Chromatography Forum

I'm looking at four hexapeptides. Crystallized monomers are in a dH20 solution (where they self aggregate) and are analyzed using ESI-MS (Finnigan LTQ, Thermo-Scientific). I first look at the full mass spectrum and then to look at each oligomer I use a function of the LTQ software called ultrazoom which is basically a high resolution scan of a small specified range.

The lower oligomers display very distinct peaks, but at the higher oligomers every other m/z complex seems to display this very odd round hill type shape



I don't quite understand what is happening. The only thing I can think of is that there are mobile protons so each peak is less distinct.

I don't even know how to search for this phenomenon in the literature as I don't know what it's called

Would really appreciate some help pointing me in the right direction

what you are seeing are multiply charged peaks, not the high- and low- oligomers. You can definitely deduce it because the difference between the peaks is less than 1 AMU. On the high mass peaks, the individual charge states are still resolved, giving the "spiky" appearance, while at the low end (highly charged peptides) the difference in m/z between the charge states is so small that it cant' be resolved by tour instruments.

A quick look hints to me that the top figure represents a charge state of +3. That means the peaks you are seeing are the isotope pattern of [M + 3H]3+ , which correspond to a peptide of 5455 Da. The figure at the bottom is harder to interpret. According to the mass, it could be the +4 peak (that means [M+4H]4+) of the same peptide. However, given the resolution shown in the picture above, I would expect to see the individual isotopic peaks resolved (you would need to fit 4 in the same space where you fit 3 , they would not be baseline resolved but still distinguishable). This makes me think that the bottom picture is a higher charged state of a heavier peptide


primer on topic

https://masspec.scripps.edu/publication ... 78_art.pdf

what you are seeing are multiply charged peaks, not the high- and low- oligomers. You can definitely deduce it because the difference between the peaks is less than 1 AMU. On the high mass peaks, the individual charge states are still resolved, giving the "spiky" appearance, while at the low end (highly charged peptides) the difference in m/z between the charge states is so small that it cant' be resolved by tour instruments.

A quick look hints to me that the top figure represents a charge state of +3. That means the peaks you are seeing are the isotope pattern of [M + 3H]3+ , which correspond to a peptide of 5455 Da. The figure at the bottom is harder to interpret. According to the mass, it could be the +4 peak (that means [M+4H]4+) of the same peptide. However, given the resolution shown in the picture above, I would expect to see the individual isotopic peaks resolved (you would need to fit 4 in the same space where you fit 3 , they would not be baseline resolved but still distinguishable). This makes me think that the bottom picture is a higher charged state of a heavier peptide


primer on topic

https://masspec.scripps.edu/publication ... 78_art.pdf

Thank you so much for your reply. My apologies for not explaining fully the first time, but the top figure is a heptapeptide with a charge state of three and the bottom figure is the same heptapeptide with a charge state of four.

The ultrazoom scan is basically a very high resolution scan of a specified m/z range, so it seems very odd that some m/z complexes have this very low resolution (bottom figure) result where the peaks are not distinct, whereas some m/z complexes don't.



Is it possible that multiple higher charged states of heavier peptides are coexisting with the lower charged lighter peptides ie the 7/4, 14/8, 21/12 complexes exist in similar intensities and therefore the differences between the charged states are small that they can't be resolved by our instruments? Is there a way to test this hypothesis?