Monday, March 25, 2013

Excessive carry-over in your LC-MS system?

This isn't news, just another random topic for discussion (monologue).  Here is the question, though:  how do you know if you are having excessive carryover in your LC-MS analysis?  There are lots of ways to check this, but this is what I do:  I run lots of blanks and I process almost all of them.

Expanding:  in between samples of importance, or in-between quantitative runs without internal controls (label-free or SRM or whatever) I inject a normal size sample load (2-10uL, depending on the LC in question).  I then process that sample using an appropriate database through my normal processing scheme.  Since I almost exclusively work with human samples these days, I simply use my Sapiens Uniprot FASTA (or IPI Human) with the cRAP database either tacked onto the end or searched in parallel.  This gives me a pretty good metric of how clean my sample is.  I don't freak out when I see some peptides.  I only freak out when I see a lot of peptides.  These instruments are so sensitive that they are going to find peptides floating in the air or reasonably fresh buffer.  They shouldn't, however, find dozens of peptides.

If I do find enough peptides to freak out, these are my steps:
Clean the front of the mass spec.  Capillary and front plate with 50% methanol should do it.  If that doesn't help, it is time to approach the LC system.  The order varies from person to person, but this is how I approach it:

1) Change my blank
2) Change my wash solvents
3) Change my running buffers
4) Run high solvent for a long period of time (a few hours to overnight, if I could possibly afford it.  Extremely rare that I could or can)
5) If none of these help, I try injecting something harsh (a full sample loop of 20% isopropanol):  Note:  This may not be appropriate for all nanoLC systems.  I don't think I've ever used bold print in all the years I've been writing in this silly blog.  But I don't want you damaging your LC system.  I don't know a lot about LCs, just enough to successfully get by and to know that I've never noticeably damaged the LC systems that I have had.  That doesn't mean that you should trust me on this one.  When in doubt, consult the manufacturer.
6) If this doesn't help, it is time to approach the guard column (if employed) and finally the analytical column.

End of tirade.

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