, and it does not like blanks in the path or filenames.  It will give you a gcc compiler and an X-server (for viewing the graphs), but it is not as complete as a Unix-based machine.  See the rather detailed instructions for the X-server at (http://x.cygwin.com/docs/ug/setup-cygwin-x-installing.html) for downloading Cygwin with the X-server as well as the Cygwin Download and Directions section below for more information which extra parts of Cygwin need to be chosen to properly run the full DEX package.  You can also try Windows Services for Unix (http://www.microsoft.com/windowsserversystem/sfu/), which is better integrated with Windows but has less functionality than Cygwin.  It is not supported directly here. 

 

 

Yes, Macintosh computers have a built in UNIX operation system that allows for direct usage of DEX.  You do not need a UNIX emulator like Cygwin.  You will still have to install fftw-2.1.5 before you can compile DEX for your Macintosh and you will also have to install GRACE if you want to view any of the results.  Once you have downloaded FFTW you can use the directions provided below under the Downloading fftw directly from the FFTW website section. 

 

To install GRACE, it is suggested the typical user use one of the porting programs (DarwinPorts or Fink) and have it install GRACE and all its dependencies automatically.  Make sure that you have the updated “Xcode” developer tools (Xcode 2.3 or higher), which are available from the system CD or the Apple developer website.  Then, download and install a program to port GRACE (DarwinPorts-1.2.1 has been used successfully) by using their installer.  Choose the GRACE graphing package in DarwinPorts and have it install it as well as the dependent packages.  Then, in the terminal, type:

Followed by:

 

In your terminal shell program file (typically called “.profile”, “.cshrc” or “.bashrc”, add the command:

Then, open up a X11 window, but don’t type your commands in it, use the regular terminal instead. 

If you have done this properly, if you type “./Run-test2 go” into the terminal, you should get the same result as shown here

 

 

You will have to rename the file by typing “mv DEX.tar DEX.tgz” while in the Unix emulator.  Then you can type “gunzip DEX.tgz” to unzip and finally type “tar –xvf DEX.tar”.  Then follow the instruction starting with the  “make-script” in the DEX-Installation/Tutorial page (or back to home page). 

 

 

Yes.  Since the beginning of 2006, DEX has been able to handle multiply charged data up to an m/z = +6.  Peptides with a m/z greater than 6 are quite difficult to separate with today’s mass spectrometers, but it could be added in the future.  To tell the software what the charge state of a peptide is, simply put the m/z integer number after the sequence when generating the natural isotopic profiles with the program isotopic-fast-profiles. 

 

 

Yes.  If your data file does not start before the monoisotopic peak, the program will automatically add the value of 0.0 for every data point before the start of your data.  Since the envelope taken for the deconvolution is 1.5 mass units before the monoisotopic peak, any value missing from the file will be replaced up to 1.5 mass units before the first peak.  As long as your data starts from the monoisotopic peak, the program will run normally, but it may be slightly different then if you were to take a longer original spectrum value.  The results won’t change much, and you should still be able to use them properly.  If the file is too short at the end, the program will automatically deal with that by padding on a value of 0.0 after your data ends.  This should not effect your data at all because the deconvolution is one directional, moving toward the higher m/z, so a padded value at the end will not effect the information you seek before that. 

 

 

Yes. The DEX resampling program can handle resampling large intervals into smaller intervals through a linear determination method.  The program will extrapolate the values from the larger intervals and resample at standard intervals, which are necessary for any FT based deconvolution.  If your data is real low resolution, and the original intervals are much larger than the resampling intervals, then the results may not be very robust and you might want to try acquiring higher resolution data. 

 

 

Yes, if you have gaps in the data, the DEX resampling program will be able to handle them and run properly.  If the gaps are fairly small, or they are in unimportant parts of the spectrum (away from the m/z value of interest) they will have little or no effect on the deconvoluted results.  If the gaps are in the middle of the envelope you wish to look at, then you might want to make sure your instrument is detecting the signal properly and you should double check to see that the results are what you expect by graphing them. 

 

 

At the moment, there are a few problems with using DEX for very long peptides (or whole proteins).  One of the main problems is the fundamental way that DEX calculates the backbone amide populations using a Fourier transform.  This method requires that the offset of the deuterated values be evenly spaced.  This offset can be approximated to be 1.00 mass units for peptides with under 10-20 deuterons added during the experimental conditions.  In reality, the offset for each deuterium is 1.0062767 mass units more than hydrogen.  After more than 10-20 deuterons, the offset is sufficiently off from the original 1.000000 mass unit spacing and causes some problems in the data calculation.  An offset of 1.005 can help extend the peptide size limit of reasonable answers for DEX, but the ambiguity of many mass spectrometry instruments in their calibration to this level prevents such an offset from being widely applicable.  You can use the peak width program original-spectrum-width (see Extra Programs) to help you determine the overall shape of the protein mass spectrum envelope.  This program will read the raw mass spectrum data and does not require any extra formatting to run. 

