Can I use a cell free extract on Mass Spec? - (Jun/16/2007 )
Hi,
I was just wondering whether I can analyse my protein on Mass spec..... I've generated a cell free extract using cell lytic reagent and a protease inhibitor (Is this the same as protein purification? 
) and want to know if it's ok to inject this cell free extract into a Mass Spec machine?
Thanks
hi.. for MassSpec protein has to have high quality.. means higly pure..
how did you purify your protein?? how is it looking at gel filtration? can you see single peak?
how did you purify your protein?? how is it looking at gel filtration? can you see single peak?
Hi again,
OK now I'm confused
I thought I already did a purification with cell lytic buffer.... I've never been told to do a purification. This is what I do in the lab:After a 4 hour expression period:
1) Pipette 5ml of each expressed culture into a Falcon tube
2) Spin the material for 15 mins at max. speed and pour out the supernatant
3) Spin the material again for an extra 5 mins at max. speed
4) Dry with tissue to remove the remaining liquid
5) Incubate cell pellets at -80 C for 1 hour
6) Leave at RT for 10 mins
7) Resuspend the pellet in 500 ul cell lytic reagent (1X concentrated) containing 1 tablet protease inhibitor/10 ml lysis reagent
8) Incubate at 21 C for 1 hour
9) Spin fragments for 10 mins at max. speed
10) Transfer the supernatant (cfe) into a fresh eppendorf tube
So now isn't this a purified cell free extract??
What I want to do next is take this cfe and react it with alpha 2,3 sialyllactose (which i need to synthesize but don't know how) to monitor hydrolysis of the substrate over 3 hours on Mass Spec.
I think you can already tell that I'm not a biologist/biochemist.... My supervisor has thrown a computer scientist into the lab!
Thanks a lot!
unfortunately the answer is a resounding 'No'.
What you have is a 'cell free extract' aka all the cell are dead and in broken down into small pieces. What you have is a soup containing eveything that a cell is made off. Protein purification is the sometime frustrating art (using a range methods) of isolating a single protein from this soup.
I am unsure of the current state of mass spec technology. Can the mess spec software and equipment resolve a known signal form the cluttered mess of a cell free extract? Sounds a bit much to me, but I haven't been keeping up to what people have been doing with these machines. Seems to me, that a method of isolating the product of alpha 2,3 sialyllactose degradation is required.
Seems to me, that a method of isolating the product of alpha 2,3 sialyllactose degradation is required.
Aaaaaargh!! So now i have to purify this cell free extract???
I've been reading about affinity chromatography.... Is this a purification method? What I'm thinking of doing now is throwing this cfe into a nickel column to purify it.... Am I right in doing this?? Does anyone have a protocol for this method?? Oh BTW, my protein has a His-tag (whatever that is). And once I get this purified stuff I'll analyse it on Mass spec. Thanks for your help....
P.S. I don't understand when you say "a method of isolating the product of alpha 2,3 sialyllactose degradation is required." - Could you please explain? Sorry for being a bit dumb about this
well, lets assume that the experiment is running, there is the protein solution. Alpha 2,3 sialyllactose is thrown into the mixture. alpha 2,3 sialyllactose begins to degrade. You take an aliquote and put it into the mass spec. But there is one problem, your protein which is soluble go in for the ride. Protein is very large, and produces noice and masks the signal. *Bad end.
You have to either immobalise your protein. Or find a way to remove the protein, or pull out the degradation product. This may be as simple as filtering the protein out. Although.... your mass spec may be able to do Tandem MS, and separate out the Alpha 2,3 sialyllactose from the protein. Can most Mass Specs do Tandem MS now?
Affinity chromatography is a protein purification method. A His-tag is a small amino acid motif that binds strongly to nickel. The His-tag allow you to do nickel column affinity chromatography. Does your protein also have a thrombin cleavage signal? This is to make it easier to remove the protein from the nickel column.
Thanks for your explanation. No my protein doesn't have a thrombin CS. Looks I'll have to purify it on a nickel column then analyse the hydrolysis reaction between my protein and substrate (alpha 2,3 SL) on Mass spec over 3 hours. I'm not too sure if our mass spec does tandem MS - If it did, then would I be able to use unpurified protein?? Thanks once again. I really appreciate your help
Sorry, I don't know. Tandem MS has come a long way, but I don't know if it has gone that far.
Perhaps you should go see the people who run the Mass spec machine in your department (I assume that is where you are doing your mass spec). Explain the situation. It is likely that some purification is needed. At least removal of all small molecules from the cell free extract.
you should be able to pick out the degradation product with lc/ms.