PROBLEM: observing a loss of signal in the hydrophobic peptides like shown below

Working hypothesis: C18 spin columns might be leaky, and C18 particles can pass through the tip upon centrifugation and end up in your sample. Some of these C18 particles then can end up getting injected into your LC. Because the Vanquish Neo needle seat is a 0.2um filter, these C18 particles get stuck on the needle seat and end up binding peptides from subsequent injections, however these peptides are not eluted during the gradient because the needle seat is not inline during the gradient phase of the run. Note, this can co-occur without needle seat clogging.

Current solution: After using the C18 spin columns (or any C18 cleanup), pass the samples through a filter plate (Millipore MSHVN4510) or filter spin column (Costar 8162) prior to running on these samples on the Vanquish Neo.

Update as of 13 May 2023 Here is the official Thermo document related to best practices and products to avoid needle seat issues:

https://assets.thermofisher.com/TFS-Assets/CMD/brochures/xx-002212-hplc-vanquish-neo-autosampler-blockages-tips-tricks-xx002212-na-en.pdf

Update as of 05 Jan 2024:

We have been running 3 Vanquish Neo systems full time for ~8 months. Every single sample run on these systems is filtered (Millipore MSHVN4510 or Costar 8162) right after resuspension for MS analysis. So far, we have not had to change a single needle seat due to the issues described below, nor have we had to replace any needle seats due to back pressure issues.

November-December 2022

Installed 3 Vanquish Neo systems attached to different instruments (Q-Exactive Plus, Exploris 480, and Lumos)
On 2 of the systems (Exploris 480 and Lumos) we are observing a loss of signal in the hydrophobic peptides like shown below (sample is Pierce HeLa digest cat# 88329)
At first we assumed it was an instrument problem, as this type of signal loss can be indicative of a dirty front source or quad, though usually it not so dramatic in the loss of signal. However, cleaning of the instrument (several times) did not resolve the issue.
Changed the column → issue not resolved
Changed the emitter → issue not resolved
Change the aliquot of QC → issue not resolved
Changed the type of vial the QC aliquot was in (using the amber vials provided with the LC → issue not resolved
Posted looking for help in twitter: https://twitter.com/dlswaney/status/1597062814400532484 and tried nearly all of the suggestions we got including:
- We ran numerous instrument diagnostics (charging test) and calibrations → none resolved the issue.
- Refreshed all LC buffers
- Purged the LC like crazy
- Ran the leak test
- Ran the back pressure test
- Monitored the pressure traces during the gradient, and the lowering of pressure suggested that buffer B was being delivered
Our focus was then on the LC. Either the sample was not being injected properly, or the sample was not being eluted off the column properly. To distinguish between these cases we took one of our n1200 LCs.
- Run sample completely with the n1200 → looks totally normal! Hurray, the mass spec is not the problem.
- Load with Vanquish Neo → separate with n1200
  - Programmed the Vanquish Neo to pickup and load the sample onto the column.
  - Then disconnected the column from the Vanquish Neo and connected it to the n1200LC
  - Had the n1200 LC run a gradient
  - Still observed the loss of hydrophobic peptides. Suggests that Vanquish Neo injection might be the problem
- Load with the n1200 → separate with the Vanquish Neo
  - Programmed the n1200 to pickup the sample and load the sample onto the column.
  - Then disconnected the column from the n1200 and connected it to the Vanquish Neo
  - Had the Vanquish Neo run the gradient
  - Hurray! the sample looks normal. This indicates the problem is the injection from the Vanquish Neo.
Ok, so now what is going on with the Vanquish Neo injection? On some injections (but not consistently) we did notice a high sampler pressure, which can be caused by blockage of the needle seat.
We swapped out the needle seat on one of the LCs were we had observed the loss of hydrophobic peptides and BAM! signal is back to normal!
Upon running more samples we noticed a few specific cases where this loss of signal issue came up soon after running sample prepared with a specific version of our phosphrylation enrichment protocol, in which the last step was desalting using individual C18 spin columns (Ultra Micro Spin Column, Silica C18, cat #SUM SS18V). However, this issue can likely be caused by many C18 cleanup where some beads pass through the filter and into the eluted sample.

Working hypothesis: C18 spin columns might be leaky, and C18 particles can pass through the tip upon centrifugation and end up in your sample. Some of these C18 particles then can end up getting injected into your LC. Because the Vanquish Neo needle seat is a 0.2um filter, these C18 particles get stuck on the needle seat and end up binding peptides from subsequent injections, however these peptides are not eluted during the gradient because the needle seat is not inline during the gradient phase of the run. Note, this can co-occur without needle seat clogging.

Current solution: After using the C18 spin columns (or any C18 cleanup), pass the samples through a filter plate (Millipore MSHVN4510) or filter spin column (Costar 8162) prior to running on these samples on the Vanquish Neo.

Note: We might be totally wrong about why this is happening, but we do know that this solution works.

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