TripleTOF 5600 Vs. Q-Exactive

Whether you are building your own mass spec, modifying an existing one, or just need help with instrument settings post about it in here.
DAI
Carbon Member
Carbon Member
Posts: 4
Joined: Thu Jul 07, 2011 7:57 pm

TripleTOF 5600 Vs. Q-Exactive

Postby DAI » Thu Jul 07, 2011 9:29 pm

Hi Guys,

Which is better for me if I'd like to quantify histone 3 modifications of cell line? We already have a LTQ-Orbitrap XL ETD.

Regards

DAI

JDRCHEM
Phosphoserine Member
Phosphoserine Member
Posts: 24
Joined: Wed Jun 29, 2011 5:35 am

Postby JDRCHEM » Fri Jul 08, 2011 10:09 am

Hello DAI,

I will preface my response by admitting I have never used the TripleToF 5600 and I've used the Q-Exactive for a grand total of 3 days. I don't know the merits of the 5600 other than 100 Hz duty cycle, but I can tell you that the performance of the Q-Exactive was very impressive. Thermo claims a 12 Hz duty cycle with with a resolution of 17.5k at m/z 200, but I think practical duty cycle was about 10 Hz for us to achieve good sensitivity (50-200 amol limit of detection in yeast lystate background) for our heavy peptide quantification experiments. The HCD MS/MS spectra were beautiful for tryptic peptides (mostly unmodified +2 and +3). There is a multiplexing feature on the Q-Exactive, but I didn't have time to play around with it, but I imagine it would be useful for high-throughput analyses.

But, I think the real question is not which of those two instruments is best, but what is the best method to do quantification of highly charged, modified histones? I think the Orbi XL with ETD is perfectly capable of doing the sort of quantification you may want. Ben Garcia's lab published a fine paper of quantifying histone H3 modifications using the very same instrument: http://www.mcponline.org/content/8/10/2266.abstract. If your goal is to see changes (maybe two-fold changes or greater) then I think this is a very elegant way to quantify H3 modifications. If your goal is too find smaller changes or find modifications present at 100 copies/cell then the Garcia method probably will not be the best. The method of quantification (global isoform percentage) has been used previously by other groups and has been analytically validated:http://www.pnas.org/content/105/11/4093.abstract, http://pubs.acs.org/doi/abs/10.1021/ac0600050

I personally would not attempt an SRM type of experiment for histones or histone tails unless you are interested in a few specific modifications or have expertise in this area. After doing SRM for a few years, I have found that label-free and isobaric tagging strategies present very good alternatives that are capable of providing similar results in a fraction of the time it would take to develop robust SRM assays.

I hope this helps and good luck!

Jason


Q-Exactive paper: http://www.mcponline.org/content/early/2011/06/03/mcp.M111.011015.long

DAI
Carbon Member
Carbon Member
Posts: 4
Joined: Thu Jul 07, 2011 7:57 pm

Postby DAI » Sun Jul 10, 2011 7:40 pm

Hi Jason,

Thanks for the reply. I've used LTQ-Orbitrap XL ETD for protein(mainly histone) characterization for about 3 years and it is clear that LTQ-Orbitrap XL ETD is very powerful for qualitative analysis. But it might not be cost/effective to use it for routine quantitative analysis. It seems reasonable to develop the method with LTQ-Orbitrap XL ETD and then transfer it to Q-Exactive or TripleTOF 5600. I noted that the high resolution/accurate mass features provided by Orbi or TOF type mass spec has led to more interesting applications in this year ASMS.

It is appreciated if you could keep us updated about your experience with Q-Excative.

Regards

DAI

tsbatth
Ubiquitin Member
Ubiquitin Member
Posts: 58
Joined: Thu Jul 12, 2012 7:26 pm

Postby tsbatth » Sun Nov 25, 2012 2:29 am

Old topic I know, but I wan't to warn anyone considering buying a tripletof 5600. DONT DO IT. Biggest piece of junk ever, highly unreliable, not robust at all and will go bad eventually like stale bread. Other than us, personally know 3 groups that have had (having) huge problems with it (ours runs worse then old Q-star pulsar, I'm being serious, and this is with contract, thankfully it's a loaner and we won't be buying). It's not a question of if it will turn to shit, rather when. Talking to the service engineers, two have personally told me it's huge problem in NA (where they work). You have been warned, test it out for at least 6 months, don't be fooled by the initial results.


Return to “Instrumentation”

Who is online

Users browsing this forum: No registered users and 3 guests