Who has the experience in top down protein analysis? I have try the C4 particle for this analysis, but no protein was detected with the CID model. Who can give me any suggestions? Thanks.
Dear Wenzue
Are you trying to detect any Ubiquitinylated regions or fragments? Did you try ETD based fragmentation approach?
How big is your protein of interest?
Regards
Prash
Hi Prash,
I'm sorry I don't why it hasn't show.
I had't try the standard protein as you mentioned and my mass spectrometer only has the CID fragmentation model. I want to separate about WM 50kDa proteins by top down method.
The PRLP-S particles have been reported for top down protein separation, so I want to know whether the C4 particles is not suit for this protein separation.
I know the CID is not the best approach for intact protein research but it also should appear a few ions to proof my LC conditions is no problem. How do you think?
Best regards.
Wenxue
Agree with Prashanth, better to use ETD or ECD. If using CID will struggle to get good fragments with anything larger than 4-5kDa
Hi Stoyan,
Although the CID approach isn't the best choose for the intact protein analysis, it also should appear big charges (more than 15 or ... now I only can see the charges 12 with the m/z about 1100) more or less. If this, I know the problem is my machine. How do you think?
Thanks and best regards.
Wenxue
Not necessarily, number of charges protein picks up will depend on many variables including native conformation, buffer composition and additives. What instrument are you using?
Are you only picking up the 12+ charge state, that's odd. Should be detecting many more charge states? Also, 12+ at 1100m/z means protein intact mass of around 13.2kDa. Is your protein a monomer of 50kDa?
Rgd your question about suitability of C4, it should work fine for a 50kDa protein. What solvents are you using?
Hi Stoyan,
The instrument is the Thermo LTQ Discovery. The separation conditions is 75um capillary with the aceonitrile and water which both containing 0.2% formic acid.
It's a mistake. The biggest charge is around 12 what I can see now. My sample has many proteins but the WM less than 50kDa for each, because it has been filtrated with 50kDa cut off filters.
I guess that the protein has been absorbed by the C4 and which could't be eluated out by mobile phase. How do you think?
I also plan to order the BSA (60kDa) for a try. Could you have any suggestions for me?
Thanks you very much.
Best regards.
Wenxue Li
Your solvents should work. First thing I would check if proteins are present after filtration (could run a filtered aliquot on SDS-PAGE). Proteins can absorb to membrane surface especially if it is polymer based.
After filtration do you further desalt the samples or straight to injection? Could be getting ion suppression if samples not clean.
Since you are working with Nano flow (75um column) need to make sure that you are getting a good spray /signal. The idea of a standard protein to test your system is good but why BSA, rather use a protein(s) less than 50kDa since will be represnt your sample better.
Rgd proteins absorbing to column, possible but you have a mixture and should thus get at least some eluting.
Hi Stoyan,
Thank you for your advices. I will try with the standard protein.
Besides, which software are you used for the intact protein searching? ProsightPC of Thermo? Do you know any other open source software for this analysis? I only have the PEAKS.
Thanks again.
Sincerely
Wenxue Li
I am not sure Wenxue, but Orbitrap and Waters must be having some OS software. I thought the instrument itself would come up with the intended software. As suggested by Stoyan, you should opt for standard protein
Best
Prash
Rgd the software, going back to the beginning of the thread, if doing CID it's unlikely you'll get good spectra for IDs no matter what software you use. Should use a high resolution instrument equipped with ETD rather. In that case Peaks should work.
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