protein-ms Files

jTraqX - Java Implementation of Quant
Readme (11/18/2012) for version 1.13 of jTraqX.

The latest version including the sources is available at 
http://sourceforge.net/projects/protms

If any questions arise, do not hesitate to contact the corresponding author, 
Andreas M. Boehm: ab@andiboehm.de

Please cite:
Muth, T., Keller, D., Puetz, S. M., Martens, L., Sickmann, A., Boehm, A. M., 
jTraqX: a free, platform independent tool for isobaric tag quantitation at the protein level,
Proteomics, 2010. DOI 10.1002/pmic.200900374 
(http://dx.doi.org/10.1002/pmic.200900374)
Boehm, A. M., Puetz, S., Altenhofer, D., Sickmann, A., Falk, M., 
Precise protein quantification based on peptide quantification using iTRAQ, 
BMC Bioinformatics 2007, 8:214. DOI 10.1186/1471-2105-8-214 
(http://dx.doi.org/10.1186/1471-2105-8-214)


jTraqX currently supports MS/MS data evaluation using iTRAQ(TM) labelling
and all other available 4-plex isobaric tags.

Prerequisites:
You need at the Java runtime of least Java 6. The Java interpreter must be 
in the search path of the system and the classpath must be set correctly.
All needed special libraries are provided with jTraqX.

Quick start:
------------
0.  Unpack all files of the ZIP container into a single folder
1.  Run a Mascot(TM) data evaluation 
2.  Copy the Mascot(TM) result file (Mascot.dat) in a folder
3.  Start jTraqX. 
4.  Make the appropriate settings. Enter the correct purity values of the 
    iTRAQ(tm) kit.
5.  Save the settings in the folder of the result file for future use.
6.  Press the "Choose" Button in the input section and select the Mascot result 
    file.
7.  Press button "Start" and wait for completion.
8.  Save the project file for future use.
9.  Evaluate the single peptide identifications and set the values in the column 
    "Validation" to appropriate values. The spectrum can be displayed by selecting
    a peptide line and then clicking on "Show Spectrum".
10. Save the finished evaluation to a new project file for future use.
11. Display the data of the protein summary section by selecting a line of the 
    protein's data, select the ratio paring and click "Show Ratio Plot". The graph
    can be saved to a PNG file in high quality resultion.
12. Draw the QQ-plot by clicking the button "Show QQ-Plot". The graph
    can be saved to a PNG file in high quality resultion.

    
In the case of problems:
------------------------

If any questions arise, do not hesitate to contact the author, Andreas M. Boehm: 
ab@andiboehm.de

In case of an error, send the console output (shell or DOS-Box) to the 
corresponding author.

Mascot(TM) version and settings:
--------------------------------

The latest version of Mascot(TM) data jTraqX is able to process depends on the version of 
the MascotDatfile library, only. This can be updated by replacing the file 
mascot_datfile-2.0.3.jar by the newest version. Do not try to place more than one of 
the mascot_datfile*.jar in the folder of jTraqX.
Mascot(TM) uses a reporter ion correction when quantitation is switched on. As far as
we could verify, the spectral data that is written to the result file is identical with 
the original data. Thererfore the data used for jTraqX evaluation is not affected by
Mascot(TM). Nevertheless, we recommend choosing "none" for the Mascot(TM) quantitation 
settings.

Peptide numbering in plots:
---------------------------

Select "Show Peptide Number" in order to number peptides from 1 onward, instead of 
using the peptide index from the original result set.

