nano LC headache

[johanski]

Hello,

I'm somewhat of a newbie to mass spec, and I'm running into a bit of a problem w/ nano LC. More specifically, we're running peptide digests from control proteins through it (BSA/cyt C), and notice that nearly all of the peptides that are coming off are doing so at above 70% acetonitrile. Consequently, we're not getting the data we want.

I've replaced the C18 peptide trap, and am using a 15cm, 77µm ID fused silica capillary (hand packed) column.

We know that our sample quality/quantity is not an issue, as we've had some excellent spectra from MALDI using the same samples.

That being said, does anyone have an idea as to what I might be doing wrong?

Any help would be greatly appreciated!

Thanks