how to remove PEG in my protein sample - (Jul/24/2007 )
Hey,
Recently I encountered a problem with my purchased protein sample. I ordered human cytokine I-309 (10e-06g) from Bachem, and it came with BSA at a ratio of 1:50 in powder. According to people's suggestion, I used Microcon YM-30 to remove BSA (FW: 64KDa) from I-309 (FW: 8KDa) sample, by adding HPLC grade water and centrifuging.
But now the problem comes: when I ran the ESI-MS, I saw lots of contamination peaks of PEG (polyethene glycol) in the mass spectrum, with a repetitive neutral loss of 44. None of I-309 signals was deteced (or they may be buried under PEG peaks).
My doubts are:
1. Where PEG come from? Since I asked the company technician, and they said no PEG added in the sample.
2. How to remove PEG peak efficiently?
3. Why with BSA, I couldn't get good spray in ESI-MS experiment?
Wait for your kind reply. Thanks a lot!!
redapple
Recently I encountered a problem with my purchased protein sample. I ordered human cytokine I-309 (10e-06g) from Bachem, and it came with BSA at a ratio of 1:50 in powder. According to people's suggestion, I used Microcon YM-30 to remove BSA (FW: 64KDa) from I-309 (FW: 8KDa) sample, by adding HPLC grade water and centrifuging.
But now the problem comes: when I ran the ESI-MS, I saw lots of contamination peaks of PEG (polyethene glycol) in the mass spectrum, with a repetitive neutral loss of 44. None of I-309 signals was deteced (or they may be buried under PEG peaks).
My doubts are: 1. Where PEG come from? Since I asked the company technician, and they said no PEG added in the sample.
2. How to remove PEG peak efficiently?
3. Why with BSA, I couldn't get good spray in ESI-MS experiment?
Wait for your kind reply. Thanks a lot!!
redapple
So, I have had the same problem now for a week or so, but I could not, for the life of me, understand where the PEG is coming from, until a few days ago. OK, here is what I did:
1. If you have a syringe pump on your equipment (I am using a ThermoFinnigan LTQ), remove some of your mobile phase buffer and inject it directly into the source. This will see whether your mobile phase is contaminated, your source is contaminated, or your emitter, all of which are easily flushed and replacable.
2. Remove any columns you have and place a line directly from the autosampler (we have a NanoLC 2D from Eksigent), and if you see it, then the autosampler is contaminated.
3. If you see nothing yet, disconnect your HPLC pump outlet and run a line directly from each of the channels to your source. The pump may be the source of the contamination.
Once you find your contamination, try a 40/40/20 mix of 2-propanol/Acetonitrile/water with 0.1% Formic acid and flush this trhough where ever the contamination is found at high volume and pressure settings. This may have to be done multiple times.
I am getting to a point now after 3 days of flushing and purging that the PEG is in the fmol level but I am still seeing it clear as day.
It will take a while because the PEG is so sticky. Also, try to run your sample through a Trap N' Tip from New Objective. BSA is notoriously known for hiding PEG because the protein is so sticky. The only problem is once you run the sample though the column to remove the BSA, the PEG shows up like it was meant to be there. Really a catch-22.
Hope this helps.
John