Top down proteomics using the Q-exactive?

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rvm
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Top down proteomics using the Q-exactive?

Postby rvm » Mon Jan 30, 2012 4:39 am

Hi,
I hope you can offer some insight into top down proteomics using the Q-exactive!??

We've been doing work on microbial proteome profiling, primarily using bottom up approaches on the Orbi classic. Its working well, we're getting as much as coverage as you'd expect from the classic. But its throughput for Quant work is pretty low, recently we've been looking at the Qexact for discover,y as well as mainly as a validation instrument, quantifying peptide concs.


However its been suggested that it is possible to perform top down analyses of microbial proteomes using the Q-Exactive for discovery work, but I'm not sure if its possible. From my (limited) understanding of the Q-Excat, is that has a HCD collision cell only, I can't find anything in the literature that describes top down approaches, describing proteome coverage, biomarker discovery, methodologies etc.. using just HCD or the Qexact?

I would like to know if its possible to perform top down protein work on the Q-Exactive? If so any pointers to any literature or posts here would be greatly appreciated!

Many thanks

JDRCHEM
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Postby JDRCHEM » Tue Jan 31, 2012 6:57 am

I've only done a couple of LC runs on a Q-Exactive analyzing intact proteins (< 30 kDa), but I have been happy with the results. The HCD data quality is comparable to Velos Orbitrap HCD data I've collected. An advantage with the Q-Exactive is that the speed permits more transient averaging in a given amount of time. This might not be that important for infusion work, but it is usually needed for LC work. It does seem that the protein upper mass limit is a little lower than other orbi platforms. I have not performed long bakeouts in an attempt to decrease the pressure in the orbitrap region, but I suspect that the upper mass limit would be extended with longer bakeouts, as this greatly helps on our other orbitrap instruments.

I used HCD collision energies ranging from 18-24 with very large AGC targets, usually 1-2E6 for MS and 2-3E6 for MS/MS. I have not found significant mass error (< 10 ppm), even with large AGC target values, as long as the system is mass calibrated frequently (every day or two). I used 140k and 70k resolution for MS and MS/MS, respectively, with 5-10 microscans.

The Q-Exactive would not be my first choice for top-down discovery work, I would prefer instruments with multiple dissociation types from which to choose. If you are concerned about proteome coverage or preserving modifications, I have no doubt that having ECD/ETD would be tremendously beneficial. But, I see no reason why HCD on the Q-Exactive can't be effective, especially for analyzing smaller proteins.

Craig
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Postby Craig » Tue Jan 31, 2012 9:05 am

This poster from ASMS 2011 supports what JDRCHEM said above: the Q-Exactive has a lower high mass limit than other instruments, and less fragmentation techniques, but it's a viable option for top-down proteomics. The Orbitrap Elite seems to be the best choice.

rvm
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Postby rvm » Wed Feb 01, 2012 4:52 am

Thanks for the responses, my feeling regarding the elite is the same.
We are in the process of acquiring a Q-exactive, which was considered primarily for validation and discovery work to complement/replace some of the work doing on the Orbitrap classic, but (very) recently top down proteomics was added to the wish list and I was not sure how the Q-Exactive would handle this.

Thanks again for the responses they were very helpful!


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