Feb 01, 2022
pasefRiQ_V1
- lcmsmethods 1,
- Yuting Yuan1,
- Namandje' N Bumpus1
- 1The Johns Hopkins University School of Medicine
- LCMSMethods.org Version 2
Protocol Citation: lcmsmethods , 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
Keywords: overall increase in peptide identification, methods for reporter ion quantification, loss in unique protein identification, reporter ion quantification, tmtpro reagent, peptide identification, unique protein identification, quantified protein, k0 of peptide, highest possible coverage of each protein, proteome standard, minute pasefriq experiment, more peptide, iterative analysis of the tmt, peptide, tmt, tmt6, protein, ion mobility ramp
Funders Acknowledgements:
NIH
Grant ID: R01AG064908
NIH
Grant ID: R01GM103853
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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.
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