Wednesday, August 11, 2021

Quantitative consequences of carrier channels in p-Tyr and endogenous peptides!


The use of carrier channels to boost peptide signal in techniques like SCoPE-MS are being explored by more than a few groups. There currently appear to be some finite limits to the amount that one can "BOOST" a peptide signal prior to there being consequences. Recently, another large study define the "carrier proteome effect" (paper here) and (video lecture by an author here). 

For a second analysis using a D20 high field Orbitrap (the little ones with the increased field curvature) specifically looking at phosphotyrosine (!!) and MHC peptide (!!!!) you should check out this recent study 

This team carefully looks at the advantages and consequences of different levels of carrier channel in two peptide populations that we'd all love to crank the signal up on.

The math closely matches the results from the Rose et al., study linked above. Which, from a technical level is really interesting because it showcases the similarity in the ion measurement and transfer levels in the Orbitrap Eclipse and Exploris 480. This was historically a headache for fine tuning experiments between the LTQ Orbitraps, Orbitrap Fusions, and Q Exactive systems. 

What we see is that while higher carrier channels in these little tiny high field Orbitraps do end up generating lots more peptide identifications, degradation of quantification becomes obvious above a 200-cell carrier somewhere, with 500 cell carrier looking pretty wonky. I would, however, urge some caution in defining this as the absolute carrier limit for all devices. Talking directly to you reviewer #2. Although the proteomics world has been dominated by Orbitraps, a roughly 2 order of magnitude intrascan dynamic range is a well characterized parameter of these devices. 
Heck, here is the original description of the device. The goal was to get the system to an intrascan dynamic range >10 and it could do that without a C-trap. It needed the curved trap (introduced later) to get to 300. 

These are not, however, the only mass spectrometers and the unique characteristics of having a curved trap to cool and compress ion packets before introducing them into a second trapping device introduces variables that can not be extended to the ions being measured by other devices. It would be silly to try to apply these same limits as universal to, for example, a triple quad device which has no ion gating or trapping at all, and therefore has an "intrascan" linear dynamic range many orders of magnitude larger.  

For a quick overview of how SCoPE-MS looks on an instrument or two that are not D20 high field Orbitraps (I'm of course, well aware that the majority of single cell data in public repositores at this point is on the D30's at Northeastern), you could check out my short (remote) talk at Single Cell Proteomics 2021 next week. 

No comments:

Post a Comment