PTM Profiling is a great new feature found in PEAKS 7.5. It is built to deal with a specific issue regarding post translational modifications. In many cases a protein is identified with both modified and unmodified peptides at some positions. PEAKS PTM profiling works with all PTMS, in this example we will look at phosphorylation. In this protein there are several phosphorylation sites. However, unmodified peptides were found at those positions as well. This leads to two questions. At what positions do the phosphorylations occur? And, if it is phosphorylated, how much of the protein is phosphorylated?
To answer the question of whether or not the PTM is true, PEAKS provides an Ascore for each proposed PTM. The A score is based on the evidence from the fragment ions, and is the probability that that modification is present at that position compared to other possibilities. PEAKS also has the ability to assign PTM confidence based on direct fragment ion proof. So, the MS/MS spectrum shows fragmentation before and after the proposed modification site above 5% relative intensity, it is considered confidently modified. In either case, if the PTM is considered confident it will appear above the protein sequence.
To answer the question of how much of the protein is phosphorylated, PEAKS uses a concept implemented in label free quantification experiments. It uses the concept of peptide features, meaning the lcms signal of a peptide. It has been proven that the area under the curve of the peptide feature is proportional to the relative abundance of that peptide. So, for a peptide with a confident modification site shown in this LCMS, PEAKS will find the area under the curve of its associated LCSM feature. It will repeat this as well for all of the modified and unmodified peptides found at this position in the protein. This table shows all the modified and unmodified peptides which were found at this position. The Ascores are reported for each modified peptide. And, the peptide feature area is given as well.
Using this information, PEAKS creates a bar chart that gives a ratio of the relative quantity of phosphorylated peptide versus unphosphorylated peptide at each identified phosphorylation position. Only fully digested peptides are used in this chart to give added accuracy. Scrolling over the graph will give the percentage of the total amount. You can also use the drop down menus at the top of the window to compare the phosphorylation ratios across multiple runs. Here you can see at some positions there is consistency across runs. At other positions the modification didn’t appear. So, you can see the similarities and differences in phospohrylation across multiple runs. You can also export the raw ptm profiling data to get more details.
To actually run PTM profiling is easy, at the top right hand corner of the coverage view, select the PTM profiling button. This will compile the data and present the PTM profiling data for all the identified modifications in that protein. Only confident PTMs are used, so be sure to select either A score or minimal ion intensity and the desired cut off using the legend to the right of the coverage view.
That is all you need to know to get started with PTM profiling with PEAKS. Thanks for listening.