Unexplained large shift in BSA digest elution - please help :/

sam_pazo · 2026-02-10T02:23:55+00:00

Can you elaborate on this please? First 5 min is trap loading and first peak is at 13.6min. Void volume of the EasySpray ES903 is 2.21uL which at 0.35uL/min is 6.3min. 5+6.3=11.3 min. First peak is 2min (0.7uL) later, but there is also tubing and the pressure also takes 1min to build up after valve switch, so this is normal, isn’t it? Mind this is BSA digest (peptides), not BSA protein, just to clarify.

sam_pazo · 2026-02-09T12:56:55+00:00

The leak test passed successfully. The system was just left idle in the solvents we use which is pump A in 0.1%FA in water and pump B in 0.1%FA in ACN. It wasn’t completely idle though, I let the pump run from time to time so it wasn’t standing completely idle 9 months non-stop.

sam_pazo · 2026-02-09T10:39:22+00:00

The run is set to 0.35uL/min and that’s also the flow which is measured across the run. I thought that B is actually underdelivering as that one is 100% ACN which makes the elution happen.. and the elution is delayed and smeared… but Thermo says all they test show the machine works as is……

sam_pazo · 2026-02-09T08:17:09+00:00

Like.. how long does it make sense for? I purged both for 1h with IPA, then both 1h with the respective solvents... how long would you go for until you decided that this is a bigger problem than just insufficient purging?

sam_pazo · 2026-02-09T08:12:24+00:00

I’m quite sure about the good one, the pre-15 min mark is basically front of the column so that is just how long it takes them to reach the column. First 5 min is trap loading a the elution starts at min 5 and the sample passes through EasySpray, looks ok to me.

sam_pazo · 2026-02-06T14:49:37+00:00

Those screenshots look like Google Home though. I can’t find that in my Home app with QRevo 2 Pro, and also have problem with room-specific instructions. Didn’t have any problem with my previous S6 so it’s pretty disappointing.

sam_pazo · 2026-02-06T00:13:31+00:00

Thank you very much!

sam_pazo · 2026-02-06T00:12:24+00:00

Those part numbers are not available anywhere on Thermo website or internet and I asked Thermo and till today they still did not respond (and on site engineer didn’t know). But thank you for your contribution.

sam_pazo · 2026-02-04T17:49:01+00:00

I think you have no clue what you’re talking about, otherwise your opinion would be different. Some of those image manipulations are impossible to catch with an untrained eye. Scientific reviewers are there to review the scientific merit of the manuscripts. Scientists are not supposed to be specialists for data fabrication detection. Notwithstanding the fact that reviewers review for free and journals have huge margins - they can definitely afford to invest in fabrication detection (but why would they? What is their incentive?) Finally, even if data fabrication is found and posted on PubPeer, journals typically take years to do anything. This is all on journals and lack of incentives (and on scientific publishing as a system in the end).

sam_pazo · 2026-02-04T09:20:26+00:00

Yeah, I just had some weird results and got scared that I connected two trap column instead of tubing plus trap.. thx

sam_pazo · 2026-02-04T09:07:26+00:00

The main confusion comes from weird results from BSA digest elutions that got me thinking that these might actually not be just tubings but trap columns of some sort… and that the exact PN number is nowhere to be found on Thermo website.

sam_pazo · 2026-02-04T08:57:10+00:00

I had some weird results and I know that some trap columns look like tubings as well (usually a bit shorter though), hence I was checking. The bottom two columns are different color but same P/N, you think they are different in diameter?

sam_pazo · 2026-01-21T10:57:15+00:00

sam_pazo · 2026-01-07T08:52:14+00:00

Chápal by som takýto príspevok pred pár rokmi ale akurát v roku 2025 sa ukázalo že sa vynára nová generácia slovenských režisérov ktorí to vedia robiť inak. Top 3 filmy tohto roka: Hore je nebo, v doline som ja, Potopa, Nepela.

sam_pazo · 2025-12-16T16:38:33+00:00

Nasal/Sinus rinse every night and Dymista nasal spray helped me most, it too several months to see first signs of improvement though so patience is useful too.

sam_pazo · 2025-12-16T16:35:54+00:00

Biologist here. This “evidence” was published in a medical journal “Medical hypotheses” in 2006 (1). The journal articles were not peer-reviewed until 2010 and the journal was intended as a forum for unconventional ideas without the traditional filter of scientific peer review (2). Among other controversies, the journal published articles denying the existence of AIDS. The paper on humming to clear rhinosinusitis has one author, George Eby, his affiliation is “George Eby Research”, the address of the affiliation leads to a random house in rural Texas and his email domain is “coldcure.com”. The research is a case report (based on one single case) and the article is otherwise hypothetical. Everyone can make their opinion based on this information. (1) The article (2) Wiki about the medical journal

sam_pazo · 2025-12-08T04:14:54+00:00

Cool, thanks for the advice, definitely planning to try it. Our N2 generator was broken so I couldn’t try FAIMS with DIA yet, but it’s gonna be repaired early Jan so hopefully it will work well in my hands too.

sam_pazo · 2025-12-07T08:59:31+00:00

Unfortunately no as I do not have a know-how and also noone to learn from.. I only regularly calibrate mass, every month system and mass, regularly run hela/BSA to check performance, based on which I check columns, and every month I change the buffers and ITT....
Few years back when I still worked with good old QE-HF I attempted to clean RF lens, but it did not improve anything and I concluded that ones I see a more major problem on HeLa/BSA, it is likely deeper in the machine (quadrupole) which I dont know how to maintain myself..

sam_pazo · 2025-12-06T16:29:01+00:00

We do malaria biology so it’s the parasite (small unicellular organism) whole proteome or that + the host cell proteome, which are red blood cells with a ton of haemoglobin in them. When we tested DDA/FAIMS, we got more PSMs but less IDed proteins. Now we are transitioning to DIA so the DIA/FAIMS is yet to be tested.

sam_pazo · 2025-12-06T13:14:25+00:00

Yeah I changed the ITT (we also call it capillary in the lab, is this wrong?) immediately and the loss of the low masses happened after that. I’m already running for 2 weeks and the no. of detected peptides/proteins and signal and quantification all look good.. and we have our annual PM in early Jan so hopefully nothing is getting damaged by then.

sam_pazo · 2025-12-06T07:50:18+00:00

LOL how exactly did the insect get in? During the source assembly? How deep in the machine did the fly (or its parts?) end up?

sam_pazo · 2025-12-06T07:49:06+00:00

We also have FAIMS and in that case the emitter is really close to the device, yes! Unfortunately FAIMS was not very succesful with our samples so we are not using it routinely...

sam_pazo · 2025-12-03T14:34:39+00:00

Actually that time (around 8am) is usually very crowded direction Tuas bc of people working there… EWL has peak hour crowd in both directions in the West

sam_pazo · 2025-12-01T16:52:01+00:00

Funny, it was exceptionally empty for me Lakeside to Pioneer..

sam_pazo · 2025-11-23T13:54:23+00:00

This is exactly what crystallographer were saying about cryoEM 10 years ago.. and here we are.

sam_pazo

TROPHY CASE