Thursday, October 25, 2012

Final opinion -- HCD vs. CID+HCD for iTRAQ

I get this question a lot:  What is the best way to do a reporter ion experiment (iTRAQ, TMT) on an Orbitrap?
In order to find out, we took human serum, depleted it, digested it and fractionated the peptides with strong cation exchange chromatography.
Each fraction was ziptipped, resuspended and separated into two identical fractions.
One half of each fraction went into a Top5 method, where the 5 most intense ions were first fragmented by CID then with HCD.
The other half of the fractions were fragmented only by HCD, and both the reporter ions and sequence were read in the Orbitrap.

The column, gradients, and applicable settings were the same for the Orbitrap Velos that was used.  The only real difference was that the samples ran on the first method were ran in triplicate.

In my mind, the results aren't even close.  In 3 times the run time, the CID + HCD method still lost.  While the majority of proteins were the same, in 1/3 the time, the HCD method turned up significantly more proteins.
This isn't the first time I've seen this kind of data.  In general, anything that increases your cycle time hurts your results -- even adding a complementary fragmentation method.
Summary:  Use an HCD based fragmentation method for iTRAQ experiments.

Of course, there is a caveat here, and an explanation for the Orbitrap technical note that endorses the first method:  Between the Orbitrap XL and Orbitrap Velos platforms, the HCD cell was dramatically altered.  Dramatically.  HCD is hundreds of times more efficient on the Velos platforms due to these changes.  This allows me to clarify this summary:
If you have an Orbitrap Velos/Pro/Elite, use HCD only.  For an Orbitrap Discovery or XL, use the CID+HCD method.


  1. Hi Ben,

    I have observed that HCD is kind of slow in Discovery/XL instruments. Why then CID+HCD works better there? Also, due to the dual pressure ion trap, the trap in Velos and other instruments is much faster, so CID scan would take much less time. Have you analyzed any pre-fractionated complex sample to compare the same?


  2. Santosh, Yes, the prefractionated sample bears out the same results. On the XL and Discovery, HCD is both slower and less efficient than on the Velos family instruments. It looks to me like you get so many fewer "good" spectra when trying to get reporter ions and sequencing in one scan with these instruments that it offsets and difference in speed.
    The inverse is true of the Velos family of instruments. While the dual pressure trap is significatnly better than the original ion trap, the improvement in speed/coverage pales beside the improvements made to the HCD cell.