Hi all,
I am doing a series of large-scale experiments to extract HLA class I peptides. After the desalting step, I elute the peptides with 50% ACN instead of 30% like most people do. The idea is that some important target peptides even though at small sizes, they are highly hydrophobic, hence, 30% ACN might not be enough to recover these peptides. But at 50% ACN, I might risk recovering bigger molecules like B2M along with the peptides.
I had tried the MCWO filter but this led to a huge sample loss (at least 30% peptides).
At the moment, I have to use a trap column (cat no. 164705, thermo) to avoid clogging our analytical column (ES902, thermo.), I did a few test runs with Hela before running my samples.
The problem is a huge reduction in signal intensity and much fewer protein IDs when using a trap column. I have never set up a trap column before. So, I only used a method that was already in our system. Could anyone please help with how to set up a trap column? And is this column a good choice for my analytical column?
Thank you so much.
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