Dear all,
Recently, I isolated a protein by LC (IEC and GFC) which showed one band on SDS-gel. Then, I sent it to determine its accurate MW using LC/MS. The brief result is attached which showed that there are two proteins in the sample with slightly different MWs, 15102.273 and 15146.292.
I sequenced the full length gene (gDNA and cDNA), and calculated the MW 15134.87 (average), 15125.36 (Monoisotopic). I am sure the sequence is correct but the theoretical MW is different from the LC/MS determined one.
I am confused.
My questions are:
1. Did the two determined MWs show that the protein I isolated is not pure?
2. Any PTMs in the protein caused the mass difference between theoretical and LC/MS determined MW? I know that the protein is a monomer and there might be one or two disulfide bonds in it.
Zha
Hi,
Based on the data and background you provide, I think your protein preparation is indeed not 'pure' as it seems that there are (at least) two variants in your sample. This however could sound worse than it is, as the difference might not be relevant for the function/activity of the protein. Considering the mass difference observed, you can expect that these appear in a single band on a PA gel, as that technique likely has insufficient resolution to separate these species.
Overall, I think your mass nicely corresponds to the theoretical mass based on the gene sequence, so that is good.
Indeed, several post-translational modifications (PTMs) can result in a different actual molecular mass as determined by mass spectrometry: in addition to mass differences that are the result of disulfide bonds, N-terminal glutamine residues can be converted to pyroGlu, for example, or modifications like asparagine deamidation and methionine oxidation can occur (which I think also happens given the presence of some less abundant species in your bottom deconvoluted spectrum that differ by 16 Da in mass).
If you would like to know more details, I think it is advised to perform a proteolytic peptide mapping LC-MS/MS experiment to confirm the amino acid sequence (and ensure that no sequence variants have been formed) and to map any PTMs that have occured, as these only show up as an accumulated mass difference when performing intact LC-MS.
Hope this helps!
Zha,
The excellent explanation provided by Eef sums up many of the thoughts I had. I agree that it would seem you have two variants of the same protein. The fact that you have only two masses would suggest it is not a highly variable modification like oxidation, but likely a very specific difference between the two. You might inquire as to how accurate / precise the mass determinations provided are so you can tell how precisely you may assess the mass difference between the two, which stands at 44.019. There are several AA substitutions that get close to that mass difference; e.g., Ser to Met, Gly to Thr, Cys to Phe which all have mono or avg mass differences of 44.0 Da.
Regards,
Andrew
Dear Eef and Andrew,
Thanks a lot for your answer. Both are very helpful. Eef's make me worried about the result. The molecular mass difference can't allow me to link it with any PTM I known. In fact, I identified the protein by 2DE , maldi tof/tof. After purification, I identified it again and cloned the corresponding gene. They matched well.
I tried to detect any PTMs on this protein by measuring its accurate MW. But it looks difficult now. As Eef suggested, I will check the tryptic fragments mass.
There are still two questions left. I tried to calculate the MW myself, according to the mass data, my results are 15111.4 and 15155.3. But the result I received are 15102.3 and 15146.3. Both have 9 Dalton different. Which is correct?
If there were two proteins in the sample with slightly different MW, according to the result I received, can I figure out which one is dominant?
Sorry for my English and best wishes
Zha
Hi,
Cloned genes can be unstable, leading to sequence variants in proteins. Consequently, an alanine to aspartic acid mutation results in a mass difference of 44 Da, for example.
So please check whether the sequence(s) of your protein(s) match the expected sequence based on the gene sequence using proteolytic peptide mapping.
With regard to your questions: how did you calculate the masses of 15111.4 and 15155.3? Using the charge states in the non-deconvoluted spectrum? If so, did you take the protons into account that are on the ions detected in your spectrum? For example, if you use the 10+ charge state, a way to calculate the molecular mass is to multiply the m/z value by its charge minus the number of protons.
It is difficult to deduce which protein form is dominant (assuming that you mean most abundant), as you'll need high resolution to separate both.. Taking your MS data into consideration, I'd say both forms are equally dominant (~1:1 signal intensities), assuming that the difference between the two is not influencing their ionization efficiency.
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