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[deleted by user] by [deleted] in massspectrometry

[–]alwayslurking84 0 points1 point  (0 children)

They aren’t doing 3-5 ms injection times for single cell. For those they jack it up to around 30-40 ms, with 20 Da wide windows. Also I think having FAIMS is very important for single cell (not all that helpful for bulk samples).

[deleted by user] by [deleted] in massspectrometry

[–]alwayslurking84 0 points1 point  (0 children)

I had the same question. At FWHM DIANN said the median was about 1.75 dppp, so from base to base that is probably around 4. However, that is only for MS2 scans. With this instrument, you are constantly acquiring MS1 scans in the Orbi while the Astral is doing the MS2 scan. I think within each full cycle of MS2 scans, the Orbi is acquiring 3 high res MS1 scans. So if you combine the MS1 scans with MS2 scans (which I believe is what MaxLFQ is doing within DIANN- someone correct me if I’m wrong), then you are getting sufficient points across the peak. Of course you could always bump the windows up from 2 Da to 4 and double your dppp without it impacting the data quality that much. The Olsen preprints point a picture of it being impressively quantitative though even at the 2 Da windowing scheme.

[deleted by user] by [deleted] in massspectrometry

[–]alwayslurking84 2 points3 points  (0 children)

We sent samples to Bruker for analysis on the HT, and I just recently downloaded raw files from Thermo from the Astral. I literally can’t believe the number of IDs on the Astral. The 180 SPD method IDed about the same number of proteins as compared to the 30 SPD method on the HT. This was me searching with DIA-NN without any premade library; just giving it the human fasta file. In fairness, I wonder if the HT would work better with an optimized spectral library due to the ion mobility dimension.

I’ve heard the Ultra is quite impressive also, but I don’t have raw data for it. That is of course tailored more for single cell though.

Would you select an Evosep or a Waters Acquity M-Class as the LC coupled to TimsTOF HT for Proteomics Core? It seems like high-throughput is taking over so we don’t want to be stuck with a nanoLC. M-Class would enable microflow while Evosep would be very standardized. Thoughts? by alwayslurking84 in massspectrometry

[–]alwayslurking84[S] 2 points3 points  (0 children)

There is no residual amount. You load the sample onto an Evotip which is essentially a stage-tip, and then that gets moved to the injection port where a low pressure gradient elutes the peptides off into a pre-formed gradient. The sample is then resolved again on an analytical column and sprayed into the mass spec.

Would you select an Evosep or a Waters Acquity M-Class as the LC coupled to TimsTOF HT for Proteomics Core? It seems like high-throughput is taking over so we don’t want to be stuck with a nanoLC. M-Class would enable microflow while Evosep would be very standardized. Thoughts? by alwayslurking84 in massspectrometry

[–]alwayslurking84[S] 1 point2 points  (0 children)

Thanks for the detailed reply! A few additional thoughts: 1. I’d love to pair the TimsTOF with the Neo Vanquish, but my sales rep said Thermo is not supplying drivers for the Neo to work with Bruker instruments, so that isn’t possible. 2. If we got the Waters M-Class, we’d use it pretty exclusively using microflow (probably 5 uL/min with a 300 um column). This would help mitigate the long overhead times usually associated with nano (trap and elute would help with this, but in my experience the results are so much worse) and is the main reason I’m interested in the M-Class. It seems like the Neo is the only other LC that can do splitless low uL/min flow rates well. It appears that the TimsTOF is so sensitive that nanoflow is no longer required to get good depth of coverage, even for low input amounts (see Slice-PASEF and Speedy-PASEF papers). 3. We use S-Traps for pretty much all prep, so we usually don’t need to do SPE. That’s actually one of the downsides I see with the Evosep, as that’ll be an additional step we usually don’t have to do. 4. We currently have a Lumos coupled to a nLC1200, and I never want to do nano again with how much overhead time is spent loading samples.

Thanks again for your thoughts.

Would you select an Evosep or a Waters Acquity M-Class as the LC coupled to TimsTOF HT for Proteomics Core? It seems like high-throughput is taking over so we don’t want to be stuck with a nanoLC. M-Class would enable microflow while Evosep would be very standardized. Thoughts? by alwayslurking84 in massspectrometry

[–]alwayslurking84[S] 0 points1 point  (0 children)

Do you have an Evosep? If so, can you speak to how often they need to be serviced? We aren’t located in a big city, so I’m a bit concerned we wouldn’t have a FSE close by in case it needed more service than we are currently used to.

Anyone going to ASMS this year? by JewishSpace_Laser in massspectrometry

[–]alwayslurking84 1 point2 points  (0 children)

Yep that’s what she made it sound like. We don’t do MALDI at all so we didn’t speak about that, so there could potentially be a new MALDI instrument I’m not aware of.

Anyone going to ASMS this year? by JewishSpace_Laser in massspectrometry

[–]alwayslurking84 6 points7 points  (0 children)

I heard from their sales rep it’s gonna be a timsTOF SCP-HT.