Hi,

I'd guess that you have a RAM issue. Are you running PP on a 32-bit system? If so, you are restricted to around 3 GB available RAM. If this is the case, make sure that only essential programs are open, so you maximise RAM for PP. I'm not sure if PP runs on a 64-bit system, but the company will be able to tell you.

You might need to use a different Pipeline if you can't get PP to behave. For such a large dataset, you really need as much RAM as you can get, so a 64-bit system is the way to go. I know Mascot, OMSSA, and X!Tandem all run in 64-bit environments.

If it's not the RAM, then I'm afraid I'm out of ideas...

Good luck,

Peter

Thanks a lot for your suggestions. I will try it.

I am agree with Peter that you should have a RAM issue. Anyway, it is good to have an alternative way to do proteomic data analysis. I strongly recommend you give a try to OpenMS and TOP (http://open-ms.sourceforge.net/), which is an opensource framework for mass spectrometry and a proteomics pipeline. They proposed some easy customizable workflows, some of which are intended for iTRAQ quantification, using a consensus search with Mascot and others search engines.

By using other tools you can always check when your troubles are software specific or not.

Good luck!

RAM Trick: if you are running PP on linux system then increase the swap memory ( to few GB), it will not make the processing faster but you PP will not fails due to low memory problem.

# dd if=/dev/zero of=/mnt/myswapfile bs=1M count=1024

1GB file myswapfile at some mount point (change it as needed )

Make this file as a swap file using mkswap command.

# mkswap /mnt/myswapfile

Setting up swapspace version 1, size = 1073737 kB

Enable the newly created swapfile.

# swapon /mnt/myswapfile

Trick work for our in-house Mascot and Phenyx servers

Try to search file one by one instead of one combined 1GB mgf file and combine the results later.

if you have sufficient computing power then go for suggestion of Amaury or Peter

or try it our at cloud server (galaxy enabled tool-set, you can see the tag "Proteomics Tools" including OpenMS, OMSSA, Xtandem along with other tools)

http://galaxy.nbic.nl/root?tool_id=masscomb_xtandem

You have zip all mzML the files to one and select mscfileset while uploading

if you are new to galaxy, http://wiki.galaxyproject.org/Learn/Screencasts

Great, thanks for everyone.

As peter guess i too feel it have a RAM issue, So you can better upgrade it as for Proteomics researcher we need to have higher Configuration.

Regarding the iTRAQ Experiment I think the best one to work is MS-GF+ (from Pavel Pevzner Lab,UCSD,http://proteomics.ucsd.edu/) Search which lead to Successfully result for iTRAQ Sample including quantitative and Qualitatively. Then for DEP Analysis you can Use isobar or iTracker which of much useful recently for my own work.

All the best.

Thanks for the suggestion from Inamul.

we are going to run iTRAQ sample with QE,could you show me the method of QE,such as some parameters.thankyou

These are the parameters.

The electrospray voltage was 1.6 kV, and the ion transfer tube temperature was 280 ˚C. The S-Lens RF level was 50.00. The data acquisition was programmed in data dependent acquisition (DDA) mode. A top 12 method was used. Full MS scans were acquired in Orbitrap mass analyzer over m/z 380-1800 range with resolution of 70,000 (m/z 200) and the number of microscans set to 1. The target value was 1.00E+06, and maximum injection time was 250 ms. For MS/MS scans, the twelve most intense peaks with charge state ≥ 2 were sequentially isolated and further fragmented in the higher-energy-collisional-dissociation (HCD) cell following one full MS scan. To reduce the interference of peptide co-fragmentation to the iTRAQ quantitation, the isolation window was set as 1.0 m/z. The normalized collision energy was 33%, and tandem mass spectra were acquired in the Orbitrap mass analyzer with resolution 35,000 (m/z 200). The fixed first mass was m/z 100.0. The target value was 1.00E+06 and maximum injection time was 120 ms. The number of microscans was 1 and the ion selection threshold was 5.0E+04 counts. Peptide match and exclude isotopes were turned on. Dynamic exclusion was set as 30 s.

thank you anyway, my tutor has run the sample and we are analyzing the data