When I ran a sample of Candida lysate in a 2D gel, I found the same protein in different points of my gel. After LTQ-Orbitrap MS/MS analysis it results the same proteins, and the tryptic fragments are almost the same. I am sure that there is no contamination at all, I think they could be isoforms, but at so different pIs and MWs?
This is a very typical scenario. From what you say it is not even clear if your protein just smears over a region and the spots you cut contain it as "contamination". There is several ways to deal with this. This depends on how interested you are, and what tools are available for this protein. The most logical step (imho) would be a WB of the 2D gel (same range/dimension) you use for the MS analyses. This will show you if your spots are really your protein. BTW, after the vlor you can still stain the original gel, depending on how much protein there is/was and your blotting conditions, there might be enough there for a stain (it will be distorted compared to the blot in any case, as gels shrink/expand and warp).
In addition (or if no antibodies are available) have a very close look at your MS data, which peptides are found? Make a graphical list, do you always find the same ones? Do a second class search, allow all sorts of modifications searching just the one protein. This will show up differential length/modification of the protein in question.
Also, cut "empty" (visually empty) regions between the spots, do you get the same protein? If yes, it smears...if not, a good hint your spots are really form the one protein. Obviously there could be any number of reasons for the different spots......Glycosylation tends to produce horizontal (pI) movement, phosphorylation usually in both directions (more horizontal) degradation obviously down, but it could be left or right, depending on the sequence. And o top of all this are anototehr 250 or so (know) PTMs....
Any Q just ask for more details....
Josip
Is a denatured reduced gel? If not, you can see the polimers of the same protein. Some times even in denaturing-reducing conditions. As regards the isoform they often are quite different in pI and MW
I find isoforms at different PIs and MW but they are always pretty close to each other
The MWs and pIs are very different, the gel is a reducing and denaturing one, and even the pattern of the spots is reproducible
What about alternative splicing? That could cause significant changes in both pI and MW.
Posttranslational modification maybe: is it a candidate for phosphorylation, glycosylation, proteolysis etc?
I have observed the same things on some of my 2D gels with a significative difference in the pI et MW. Finally we have shown that all these "proteins" were in fact the same with high susceptibility to proteolysis...all these spots were different products of the complete protein...and the peptides found by mass spectro was allways the same in the "central" part of the protein. The presence of protease inhibitor is sometimes unsufficient and lead to "controlled" proteolysis.
Sounds to me that you have splice variant or initiation of transcription/promoter at different place on the gene!
For my case, no splice variant is described and nucleotidic sequence did not show any consensus sequence for initiation site.
I suggest you repeat the same experiment at least four times until you are sure of that.
I found that in 6 gels that have been reproduced in 4 different experiments and protein extracts.
My guess would be that your protein of interest is being cleaved by an enzyme, and so while your antibody is recognizing only one specific part of the protein, the full length of the recognized peptides differs.
As mentioned, post-translational modifications may be the culprit. I have seen PI shifts caused by phosphorylation. Stains for such PTMs are available and may be able to help you narrow down what's going on.
Dr. Alberto Vitali
I woul like to suggest that the protein is modified by phosphorylation or glycolysation. There is also a possibility that this proteins are members of the same protein family which share functional domains and this is recognized by the antibody.
If you can cut out the protein from a 2D, gel you can identified it by microsequencing. You probably need to add spots together from several gels to have enough matherial for this.
Best regards
Borbala Gesser
@Borbala Gesser: A. Vitali wrote, that
1) the differences are big, thus it should not be phosphorylation/glycosylation
2) he identified the protein by MS, not with antibody, that should rule out the possibility of isozymes
I did not think about an isoform of the protein. But, it might be a precursor protein, test the protein by Western blotting. Proteins might share the same functional domain. Use also more protease inhibitor.
The proteins may be isoforms, or they may be the result of post-translational modification. There are many post-translational modifications that occur - proteins can be acetylated, methylated, phosphorylated, glycosylated, internally cleaved, N- or C-terminally truncated by exopeptidases, and linked to other proteins by disulfide or gamma-glutamyl links which are quite stable.
This is a very typical scenario. From what you say it is not even clear if your protein just smears over a region and the spots you cut contain it as "contamination". There is several ways to deal with this. This depends on how interested you are, and what tools are available for this protein. The most logical step (imho) would be a WB of the 2D gel (same range/dimension) you use for the MS analyses. This will show you if your spots are really your protein. BTW, after the vlor you can still stain the original gel, depending on how much protein there is/was and your blotting conditions, there might be enough there for a stain (it will be distorted compared to the blot in any case, as gels shrink/expand and warp).
In addition (or if no antibodies are available) have a very close look at your MS data, which peptides are found? Make a graphical list, do you always find the same ones? Do a second class search, allow all sorts of modifications searching just the one protein. This will show up differential length/modification of the protein in question.
Also, cut "empty" (visually empty) regions between the spots, do you get the same protein? If yes, it smears...if not, a good hint your spots are really form the one protein. Obviously there could be any number of reasons for the different spots......Glycosylation tends to produce horizontal (pI) movement, phosphorylation usually in both directions (more horizontal) degradation obviously down, but it could be left or right, depending on the sequence. And o top of all this are anototehr 250 or so (know) PTMs....
Any Q just ask for more details....
Josip
@Josip Lovric: didn't you mean that glycosylation causes mainly change in weight?
well, yes, but weight is not measured in gels, it is mobility. most glycosylations casue a train of spots in the pI dimension. A proteins gets heavier with more sugars on it, but its mobility is not affected (2nd dimension) in most cases, as the sugars are charged. Phosphorylation charges a protein stronger, but it also changes its conformation and displaces SDS, so some (no all!) phosphorylations make the protein appear "larger" on the SDS PAGE gels, but this is a change in mobility, not mass (e.g. 10 phosphorylations add 800 da of mass only, but the protein might shift from ApMW=70kDa to 130kDa)
Josip
The closly related protein in different spots mighe be due to the proteolytic cleavage of the same protein.
Is it not possible that the higher molecular weight protein is a precursior molecule of the lower molecule weight protein. You should test by 2D Western Blotting if an antibody recognizes the two spots. That meens that they may share a common sequence, they can belong to the same protein family, It can further confirm your MS analysis results.
@ Josip,
Your suggestions for shifts in mobility and pI are very interesting. Can you also provide a manuscript with such examples?
I have come across a study where they identified same protein in different spots and they used Scaffold to visualize and compare the peptides from the different spots: http://www.mcponline.org/content/early/2012/10/01/mcp.M112.019372. Scaffold has very useful visualization of the sequence coverage which can help you as suggested above (peptide maps).
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