Wednesday, March 11, 2015

Do you love parallel reaction monitoring, but hate the low number of targets?

Parallel reaction monitoring (or quantification via targeted-MS2 fragments) seems to be rapidly increasing in popularity.  There is, however, a cap on the number of targets you can look at.  This cap is much lower than more traditional targeted quan instruments like the triple quadrupole mass specs (QQQs).

What if I told you that someone just wrote custom software that blows that limit away?  Now, instead of doing PRM on 20 or 30 peptides at a time, you can do PRM on over 600 peptides?!?!

Well, that is exactly what is in this paper in press at MCP from Sebastien Gallien et al., out of the Luxumbourg Clinical Proteomics Facility (btw, the LCP needs a cool logo for me to put up).

How'd they do it?  First of all they wrote some sweet custom software, I think they said its all in C#.  Second, they spike in an isotopic labeled peptide for each of their peptides of interest.  Each method is based around a full scan followed by PRMs.  The full scans are rapid and are looking, not for the peptide of interest, but instead for the isotopic spike in.  When the instrument sees the spiked in peptide which will be eluting at the same time as its endogenous friend, then it starts doing PRMs for the endogenous peptide.

Is that crazy smart or what?!?  Think about this.  You can spike in your standard at a high level if you want.  High enough that the less sensitive full scan can easily see that its time to start monitoring for your peptide of interest.  Then the super sensitive PRMs get to work.

Did you change your column and experience a 2 minute retention time shift?  This method doesn't care! You aren't trapped into a finely tuned window that was designed on the last column. Because the retention times of your standards are going to shift, you've intelligently shifted your target windows.

 Now, I do have to throw a caveat out there.  The 600+ peptide targets were achieved on a QE HF. Using this method on the slower QE or QE Plus would probably mean about half the target list...but still!  300 PRM targets on a QE?  Heck of a step in the right direction!

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