453 and 679 m/z peaks on every run

[RochesterProteomics]

Hello everyone. First post.

We are getting 453 and 679 m/z peaks on every single run using our Thermo LTQ trap. I have done research and noticed that it could be a cyclic oligomer of polyamide 66, which is present in membrane filter disks. However, we do not use filter disks, and use no other type of nylon throughout our digestion or our LC-MS runs. I have run blanks after some of the runs to see if the two peaks are still present and they are not. This makes me think it is nothing within the system, but instead something that is occuring in every single sample. PEG also occurs at these masses, but we always use LoBind tubes that are made of PPG and tend to leak plasticisers far less than standard tubes. If anyone has any ideas on what this could be that would be great. Thanks a lot.

PS: We pack our own columns using C18 to 5 cm in 75 um ID fused silica if this makes any difference.

[Kostas Petritis]

You might have to describe all your sample preparation and vials used. It might something like use of acetonitrile with eppendorf tubes or similar (do you do SPE)?

Were the blank runs just water or did you use water and did the whole prep? Are you sure that all the reagents used are clean?

[Pepter]

http://www.waters.com/webassets/cms/sup ... r_list.pdf

Maybe this will help a little.

[bhuvfe]

Do you see a trend in these contaminats? Or their concentration is constant in every run?

When you run the blanks did you use the same solvent as in your samples?

If you change column do you still get these masses?

[RochesterProteomics]

Thanks for the replies. I will try to detail my experiments better in the future.

We do standard tryptic digests, both in gel and in solution, using 50 mM ammonium bicarbonate, reduce and alkylate using DTT and IAA, and then quench the trypsin reaction using formic acid to .1%. All of this is done exclusively in LoBind tubes from eppendorf. After digestion is complete I dry down the samples and bring them up in 95/5 water/acn with .1% formic acid. I then pack ~5-10% of the sample directly on a tip using a pressure bomb. This greatly reduces the amount of carry over from previous runs because the sample is not being injected into the sample loop. We have been getting great results, except for the very large contaminant peaks.

Since we pull and pack our own columns, and it is very inexpensive, we use a new column for every sample to ensure the greatest sample integrity possible. The 453 and 679 are by far the 2 biggest peaks in every run, sometimes baselining other peaks that otherwise would be considered quite large. Also, the concentration of the 2 contaminant peaks holds relatively steady for each sample run.

When I run a blank after a sample run using the same column, the run is completely blank, with no peaks showing up. The method I use for the blanks is the exact same method I use for the samples so I can see if the peaks still come out. This is why I believe it must be something in my sample and not the LC solvents.

Also, when I was troubleshooting the problem, I packed a C18 column with no sample packed on it and ran my method to check if it could possibly be a contaminant within the C18. However, this came back completely blank. I also packed 95/5 water/acn with .1% formic acid on the tip and ran that to see if it was a contaminant within the buffer I use to reconstitute my sample. Again, this came back completely blank.

All of this leads me to believe it must be something that happens while digesting, but I cannot figure out what it could possibly be, since we do not use nylon at all in our digestion protocol.

I hope this helps describe my problem a bit more. If you have any more questions please post them and I will try to give you more details. Thanks a lot for the responses.

[JMB]

Finding the source of a contaminant is easier if you can ID it.
It looks like you have

[M+H]+ m/z 227 (unseen, below scan range), [2M+H]+ m/z 453 and [3M+H]+ m/z 679

where MW 226 Da.

"in-source" CID on m/z 453 should take you to m/z 227, then MS/MS on 227 should initially eliminate CO to give m/z 199 if you have the cyclic mono-oligomer from nylon 6,6.

Accurate mass on m/z 453 would help to confirm/deny the 6,6 oligomer theory.

JMB

[yangz00g]

I suspect two things based on your description:

1) something from the digesting/reduicng reagents, or
2) the samples carry sth out of the column, while water, mobile phase only don't