Hi Antonio,

I am using 0.1 M glycine/HCl buffer with a pH 2.5-3.0 for the elution of proteins in a pulldown experiment. Because some of your eluted proteins could be damaged by low pH, it is better to neutralize your eluate immediately after recovery by addition of 1/10th volume of alkaline buffer such as 1 M Tris/HCl, pH 8.5 to minimize the duration of time in the low-pH condition.

https://tools.lifetechnologies.com/content/sfs/brochures/TR0027-Elution-conditions.pdf

After digestion you can desalt your samples with stage tips and reconstitute your sample in  a MS compatible buffer.

Best,

Murat

Hi Antonio,

I think volatile buffers could work better with the ESI source.

Take a look at the answer to this question:

https://www.researchgate.net/post/What_are_good_volatile_buffers_for_use_in_protein_analysis_by_ESI-IMS-MS

And good luck!

Yes ok, but does glycine-HCl give issues with mass spec?

It will most probably foul your ESI source and mass spectrometer inner parts; then it will probably suppress (most of) your signal.

In case the signal suppression is not that strong, you might not notice it in a first round of analyses, but it can progressively decrease the performance of your instrument and it will require frequent cleaning of both the source and the mass spectrometer.

Once again, I really suggest that you use a volatile buffer or that you add a second column elution with a volatile buffer system.

ok, thank you

Hi again, where do your proteins come from? Interaction partners from a pull-down experiment? then you may need the low pH for the elution of your Proteins of interest.

But like I have mentioned before you can get rid of the glycine-HCL afterwards, by solid phase extraction with reverse phase material and reconstitute your sample then in a ESI compatible buffer like ACN containing 0.1% FA.

I totally agree with Leonardo that the usage of glycine-HCL directly into the MS could harm your MS system.

Best,

Murat

Are you contemplating to infuse this 'sample' directly into mass spectrometer? Directly infusing it for a long time is not a good idea.

What is the objective? Is it simply measuring the MW of one or more proteins? Will your sample have a single protein or is it going to be a mixture of proteins? Assuming it to be a mixture of proteins, then directly infusing even from volatile buffers, may not provide you useful information as the mass spectrum is going to be complicated from the presence of multiply charged ions from each protein.

One option would be to separate the mixture by reverse phase chromatography and generate lc/ms run. In this scenario, the glycine elutes early and can be diverted away from the mass analyzer.

The other option is to analyze the protein mixture by MALDI-TOFMS as it is more tolerant to sample buffers and interpretation of the spectrum is simpler.

I would probably just Zip-Tip (or stage Tip) your sample after elution before putting it through LC-MS.