My first thought is that you have insoluble material in your final sample. After your final desalting step, do you centrifuge your samples? And I mean 10 min at 18,000 g as a minimum. If not, give this a go. I'd suggest you transfer the supernatant to a fresh tube even if you can't see a pellet. Just avoid the part of the tube where the pellet would be found when transferring the sample.

If you do centrifuge your samples prior to loading, I'd guess you are overloading your column and it's possible the sample is precipitating on the column, hence blocking it. I'd suggest the first scenario is more likely.

Finally, do you use a pre-column? Do you reverse the flow through the pre-column when loading onto your analytical column? This all assumes you are performing nanospray MS. If not,forget I asked.

Thank you Peter! We do use nanospray, I am currently using the Q Exactive nanospay with a 50 cm viper column.  I do not spin my samples after the desalt, we usually proceed directly to speedvac and then resuspend in 0.01% FA in HPLC grade water at 1mg/ml.  I am loading 5ug on the column over 3 hours and the column seems to clog after 2 samples.  I don't have this problem with any cell lines or primary animal cells, only human.  I figured the sodium deoxycholate would solve any lipid/membrane issues.  I will try spinning and see if that helps.  Thanks for the tip!   

I find this kind of thing myself on occasions. I only start to have problems when I get to the more concentrated samples. Given it happens with liver, it could well be a lipid issue. ie. despite your best efforts, some lipid is still present in the sample.

There are two ways you can deal with this. Once the sample is desalted make up the sample in 1% (v/v) formic acid and spin for 15 min at 18,000g. The 1% acid precipitates the lipid, but the vast majority of your peptides should solubilise. Collect the supernatant, speed-vac to a volume you are happy with and load. Or speed-vac dry, resuspend in whatever you usually use and load.

Another thing worth considering is Lipid Removal Agent from Sigma (cat No: 13358). I use this at 0.1g/mL lysis buffer with rat brain. You'll need to determine what ratio works best for your sample. Add this to your lysis buffer, incubate for ~30 min with mixing, then centrifuge it out. It doesn't get any easier than that.

Yes, some proteins will bind to this, but it really does wonders in removing lipid. You'll need to check if the protein loss is acceptable in your case, if you go this way.

Thank you, I'll let you know if it works 

What you want to see is protein or pepties or some small drug molecule (less than 1000 amu)?  I am a residue chemist (less than 1000 amu). I don't see you have steps to precipitate protein (if you are looking for a small molecule). Protein can be binded on the column and block the column.  If you are looking for the protein or peptides, please ignore my suggestion.

Thank you Narong, I am looking for peptide/proteins.

Depending on what proteins are causing the clog and which ones you are interested you can use a molecular weight cut-off spin filter to remove insoluble garbage. I've used Pall 10,000 kDa spin filters to prep samples before HPLC and LCMS but there are options with a larger cut-off if your peptide/protein is larger.

If you can't remove the clogging agents this way you can (and should in either case) have both an in-line filter and a guard column on your machine up-stream of the column. The filter removes insolubles larger than 0.22 um and the guard column acts a a sacrifice for anything that gets by the filter that would otherwise clog your column. This way you can throw away these relatively cheap parts instead of your column.

Hi Crystina

You don't give too much detail about what you are actually doing or what you are looking for. How are you separating your cytosolic and nuclear fractions? Are the membrane proteins from plasma membranes or intracellular membranes and how are you isolating these?

You could be precipitating the proteins of interest on the column if your mobile phase does not contain sufficient detergent or chaotropic agent to maintain solubility, both of which are not great for the MS system as they will contaminate your source and first quadrupole.

The blockage could be from excess lipid that can be removed by a number of methods of which the easiest is using adsorbent materials like LRA and mentioned above by Peter. This does however also take out highly lipophilic proteins (like membrane proteins which you seem to want to assay). Solvent extraction is more tedious but tends to leave more of the proteins.

The one component not mentioned so far is the cellular nucleic acid burden. These can be a nuisance, adding to the viscosity of the sample and causing all kinds of strange chromatographic effects that can only be eliminated by removal prior to chromatography. By using polyethyleneimine (0.1% (w/v))  or protamine sulphate (1% (w/v)) followed by high g centrifugation or use DNAse 1 at 1 ug/ml incubating for 10 - 20 minutes that cuts the DNA into subunits but does not remove these from the sample.

You should be using an online filter and or a guard column especially if using poorly processed samples as the analytical columns are not cheap.

Many things to think about.

Good luck

Thank you Duncan I will keep these in mind.  We do use a guard and filter.  The samples run ok on the Orbi but the nano-column just gets junked up.  I am trying a lipid removal technique first to see if this helps.  I have large amounts of lipids in both the cytoplasmic and nuclear fractions and my best guess is that this is the reason for my trouble.