Friday, December 8, 2023't use acetic acid if you are using a trap column!


Since I've been even more short-handed than usual that last month, I've had to get some weekend time in the lab around grants and hunting a permanent position. Funny story that is probably obvious -- on a PepMap trap, 0.5% acetic acid causes an impressive lost of the more hydrophilic peptides! 

Actually, what I'm talking about is this original work

and my work that suports these findings

While it might seem crazy to be even trying to use a trap for ultra-low input or single cell proteomics, Claudia Ctortecka has shown some great data in recent talks that having a higher volume sample pickup + trap improves the reproducibility from sample to sample (vs trying to pick up half a microliter or whatever your autosampler can do). As crazy as that sounds (sample loss by trap, right??) on a normal 0.1% formic acid EasyNLC pepmap setup, it seems to work in my hands as well -- 

Till you wash that trap with 0.5% acetic acid.... (spectronaut presursors shown per cell/library free/30 min LC method at 300nL/min) 

I tell you what - Microsoft has the clear advantage of the Snipping tool for this kind of stuff. What I'd like to do is circle all the cells on the left side of that thing above and write "formic acid" on them. There is a gap in the middle where there are two empty cells - those are method blank controls, and then the right is the acetic acid trapped cells. The next to last one doesn't look that bad.

I already tried a different plate of cells just in case there was a bad plate, but it looks pretty consistent that the acetic acid trapped cells are missing a lot of stuff in the early elution range. 

Screenshot grapped from DA - the green is the acetic acid trapped peptides - later in the signal intensity looks really similar in this run, but look at how much less complex it looks vs the one formic acid run in purple. 

Weird, right? 

1 comment:

  1. Hi Ben, interesting data. I think the loss of hydrophilic peptides should not be that dramatic ~0.7 % ACN reduction in hydrophobicity is ~the same 0.7-0.8% in the number of detectable peptides. It would be nice to compare hydrophobicity of your detected peptides at both FA and AA and see, which population is missing. Another question here is the consistency of your LC / eluent system: the major peak on chromatogram shifted right under AA conditions, plus all peaks appeared to be significantly wider...this is very unlikely with the system working properly - retention should be lower.
    Thanks! Oleg