I am performing mass spectrometry on an intact, denatured protein, and I am being thwarted by polyethylene glycol (PEG) contamination. I would like to know where the PEG is coming from so that I can avoid encountering it!
Details: I am looking for a post translational modification that is too labile to assess by bottom-up proteomic methods. Instead, I have opted to look for the PTM in an intact protein. The PTM in question is S-nitrosylation, but this is not relevant to the problem I am facing.
The method commonly employed for measuring intact proteins by mass spec is to obtain a sufficiently pure protein sample, dialyze it into a volatile solvent (ie, 20 mM ammonium acetate) to remove buffers/salts, and dilute the dialysate into acidified methanol to denature the protein. I have used this method in the past, with other proteins, on the same mass spectrometer (an Orbitrap Fusion Tribrid), and I have not had any problems.
Shockingly, the quality of my last few protein preps has been impossible to assess. When I inject the sample on the mass spec (direct injection), all I see is a charge envelope from 500-3000 M/Z with repeating units of 44 Da. This is the mass signature of PEG. Because the instrument is an Orbitrap Fusion Tribrid, the PEG signal is beautiful. But, because PEG competes for the same charge states a protein would, it literally masks any signal the protein could give me.
I know that I have protein. My protein is an enzyme, and it has very robust activity. It is spectroscopically visible at 280nm, and it makes a beautiful single species on a Coomassie-stained gel. The sample I assess by mass spec is ~3-5 micromolar, by the Bradford assay.
I have checked my ammonium acetate and methanol on the mass spec. They do not have any PEG.
I was previously using Triton X 100 detergent to lyse my cells. I worried that Triton detergent may contain PEG, so I left the detergent out. This made no difference! Protein purified in the presence or absence of Triton still has a huge PEG signal.
Thus far, my best lead is that the plastic microcentrifuge tubes I am using (Axygen brand) may be leaching out PEG. The solvent used for injection is 49 % (v/v) water, 47 % (v/v) methanol, 4% (v/v) acetic acid, 10mM ammonium acetate. This solvent is prepared fresh in glass beakers prior to mixing it with the protein, which is performed in a microcentrifuge tube. I tried a "blank," wherein I added the solvent components in a microcentrifuge tube, but without protein. To my horror, when I injected it, there was a PEG signal. Not as strong of a PEG signal as my protein, but definitely PEG, and definitely abundant. Remarkably, if I added these components together in a different brand of microcentrifuge tube (USA Scientific brand), there was no PEG signal. This result suggests that Ayxgen tubes may leach PEG, but it does not conclusively prove that this is the *only* source of PEG.
I am at my wit's end, because I do not understand why PEG contamination was not a problem before, and now, all of a sudden, is.
Could it be my Millipore amicon concentrators? I typically rinse these out once or twice with water before applying my protein to them. i do not know if there is any PEG on these filters. Millipore says the filters contain glycerin (glycerol). But glycerol would not be responsible for the PEG signal... unless the filters also contain PEG in trace amounts. But again, this seems odd, because I used Millipore concentrators with previous protein preps, and they did not give me any trouble before. It's even the same BOX of Millipore concentrators!
Admittedly, we have changed our microcentrifuge tube vendors. We were using Starstedt, and then we switched to Axygen.
My apologies, dear reader. I am sorry for seeming to answer my own question in this post. But honestly, I do not know, and I am wondering if anyone else has experienced this problem before.
The only thing--the ONLY thing--that is different, is I am now looking at a mutant protein. Before, I was looking at the wild-type. That is the only thing that has changed.
Oh, and other people have used the mass spec for the same kinds of experiments, and they have not had any PEG problems. So, the PEG contamination is not intrinsic to the instrument.
Any ideas? I would really appreciate some guidance here! Thank you!
We have systematically checked our lab consumables for contaminants some time ago and found that three out of four membrane filters (0.2 or 0.45 µm, as used for the filtration of HPLC samples) gave rise to PEG and also fatty acid amides in LCMS. Fatty acid amides were also common contaminants of plastic tubes and pipette tips.
PEG contamination could be in the MS system. Did you try to change peek tubes in the ion source? I had a similar problem (in my case doing metabolomics by LC-MS), and I solved it by changing plastic pieces in the ESI sorce that could be contaminated or deteriorated.
Thank you for this suggestion. I do not think it is the tubing though, because solvent alone injected through the same tubing does not result in a PEG signal. HPLC-grade methanol is clean, my ammonium acetate buffer is also clean, only my protein sample gives PEG.
Triton X-100 is a polyoxyethylene-type detergent, so it has the same 44 Da -O-CH2-CH2- repeating unit as PEG. My guess would be that the Triton detergent you used in the earlier purification has contaminated your system.
Hi!
Should be the system. Clean the injector with a solution 1:1:1:1 H2O, Isopropanol, ACN e MeOH (HLPC grade). All bottles first clean with water miliq 5 times for two minutes in sonication and after three times with MeOH and Isopropanol. After take out the column and clear the system.
Inject a standard and see if show the PEG. After a white. If you try all things and not get nothing try subtract the PEG of spectra.
Good luck!