 

 

There is a sample file in the data_lib folder called Isotope-list.txt that will give you a template to follow.  Only the higher isotopes of a particular atom should be listed.  For example, 13C and 14C can be listed but 12C should not.  The offset should be an integer over the lowest isotope and the Mass_zero should be the mass of the lowest isotope, not the higher one listed.  The abundance should be anywhere

from 0.000 to 0.999.  Changing the abundance or composition of atoms will affect all the atoms of this type.  Thus changing the abundance of 13C will affect all the carbon atoms in the peptide. 

 

Cygwin Download and Directions

 

For a walk-through of downloading parts of Cygwin, go to http://x.cygwin.com/docs/ug/setup-cygwin-x-installing.html

and follow their instructions to get you to the point where you need to select the download packages.  You are downloading the packages when you click on the name and a number is shown under the “New” column.  Some of the parts below are automatically chosen when others are selected, so just check to see that they have a number in the “New” column. You are welcome to download any extra parts of Cygwin, but at a minimum, you must download all the default pieces as well as the following parts:

 

Base section

diffutils

gzip

man

tar

which

 

Devel(opment) section

autoconf and autoconf2.1

automake1.9

binutils

gcc

gcc-core

gcc-g++

gcc-g77

gcc-objc

make

 

Editors

gvim

vim

 

Libs

XmHTML

gtk2-x11

gtk2-x11-runtime

jpeg

libXft

libXft2

libpng12

libpopt0

libtiff5

libxml2

ncurses

opengl

tiff

 

Net

autossh

openssh

openssl

 

Publishing

psutils

 

Shells

bash

bash-completion

tcsh

zsh

 

Text

a2ps

less

more

 

Utils

cygutils

d

time

 

X11

X-start-menu-icons

X-startup-scripts

grace

xorg-x11-base

xorg-x11-bin

xorg-x11-bin-dlls

xorg-x11-Indir

xorg-x11-etc

xorg-x11-fenc

xorg-x11-fnts

xorg-x11-libs-data

xorg-x11-xwin

xpdf

xterm

xwinwn

 

Fftw version 2.1.5 should be downloaded separately, as the version supplied in Cygwin is not the same used in DEX and may not work.  See instructions below on how to download and install fftw. 

 

If you are not familiar with Unix like commands (Cygwin emulates this within MS Windows) please see this simple reference page for a few of the commands you will need to use to effectively run the software.  You can also find many websites that have much more information about commands such as this site

 

Once you have downloaded and installed Cygwin, you will need to open the program and type: “startx” to start the x-server that will allow you to run the GRACE graphic window and view the results. 

 

Running DEX scripts and programs on Cygwin

 

You may have to change the compiled versions of some programs, such as any file that ends in “.exe”.  See the DEX-Install-Tutorial for more information. 

 

Downloading fftw-2.1.5 directly from the FFTW website (http://www.fftw.org)

 

Click on the download tab at the top of the page.  Choose to download file “fftw-2.1.5.tar.gz” to your directory.  If using a Windows machine, the file might be converted to fftw-2.1.5.tar.tar or fftw-2.1.5.tar, which can be renamed in Cygwin

by typing “mv fftw-2.1.5.tar.tar fftw-2.1.5.tar.gz”. 

 

Unzip and unpack the file fftw-2.1.5.tar.gz by typing:

 

 

To configure and install FFTW type:

 

 

You can test the program by typing “make check” and look to see that everything good or ok.  The location that you type after the --prefix will also be the location use for the “make-script” arguments.  For example, if you typed:

 

 

Then when you compile DEX using make-script in the DEX-Install-Tutorial, you will type:

 

 

Downloading GRACE directly from the website (http://plasma-gate.weizmann.ac.il/Grace/)

 

Click on their download link near the top of the page.  Click on the “src” folder and then on the “stable” folder.  You can choose any of the files that are not labeled alpha or beta.  For example, choose “grace-5.1.9.tar.gz”.  Save this file and unpack it like you did with the DEX gzipped and tarred file as explained above or in the DEX-Install-Tutorial under

“Installation of DEX.”

 

Update

Written by Matthew Hotchko and last updated last on February 7, 2006

 

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