Symptom: Early eluting peptides are missing (based on monitoring PepCalMix LC-MS test or observing a change in the shape of the proteome TIC).
Test: Use PepCalMix LC-MS test to monitor binding of early eluting peptides. There are a number of very hydrophilic peptides in the PepCalMix which are great for testing the quality of trapping for your LC-MS experiment (IGNEQGVSR – 485.253 m/z, LVGTPAEER – 491.2656 m/z, AETSELHTSLK – 408.5501 m/z).
- Ensure a few conditioning injections are done on a new trap and column, to reduce any non-specific binding
- Ensure the loading buffer is 100% water with 0.1% formic acid
- Do not use DMSO in loading buffer, we have found that this can sometimes reduce binding of hydrophilic peptides on the trap. If you are using DMSO, you just need to have it in the analytical gradient buffer.
- Ensure the trap flow rate is as defined in SOP, flowing at too high a flow rate can cause isocratic elution or break-through of weak binding peptides
- Install a new trap and condition it, then repeat the PepCalMix LC-MS test
- If there is still an issue with trapping hydrophilic peptides, contact your service representative