So to clarify, you've got 4-plex labelled peptides, you've done the LC-MS/MS and now you want to analyse the data? It sounds like you want separate ion intensities for each reporter ion, and not a ratio. Is that correct? If so, then that limits your software choices.

I've had good success with i-tracker, so I'm surprised it didn't work for you. How are you processing the data?

IDPicker is for processing the matched spectra into confident ID's and determining an FDR. How are you using this in your workflow?

Once we have some more information, we can work out exactly what may be going wrong and how to proceed from there.

Peter

Thanks for your reply, Peter Hains, As i wish to get reporter Ion ratio.

Let me make it clear I have 2 sample one is control and other is Diseased one. Here i have used 116/114 tag as control and 117/115 tag in Diseased sample. Now i need to quantify protein Expression level between control and Disease.

Till Yesterday i was Struggling With i-Tracker but I dont find much useful results. As it give information "Reporter ion peak collection" and "Reporter ion area calculation" but i dont know how i can interpret the data from i-Tracker to get the Quantify protein Expression level between control and Disease.

As for IDPicker it give complete information on Protein Clustering and Protein Grouping.

Please Suggest.

So you are using an MGF or DTA file as input?

And as an output from i-Tracker you get a *.hum and a *.mcn file?

It's all explained in the user_instructions.pdf that comes in the ZIP file with i-Tracker.

Am using MGF file from Proteome Discoverer. Yeah in User Instruction its give little description on the results But not clear.Such As (Areas,114, 115, 116, 117,, Ratios, 114, 115, 116, 117,,Errors,114,115,116,117) Its all related to Area but not the Complete results for Reporter ion intensity ration. As am new to this Topic so i feel difficulty in interpreting and calculation this Method with that of Area based Quantification.

Okay, I've worked with the 8-plex output and that may be slightly different to the 4-plex output. I'm referring to the *.hum file. I used the "Norm" row for my calculations. I took those values and did some simple calculations. I did not use the ratios reported by i-Tracker as my labelling regime was not compatible with the simple choices it offers for ratio calculation.

You simply match the appropriate "Norm" row value with the matching "Areas" row value.

An example output is like this (comma delimited);

Areas,113,114,115,116,117,118

Norm ,0.146,0.138,0.015,0.171,-0.018,0.213

I've omitted all the other information that i-Tracker gives as we won't be using it now. The "Areas" row actually refers to the iTRAQ label and the "Norm" row refers to the value for that reporter ion. In the above case if I wanted to know the ratio of 114/113 I'd do 0.138/0.146. Easy as that.

Good luck,

Peter