Not getting enough phosphopeptide IDs? Try stepping your collision energy

I stole the above figure from this not-quite-new-but-new-enough-for-this-silly-blog paper from JK Diedrich et al., out of John Yates's lab.

In this study they looked at the utility of the stepped collision energy function on my favorite instrument, the great Q Exactive.  The paper is open access and definitely worth a read.  I see the small molecule people making use of the stepped NCE function, but rarely us proteomics people.

On the QE, when you employ the step function, your AGC target is divided into 3 parts.  The first 1/3 of your target is isolated through the quadrupole, fragmented and stored in the HCD cell at the lowest collision energy.  The next 1/3 of your target is gathered at the central collision energy and the final part is collected at the highest collision energy on your step.  All of these fragments are deposited back into the C-trap and go in for one single Orbitrap scan

If you have older QE software, your fragmentation energies must be symmetrical (you set the central, and then a percentage above and below of the central normalized collision energy).  On the newer builds (QE 2.3 and up) you can set all 3 energies to whatever you want.

I've used this function when doing top-down and when looking at glycopeptides, but this paper shows clear utility for this function when doing phosphopeptide analysis and also for liberating TMT reporter ions from peptides. On the histogram above, the x-axis is the a-score of the phosphopeptides.  The sum column shows that they found hundreds of new phosphopeptides by simply checking the stepped NCE box and re-running.

 It may be worth taking a second look at on your less ordinary samples!