H-T Lau

I think reviewers are right to ask for Western blot to confirm the change in protein abundance now

Is this a Seer competitor?

Sadly Sciex doesn’t even want Skyline support

Has anyone tried CDMS/Stori analysis on the QE classic? They said that it can be done on all Orbitrap instruments. Is that true?

This is very nice. Thank you. I started with MaxQuant and always use narrow tolerance to search.

Thank you!

Can you explain this? Are you guys talking about instrument settings or search settings?

How many peaks across peak can you get from this kind of runs? I need to have a look at the data…

I found it interesting that you were very kind when interview Nautilus’s founder. Perhaps the interview was not about their technology.

I think people will move away from mass spec with these platforms promising they can “quantify” thousands of protein in every run/sample.

I always wonder about this. Like those spatial proteomics platforms. Do they provide information saying they have verified every antibodies? I always feel suspicious about aptamers, probably because I liked the GDF11 story in 2010s so much and it turned out to be not reproducible…

MS vendor - I don’t think so.
WhateverScan - Maybe?

They used to only have 3. I understand why your mind blown now. 🤯

You should test those that you are interested in for different applications. I think they are cheap and last long enough (I stagetip my samples). Let me give you a email to get quote when I get back to work tomorrow.

I started with the 25 cm 75 mm ID and a 60 min run, then shortened to a 30 min gradient. I think I lost ~20% peptide counts, but not that much on protein. Then I talked to Bruker about fast gradient, they suggested me to use the 15x150. I think there isn’t much change in ID if I remember correctly

@proteomicsmarburg.bsky.social I have not tested them extensively. I mainly run whole cell lysates and I found them giving me similar peptide IDs. So I mainly use the 150um x 15cm for faster runs.

I am still on this problem and Thermo is not fixing it for me. What a nightmare.

Have not tested nanoflow, but I think it will be worse.

2/ Peptides are eluting at different time because the path in the Flex is much longer. With 8 injections, the peak area CV centered at 5%. From the Neo, %CV centered at 15%. I am going to call Thermo.

1/ OK, I am convinced that it is the Neo. I tracked the same enolase peptides using Skyline. All of those have good peak shape so integration is not an issue. Use the same PepMap 1 mm ID on both the Vanquish Flex and Neo, 50 uL/min at 40oC. Same gradient.

I am doing more tests now. Digested some a protein, run it with a Vanquish Flex with Waters CSH column. The peptide peak areas are 5-6% CV. So I think I can rule out the mass spec at high flow condition.

Will do more tests and update.

It is going up and down. I did 12 runs and there isn’t really a trend. The patterns of the peptides are also different. Like the first 5 can go up but the rest can go the other way…

No. It is going up and down

I injected from the same vial too.

I did a 7x eq volume. Direct injection, so no trap. I also started with the trap, but switched to direct injection because the intensity is much higher in direct injection.

I use Waters total recovery vial. The standard is Pierce PRTC 5 pmol/uL. I diluted 1000x and injected 10 uL, so 50 fmol.

TimsTOF people, if you inject a peptide standard for 5 times, what would your peak area %cv be?
I have been getting >20% for some peaks. Can it be the MS or it has to be the LC? The RT is reproducible.

We have a Neo, running a 10 cm 150 ID Pepsep at 300 nL/min, inject 10 uL.

One-Tip enables comprehensive proteome coverage in minimal cells and single zygotes bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Will this work?

Mass spectrometrists should search only for peptides they care about - PubMed Analysis pipelines that assign peptides to shotgun proteomics mass spectra often discard identified spectra deemed irrelevant to the scientific hypothesis being tested. To improve statistical power, I...

I still wonder why we search for contaminants in DDA. Doesn’t it add stuff that we don’t care about?

There is really no support. I often want to tell new user to use Fragpipe, but also don’t feel right to turn people away in their group…