I have a question regarding the result of MS (protein sequencing). We made the recombinant r- IgT (unique immunoglobulin in fish) in mammalian HEK cells, we want to characterize the r-protein, we made SDS-PAGE and cut the band and sent to the MS for sequencing, I got the result from MS stating that the r-protein is not the one we expected, it was cloned from salmon, any suggestions.
Hi,
As you are working with cell content it does make sense that you do immunoblotting for your protein of interest to ensure the presence and also expression of your desired protein. Because there are large number of proteins in cell content how can you be sure that you you cut the correct band? Maybe there are lots of protein with the size you expect!
It's most likely that your unique immunonglobulin in not in the database, it gave the next most likely match (I assume the MS result was a fish ig).
Thanks Vince, actually it didn't as the result suggested that we have ribosomal protein.
Thanks Amin, of course I did, and I found a band at the expected size but also we have a smear of big size above our band it means that the band we cut was't the band we seek, Moreover, results of ELISA showed that the supernatant of mammalian cells (containing the His tagged protein) reacts well against the anti-his tag.
I could imagine that immunoglobulin will be tricky to work with on MS. Did you tell them what you expect (also provide sequence to avoid what Vince suggested) so they take that into account?
I'd assume, if you have signal on blot, yet MS did not show your protein, that either they just co-migrate (have the same size) or they bind to each other, even on SDS-PAGE (cystin bridges?).
In either case, either the ribosomal protein is present in much higher quantities (that would suggest rather option 1) or they got the peptides, but were not able to assign them to your protein (if they didn't have the sequence).
Was the protein purified or was it crude extract? What about MS analysis of the whole sample (without SDS-PAGE and single band cut-outing)? That will tell you, if they are able to detect it at all.
Dear Tomáš Hluska, thanks for the good ideas you pose here.
I agree with you considering the MS of the Igs is quite tricky, particularly If it was a unique IgT from fish species. I told them that I expect IgT from salmon but unfortunately, I didn't give them the sequence.
Yes, the protein were recombinantly produced in mammalian suspended hek 293 cells and purified the supernatant (culture medium) using anti-His-tag column.
I was thinking in using the whole sample rather than using the cut band.
Thanks one more time for your help.
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