...and use always low binding tubes for your sample preparation ! Good luck !
I think if the solvents did not show a PEG signal it should not be the system itself. I go with the microcentrifuge tubes.
Do other people use the Fusion at the moment with the same injection system?
The system is not contaminated.
Today, we ran several controls:
1.) Solvents alone. There was no PEG signal.
2). Model protein lysozyme in solvents. There was a beautiful protein signal from lysozyme, but no PEG.
3). Model protein lysozyme after being mixed with the buffer I purify my protein in. There was a beautiful lysozyme signal, but no PEG.
4) Model protein lysozyme mixed with my protein recently eluted during a purification. There as a very strong lysozyme signal and a very weak signal from my protein. There was no PEG signal at all.
5) My eluted protein alone. PEG!!!!! And *only* PEG, and *no* protein signal.
So, why is this happening?? Why is there PEG in my eluted protein, but not in lysozyme, even though all of the same glassware was used for both lysozyme and for my eluted protein?
Here is one possibility: For lysozyme treated samples, including the one that included eluted protein (sample 4), buffer exchange took place in 0.5ml amicon concentrators. For eluted protein alone, a 4ml amicon concentrator was used. The EMD Millipore website says that 4ml concentrator caps and liners are composed of polyethylene. In contrast, the 0.5ml concentrators do not have polyethylene. Does anyone think that this polyethylene could be the source of my PEG contamination? Because as far as I can see, this is the only variable that is different.
I don't think polyethylene plastic is the source. I would look to the chromatography materials used in the protein purification.
Thank you, Adam. But if it were the chromatography materials, I would expect to see contamination in my control sample 4 (see above). But control sample 4 is clean.
Try to do this. Do a sample purification using SPE of 100 mg with 40/60 MeOH-Water. The PEG will get out and you will have just your sample. Did you agree Adam?
Best regards
Hi Damien,
I ran into this issue as well and determined the source was coming from my concentrators. Mine are not EMD Millipore however, so I'm thinking this may be a contamination issue with the polyethylsulfone manufacturer. What do you think? Did you find out any more information?
Thanks!
Hi, check this paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949484/
I was having a problem with PEG in some clinical samples - we decided it was most likely coming from lubricant or tubing used to acquire them. I solved it by running the samples into a stacking gel of pre-cast SDS PAGE gels (
BTW, only 10KDa MWCO Millipore amicon concentrators is coated with PEG and not glycerine as stated by Millipore!!
Hi Samir, that's really interesting. Can you provide the literature about Millipore filter coated with PEG? Thanks.
Hi Damien,
I realize this is a very old post, but I am having a similar problem now and I was wondering if you found out what the problem was.
thank you!
I recently had a problem with low molecular weight adducts of my protein when analyzed by intact protein mass spec. The protein had been in contact with a Microcon YM-10 to do buffer exchange. The problem was eliminated simply by running water or buffer through the Microcon before using it with protein.
Mays, I would recommend minimal use of detergents during purification and ensure that common equipment is kept detergent-free (or nearly detergent-free) also. In addition, you should ensure that you have a more or less electrophoretically pure protein species that is abundant enough to visualize by Coomassie. Adam's suggestion of running water through the microcon is also advisable since it will remove most of the water-soluble PEG/PPG.
Article Direct Measurement of S-Nitrosothiols with an Orbitrap Fusio...
This article details multiple strategies to mitigate PEG contamination.
We came to the conclusion that PEG from hand creams used by scientists in the lab was finding its way into samples a couple of years ago...
Damian, did you ever get the reference for the peg contamination in the Millipore filters? If so, will you please pass it on? Thanks.
I am currently having this problem with off-line HILIC. The fractions obtained have a high content of Acetonitrile. The tubes we use are Low Binding SafeSeal microcentrifuge tubes from Sorenson BioScience. Has anyone experienced this and how did you solve it? Thanks in advance
Ignacio, is there any possibility of performing the experiment in-line instead of off-line? Hydrophobic interaction chromatography is a good traditional tool for analytical separation, but if you test fractions by mass spectrometry off-line, you will not know where PEG elutes relative to your protein of interest. I would not predict acetonitrile by itself to be a problem since it is routinely used in LC-MS. If you are concerned that acetonitrile is causing co-elution of PEG with your protein, you could always modify the gradient.
We have systematically checked our lab consumables for contaminants some time ago and found that three out of four membrane filters (0.2 or 0.45 µm, as used for the filtration of HPLC samples) gave rise to PEG and also fatty acid amides in LCMS. Fatty acid amides were also common contaminants of plastic tubes and pipette tips.
I tend to agree with Jandirk. I have went as far as to purchase prepared solutions of ammonium formate to eliminate filtering the eluents. Half hour in the sonicator to degass.
I´m having the same problem , I guess It could be millipore filters because is the only difference with a colleague that follow the same sample treatment, she have no PEG contamination. Otherwise I use vidrio material instead plastic for all the process. I'll thank you if you share any experience with PEG contamination due to millipore filters.
Just found this post after searching the same problem online. For us it was the syringe filter. We recently ordered some hydrophobic PVDF filters from a different brand and since then the PEG signals appeared in every sample...
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