...iHUPO single cell initiative TIMSTOF Ultra2 methods don't seem to perform super well in my hands.....

 

I'm back at this great iHUPO single cell initiative paper again, I guess. Sorry if you aren't interested, but it's TIMSTOF Ultra2 vs Astral on standards and real single cells, so I'm interested. 

Another thing that caught my interest was how very different the instrument method on the TTU2 is compared to the basically default instrument method we have ran for single cells for about 18 months.

Above you can see our method on the left and the method from the iHUPO paper on the right. The format is a little different since I had to cut the darned table out of a PDF. Which is always a joy. 

But you'll see that we use 8 cycles and we run from something like 0.65 to 1.4 and the iHUPO method runs 5 cycles and from 0.65-1.3. 

I feel like we tried a ton of different methods and ramp times and finally ended up with basically the default method plus 50% more ramp time worked better for everything in our hands. But we optimized on mouse hepatocytes last year, but those are kind of big and also kind of friendly (there are like 550 proteins that you can detect even if the instrument is vastly underperforming, because they make up about 90% of the total protein content. Compare that to human plasma where 1 protein makes up about 90% and you'll see what I mean).

We run a longer ramp time which gives us a full ramp at a speed of 6.37 Hz according to the software, so each precursor gets measured over 6 times/second, right? 

The iHUPO method is faster and has smarter looking windows 

It gets something in the 9.34 Hz and uses bigger windows at the low and high end of the mass range. 

I was super pumped to run something that looks so good on paper, and....it's kind of meh....

To be fair, this is just K562 standard at 200pg. BOOOOOORRRRING, but we get a steep drop off when we go from 40SPD to 80SPD on the EvoSep One. And I've never even tried 120SPD until now. Maybe with a 50% faster method it makes sense? 

This is a terrible way to display data, but I've got like 15 free minutes on a Sunday night to type this up. 

There are only 2 replicates of the 80SPD for each. EvoTips are expensive, yo. But we run the 80SPD standard method all the time. It's...like...our standard method.... and these results look normal. 

At 80SPD the iHUPO method drops off nearly 4k precursors and 450 protein groups? Ouch. 

I ran more of the remaining tips on the instrument in 120SPD because we don't have benchmarks for this and it's not ...as...bad....but ouch. It's still totally worse. 

Obviously, we're using an EvoSep and they used a funny preformed gradient on a Vanquish Neo. And quantitative accuracy measures more than protein numbers. 

BTW, this is just command line DIA-NN running at the end of each file. Nothing special. No MBR, no whatever. 

But if you're looking at this paper and thinking WHOA, the Astral gets better data than the TIMSTOF, I'd take it with a grain of salt. This doesn't look like the best method to me, and instrument comparisons are dumb anyway.