amount of protein for MALDI-TOF - (Nov/28/2007 )
Hi people,
I would like to ask anyone for experience with MALDI-TOF analysis and detection limit of amount of protein.
Our problem is, that we partially purify a protein, then we can nicely detect this one on PVDF membrane using very specific monoclonal antibody (+secondary biotinylated antibody+avidin+biotinylated HRP+ECL plus), but when we cut parallel the area from PAGE where this protein should be (in several small pieces of gel) and all these pieces where submerged to trypsin digestion and analysed with MALDI-TOF MS, people from proteomic department did not find our protein - they had either very bad spectra, probably because of low amount of protein or they found something else.
So, we think:
a) ECL plus (GE Healthcare) system has detection limit of 40pg, but for that MS we would need approx. several hundreds picograms (may be some nanograms). Do you have any experience, how much of protein did you need for clear identification by MALDI-TOF (our protein is 70kDa)? 
The gel is stained in CBB and than de-stained with 50% methanol, 5% acetic acid and than stored in distilled water at 4dgrC (before cutting for tryptic digestion). Would it be possible, that our protein is washed out from gel when gel is in that distilled water?
c) any other suggestion?
Thanks very much.
vic
Hi,
You didn't say if you can see the band by coomasie blue or not.
Stain the gel shortly before going to MASS-SPEC.
If you can see it by Coomassie you really should get it by mass spec.
If it is siver-stainable then maybe.
I would like to ask anyone for experience with MALDI-TOF analysis and detection limit of amount of protein.
Our problem is, that we partially purify a protein, then we can nicely detect this one on PVDF membrane using very specific monoclonal antibody (+secondary biotinylated antibody+avidin+biotinylated HRP+ECL plus), but when we cut parallel the area from PAGE where this protein should be (in several small pieces of gel) and all these pieces where submerged to trypsin digestion and analysed with MALDI-TOF MS, people from proteomic department did not find our protein - they had either very bad spectra, probably because of low amount of protein or they found something else.
So, we think:
a) ECL plus (GE Healthcare) system has detection limit of 40pg, but for that MS we would need approx. several hundreds picograms (may be some nanograms). Do you have any experience, how much of protein did you need for clear identification by MALDI-TOF (our protein is 70kDa)?
The gel is stained in CBB and than de-stained with 50% methanol, 5% acetic acid and than stored in distilled water at 4dgrC (before cutting for tryptic digestion). Would it be possible, that our protein is washed out from gel when gel is in that distilled water?c) any other suggestion?
Thanks very much.
vic
[quote name='mikew' date='Nov 28 2007, 03:14 PM' post='118358']
Hi,
You didn't say if you can see the band by coomasie blue or not.
Stain the gel shortly before going to MASS-SPEC.
If you can see it by Coomassie you really should get it by mass spec.
If it is siver-stainable then maybe.
Hi mikew,
I do not see band after coomassie blue staining. The reason why I do this staining is firstly that it is well compatible with MALDI-TOF and the secondly the protein of my interest is not absolutely pure so there are still some other proteins and these I can see with commassie blue, so I can better estimate where approximately my protein is in gel.
Anyway, as I said, I cut whole area (in several small pieces of gel) where my protein should be.
The MALDI-TOF is capapble to detect femtomol, so if the protein of interest is in the slices it should be normally detected.
I think the problem is with the destaining... You destain with acid methanol, so I think the protein is precipitated.
I think the problem is with the destaining... You destain with acid methanol, so I think the protein is precipitated.
Thanks for suggestion,
the CBB de-staining in 40% (50%) with 10% (5%) glacial acetic acid is a conventional protocol or not? Moreover the people from proteomic department said that once proteins are fixed in PAGE (by methanol, acetic acid that are already components of CBB staining) they should not be eluted by distillate water (isn`t it?).
The detection limit of MALDI-TOF should be tens of femtomols which should be I think at least 700pg of 70kDa protein, but I think for analysis they use less.
My experience with MASS-SPEC is that companies and people
CLAIM they can detect bands at extremely low sensitivity. I recently
worked with a machine that was supposed to detect picomolar ranges.
The reality is that if you cannot see it by Coomassie you need a little luck.
If you can't see it by silver forget it.
Scale up you experiment.
It is also possible that your protein is being "masked" by another plentiful protein.