Feb 01, 2022
pasefRiQ_V1
- lcmsmethods orsburn1,
- Yuting Yuan1,
- Namandje' N Bumpus1
- 1The Johns Hopkins University School of Medicine
- LCMSMethods.org Version 2
Protocol Citation: lcmsmethods orsburn, Yuting Yuan, Namandje' N Bumpus 2022. pasefRiQ_V1. protocols.io https://dx.doi.org/10.17504/protocols.io.b4ifqubn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 01, 2022
Last Modified: February 01, 2022
Protocol Integer ID: 57639
Funders Acknowledgements:
NIH
Grant ID: R01AG064908
NIH
Grant ID: R01GM103853
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
We have developed optimized methods for reporter ion quantification on a TIMSTOF Flex (TIMS B configuration) by iterative analysis of the TMT triple knock out yeast standard and an in-house developed two-proteome standard. For the greatest reduction in background coisolation interference in the TMT6 and TMT10/11 plex standards, 10 ion mobility ramps from 0.8 to 1.3 1/k0 provides the greatest reduction in interference on 60 minute LC gradients when using a 25cm IonOpticks aurora column. Reducing the number of ramps to 5 leads to an overall increase in peptide identifications with a loss in unique protein identifications. It is worth noting that the distribution of 1/k0 of peptides labeled with the TMT6/10/11plex and TMTPro reagents have somewhat different overall distibutions. For the maximum intrascan linear dynamic range. For 30 minute pasefRiQ experiments, 10 ramps at a 2.2 second cycle time has a detrimental effect on the overall identification of PSMs, peptides and proteins.
For the highest possible coverage of each protein identified with the TMTPro reagents on 30 minute gradients we recommend 5 ramps using a 1/k0 range of 0.7 to 1.4.
Typical results for pasefRiQ using a two proteome standard as described is 3,600 quantified proteins in 60 minutes with a 240ng total load on column.
At 24ng load on column, this same method with no alterations will return approximately 2,100 quantified proteins. Increasing the multiplier voltage with the high sensitivity model will result in an approximately 15% more peptides and proteins identified at this load.
Attachments
