Monday, June 24, 2013

Carbamidomethyl @ C, D, E, H, K, N-term

Recently I worked on an ETD dataset generated from Orbitrap Velos. The user mentioned that Carbamidomethyl on Cysteine and some phosphopeptides are expected but he was not able to get any good identification results using Mascot.

My first attempt is to use the information provided by the user and run the PEAKS DB search. The result shocked me as under 1% FDR, PEAKS DB only reported 170 PSMs. What could possibly go wrong?

Looking at the PEAKS DB result, there are many "de novo only" peptides, which means many spectrum can produce confident de novo sequences but they do not have a confident database hit. This could be a result of unsuspected PTMs and mutations.

So I decided to do a PEAKS PTM search on the db result as my second attempt. PEAKS PTM reported 582 PSMs under 1% FDR. In the summary view, PTM profile section, there are many PSMs found with the PTM Carbamidomethyl on D, E, H, K and N-term. This could be an indication of excess iodoacetamide during alkylation procedure.

Now I know more PTM information of this data. In the last attempt, I added Carbamidomethyl @ DEHK,N-term and Dehydration @ DST into the variable PTM list along with Phosphorylation @ STY. PEAKS DB search was then performed with PEAKS PTM option enabled. This time, under 1% FDR, 736 PSMs were reported, more than four times the number of the initial search.

PEAKS PTM is a great tool to find unsuspected PTMs thus help explaining more spectrum.

Monday, June 17, 2013


It was a very successful conference! There are many exciting new instruments, interesting research and talks.

PEAKS user meeting was very well attended. Researchers from the globe got a sneak peak of our latest research and enjoyed the lunch buffet.

Our booth is always crowded with people interested in our solution or service.
Many people that missed our user meeting are asking for the slides of the talks. We will polish the slides and post them online as soon as possible.

Monday, June 3, 2013

Stop Guessing, Start Knowing - Score threshold and FDR control in PEAKS

In an MCP guideline published in 2004, "a significant but undefined number of proteins being reported as identified in proteomics articles are likely to be false positives".

To tackle this problem and control the "quality" of the identification results, over the past decade, false discovery rate (FDR) becomes the most accepted result validation method.

Although many search engines have the FDR estimation integrated into their package, in order to get the result under a particular FDR, e.g. 1%, the researchers usually have to do several round of "trial and error" on the score threshold used for filtering the result. Even worse, this process may have to be performed on every results.

PEAKS introduced a score selection tool to make this process very simple. Normally, it only takes three mouse clicks. Here is how to do it. On the top of the result summary pane, there is a FDR button.

Simply click that button will give you the score selection dialog. In this dialog, you can quickly select the commonly used FDR values on the right by a single click of the corresponding button. Or you can hover the mouse cursor on the chart to find the desired FDR value. When the value is found, right-click and then select "copy score threshold", the score to be used for the desired FDR will be filled into the score filter automatically. Click the "Apply Filters" button, you will get the result under the desired FDR.