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Topic: Few MS Annotations (Read 3574 times) previous topic - next topic

Few MS Annotations

Hello all,

I'm trying to process a DDA lipidomics sample set I have and after running identification, I'm getting a lot a MS1 annotations (without MS2), but very few assignments that use the MS2s (164 vs 8). Looking at the data by hand, the MS2 look to be of at least moderate quality, so I'm not sure why there would be so few MS2 annotations.
Has this happened to anyone else? Do you know what parameters might need to be adjusted? I'm using an LTQ-Orbitrap in case that's relevant.

Thanks,
~Matthew

Re: Few MS Annotations

Reply #1 –
Hi, if you use LTQ-Orbitrap, in the start up project window of msdial, please set
1. data type for MS1 as "profile"
2. data type for MS/MS as "centroid".
According to my understanding, the orbitrap detector and LTQ detector are used for MS1 and MS/MS scanning, respectively, in LTQ-Orbitrap.
Thanks,

Hiroshi

Re: Few MS Annotations

Reply #2 –
Hi Hiroshi,

Thanks for the advice. We do have the LTQ scanning the MS2 and the Orbi for MS1. However, in our setup, both the LTQ and Orbitrap are in centroid mode. I've tried various values for the MS2 accuracy and identification parameters, but haven't improved things. Is there a guide or something regarding how to optimize parameters for MS2 level annotation?

Thanks,
Matthew

Re: Few MS Annotations

Reply #3 –
Hi Matthew,

>>Is there a guide or something regarding how to optimize parameters for MS2 level annotation?
Although there is no guide yet, I can help you. Please send me an abf file. And I will check the data.

TIPS:
(1) please check if the adduct type "ammonium formate" or "ammonium acetate" is correct in the identification setting for lipidomics.
(2) check the mass accuracy of MS2. Maybe, 0.25 Da is fine for LTQ detector.
(3) check the retention time setting. Usually, untick "use RT for scoring" and "use RT for filtering candidates".
(4) in identification setting, open advanced option, then, untick "only report the top candidate".
I am not sure if you have already done the above things, but I hope the above information is helpful for users.

Hiroshi

Re: Few MS Annotations

Reply #4 –
Hey Hiroshi,

Thanks for the advice, opening up the MS2 accuracy helped a lot. A separate issue I had is that MS-DIAL seemed to be picking some shoulders and dropping the rest of the peak. Do you have any suggestions for that? For some of these, it looked like MS-DIAL was expecting the peak widths to be shorter than they were, is there a way to change that?

Also, I would have uploaded an .abf in case that would be helpful, but I wasn't sure how to do that.

Thanks,
Matthew