Saturday, June 23, 2012
This entry is also probably a bit premature, because it looks like I've only scratched the surface of what Pinpoint is capable of. But after several days of using it, I'm beginning to become very comfortable with the basic functions of the software.
First of all, Pinpoint is for targeted proteomic studies. Specifically, if you have a protein (or, more importantly, protienS) of interest and want to perform targeted qual/quan analysis on different samples for the specific peptides, Pinpoint is your software.
When I participated in targeted studies at the NIH, we always felt that we were limited to either peptides that we had identified in discovery runs. When we specifically went looking for a protein or peptide that had not shown up in a previous experiment we went through the following steps:
1) Looking up the protein sequence through NCBI (and trying to guess the right one, because the NCBI is almost at the point where it has too much information to sort through)
2) Taking the sequence (that is hopefully correct!) to the UCSF protein prospector and performing an in silico digest of the protein sequence. Since you can't save your settings, every modification and setting has to be re-inputted every time you reopen the webpage. (Please don't think I'm knocking the Prospector project, btw, I will never fully express my gratitude to the University of California for setting up and maintaining that site! See the lavish praise I showered on this site in my first book for more information!)
3) From the Prospector output, manually remove all singly charged, redundant, and unlikely peptides from the massive list of possible peptide masses that resulted
4) Move that list into the Always Include box within your Xcalibur method.
5) Run the sample
6) Manually extract the peaks and areas for each peptide that you found using Xcalibur and plot your standard curve using Excel
7) Make a professional looking output graph in Powerpoint. The trick is adjusting your peaks for the inevitable little shifts in retention time (or big shifts, if you are using and Eksigent...)
And this way works. I know that some of my readers are still doing it this way. After you've done it a few times, it doesn't take all day to show that your protein is upregulated after drug treatment, just most of a day.
Pinpoint is so amazing because it does all of it for you. Every bit of it. In about a minute.
This is the order of events
1) You tell Pinpoint what protein(s) you are interested in. It can be from your own FASTA file, or it will look it up for you and it inputs the sequence.
2) You tell Pinpoint what you want to digest it with. You can save these settings, so it knows you use trypsin and you expect no more than 2 missed cleavages, and you use iodoacetamide.
3) It digests your protein and gives you only the good, relevant peptides that you will be able to get information on.
4) It also predicts the RETENTION TIME of said peptides. No joke.
5) You export your file and copy it right into your Method file.
6) You make your runs
7) You drag the completed .raw files into Pinpoint
8) It plots the abundance of all of the peptides from your theoretical digest from each of the samples, lining up the peaks and making all the small adjustments in retention time. Resulting in an absolutely gorgeous output file.
If you are doing targeted proteomics studies the way that I mentioned above, you want this software. But, seriously don't take my word for it. Go to the Thermo-BRIMS portal and download the free trial version.
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