Description of the iTRAQ result plots (error plots):
----------------------------------------------------

a) The boxplot consists of only one horizontal bar, if only one ratio is plotted.
b) The boxplot consists of only two horizontal bars, if only two ratios are plotted.
c) In all other cases, the boxplot consists of a box, showing the IQR and the two 
whiskers, that mark the data range and are limited to 150% of the inter-quartile-range.
Outliers will be marked in red. If you move the mouse over the outliers, the peptide
sequence, the block number and the peptide number are displayed.
In the error plot, the determined ratio is marked by a diagonal cross (X), the upper 
and lower bound of the absolute error interval are marked by a horizontal cross (+).
The horizontal green line is always at y=1.
The blue line marks the determined ratio of the protein. Therfore, at least one X lies
is on this line, if and only if the number of peptides is even.
If you move the mouse over diagonal crosses (X) or horizontal crosses (+), the peptide
sequence, the block number and the peptide number are displayed.
By doubleclicking on the crosses, the spectrum viewer will open for the underlying 
spectrum.

Description of the iTRAQ QQ-plots:
--------------------------------------

The QQ plot show the ratios of each reporter pair plotted against the log-normal
distribution. A good linear regression suggests equal distribution and therefore
the log-normal distribution of the ratios can be assumed. Naturally, at least two 
ratios are needed for a regression. 
The Shapiro-Wilk-test is known for quickly rejecting the null hypothesis, especially 
if there are too much data, say more than 30. The test data set contains more than 
1700 spectras that represent too much peptide data for a Shapiro-Wilk-test, that will 
reject the null hypothesis "data is normally distributed" in this case, while not 
rejecting only means that the null hypothesis cannot be rejected. We recommend to 
perform the distribution test visually by using the NP-plot, to use the result of the 
Shapiro-Wilk-test as rough guide, and also to have a look at the result of the linear 
fit. For more information on this issue, refer to the publication of Quant.
If you move the mouse over the data points, the peptide sequence, the block number 
and the peptide number are displayed.
By doubleclicking on the data points, the spectrum viewer will open for the 
underlying spectrum.


Configuration Input Fields and Buttons:
---------------------------------------

The main user interface
-----------------------

About jTraqX: 
This button will show the copyright and some more information.

iTRAQ Reporter:
Select the reporters that were used and enter the exact m/z value of each.
The "reporter tolerance" is the left and right interval, jTraqX uses to find reporter 
signals around the entered m/z values. The Noise Filter lets you define a criterion
for signal filtering, either none, a percentage of the maximum peak or an intensity
range. A zero here value means "no filtering".
The value of "Absolute Intensity Error" denotes the absolute intensity error of the 
the mass spectrometer in the units of the y-axis of a mass spectrum. The value of the 
"Experimental Error" is an estimation of the uncertainty occurring in the lab. Leave 
it at zero, if you are unsure. The checkbox "Only Unique Peptides" tells jTraqX to 
only use unique peptides and to skip the multiple ones. 
The "Threshold" section defines the thresholding for the Mascot results. The 
"Significance" tells jTraqX to use an entered p-value that must be fulfilled 
by the results being processed. You can also define a minimum "Ion Score" that 
must be reached by each peptide result or a lower limit of the  "Idendenty" score.

Reporter:
Enter the purity values provided with the iTRAQ kit here. Select the reportes that 
should be used for evaluation.

Logarithm:
Here the base of the logarithm can be set, that is used for calculations.
The choice of the log type provided is a matter of preference for the user and does 
not affect the results, because log tranformations are linearly related.

Configuration:
Here the configuration can be loaded, saved or set to defaults.

Projects:
Here a project can be loaded for further processing.

The project window
------------------

Table options:
Select the selection type in the table.

Box/Error Plot Options:
Select the ratio pairing used for the ratio plot. Here, also the peptide numbering
can be changed and the ratio plot can be drawn.

QQ Plot:
Clicking this button, the QQ Plot will be shown.

Spectrum:
Clicking this button, the spectrum of the selected peptide will be shown, after
the Mascot result file has been asked for.

Project Options:
Here the project can be saved or loaded.

The License
-----------
Licensed under the Apache License, Version 2.0.
http://www.apache.org/licenses/LICENSE-2.0

End of the documentation file of jTraqX