What could be the source of a broad MS peak of a protein and any ideas how to make it narrow (see attached spectra)? FWHM for the peak on the 1st picture is in the range of 30-40 Da, and resolution is about 2000. It's a recombinant (expressed in E.coli) protein, MW in the 50-90 kDa range (cannot be disclosed). No PTMs are expected. No more structural info is available. The protein sample was originally in a buffer containing Tris-HCl, 150 mM NaCl and TCEP, pH 7.5. Mobile phase was water/ACN/0.1% FA; C4 column; Agilent qTOF MS detector. Long-time and shallow gradient revealed adducts of approx +22 Da (shown on the 2nd spectrum), suggesting Na+ adducts, yet desalting did not help. Column heating, TFA addition and changing various TOF parameters caused no effect on the MS peak width.
Any suggestions would be highly appreciated.
Hi Anna,
It indeed seems that your analysis is hampered by residual (Na) salts. Were the declustering/desolvation potential parameters amongst those your tried to optimize? Or the in-source collision energy?
Or you could consider exchanging the protein to an ammonium-based (volatile) buffer, e.g. by dialysis?
I didn't see information regarding the concentration of your protein in your question, how high is it? You also write that your NaCl is 150 mM, that can already be too much.
ESI is already sensitive at very low concentrations. My former labmate Mo Winghart used concentrations around 1mM e.g. here:
Article Time-resolved photoelectron spectroscopy of a dinuclear Pt(I...
That's already enough to get a good ESI signal, otherwise you might generate adducts that decompose randomly or also your ESI needle might get partially jammed and start producing weird things.
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