I am using a low pH elution buffer (pH 2.8) for eluting an antigen of interest. There's heavy contamination of antibody in the elute as detected by Western blot. Can I submit this elute (as such or after neutralization) directly for Mass Spectrometic analysis? The most common method is running a SDS PAGE to separate antigen from antibody and then cutting a band of interest. This method, however is unsuitable for my purpose as I am interested in PTM of protein for which I need large quantity of protein. Therefore, I need large quantity of elution buffer which I cannot subject to SDS PAGE. Moreover, using elute directly for MS will give an advantage since the protein of interest will be in solution form and can be subjected to various digestive enzymes. Another solution to this problem involving cross-linking of AB to beads (apparently) affects the affinity of antibody for the antigen. Can elute containing antigen and antibody be subjected to MS?
Hi Tushar,
The SDS PAGE will not help as the amount of interacting protein is too low to be visualized on the gel. You may need to pool 10-15 IPs to get enough material for digestion. On-bead digestion as suggested above will be useful but you will have contaminant from the Antibody and bait protein. Since the sequence of these two proteins is known, you can develop the method and selectively exclude all possible peptides from these two proteins and selectively fragment only the peptides coming from your interacting proteins. The problem here is that since Antibody and Bait protein are in huge amount, they will use up all the enzyme and will prevent digestion of the other proteins.
Alternatively, you can Elute the Protein using your elution buffer and then precipitate using cold Acetone thereby removing all interfering(MS incompatible) compounds and proceed for digestion .Anyways all proteolytic digestion enzymes require pH of around 8 so no digestion will happen in glycine buffer.
Two very important things
1) Whatever you do , process all your controls and experimental samples in duplicates under same conditions.
2) Since you are targeting a protein mixture , you will need MS/MS data to identify protein. A high sensitive instrument like QTOF and/or Orbitrap will only generate this type of data. MALDI TOF or stand-alone ESI TOF will not work.
Hope this is useful.
it is 0.1M glycine HCL solution, We never had any success in MS analysis when eluted the IP product with 0.1M Glycine-HCl ph 2.8. we tried alternatives such as 0.1%TFA, 1mMHCl, 1%SDS, with no success. if the ab and ag are in equal concentration in the solution yes you can. you need to ask this que to the core facility who will be doing the MS analysis for you.
In short, I'd have to say no, you can't use your eluate directly for MS analysis. MS does not like salts, especially ESI. It sounds like you want to do an enzyme digest and then MS? Are the enzymes going to be active at pH 2.8? Most likely not. You'll need to do some form of buffer exchange, whether it be speed-vac, SPE, dialysis, centricon etc, to get the sample into a buffer that will allow you to digest it. At this stage, you may have the option of increasing the concentration of your sample. ie. if you use SPE, you can load 5 mL (for eg) and elute in 300 uL. Having digested the sample, you'll then need to do another clean-up prior to MS analysis. Again, MS does not like salts. Hope that helps.
I used a Dynabead-Antibody system to do IP and then digested the antigen directly from the beads, without an elution step. Then a TiO2 enrichment of the digest for phospho-peptide enrichment.
If your antigen is phosphorylated then it'll bind to TiO2 and your antibody will be in the TiO2 column flow-through (as was the case with my IP).
Both the TiO2 column eluate and flow-through can be directly subjected to MS/MS analysis after C18/Oligo R3 cleanup.
Hope this helps :D
1) Thank you Dr. Purohit. Does by "We never had any success in MS " mean you were not able to detect your protein of interest with 0.1 M glycine buffer? May be that protein is not soluble in this buffer (as there are no surfactants. In IP buffer we always have non ionic surfactants). You pointed out correctly, the antigen has to be in in excess or at least equal to antibody in terms of abundance in the elute.
2) Thank you Dr. Hains. I don't know the composition of elution buffer (its from Thermo Scientific). I guess it is different that traditional glycine buffer. In order to make the protein susceptible for enzyme digestion, I can neutralize the elute. Again it will create salts. As you suggested, I will have to perform dialysis so that the elute is compatible with MS.
3) Thank you Dr. Almazi. Doesn't direct digestion on the beads create products from antibody and the beads (protein A or G)? Moreover, I would like to have non-phopshorylated protein in the elute in addition to phosphorylated one. So I won't go for phospho-peptide enrichment.
Yes we could never detect any peptide. the reason is most of the time we were keeping the IP antibody at 0.5ug, assuming a 30% efficiency (IP is not a very efficient process), the losses in the subsequent steps were the one of the reason that we could not identify. We wanted to develop a high throughput approaches for protein identification using MS.
My other suggestion would be in you case and in case if you only have 1-10 IP reactions is to do a SDS PAGE of the IP product without any beta-ME or DTT, that way your antibody band will be in 150-200kD Range, use a 1.5mm thick gel to increase the loading volume. you can even tape two wells together to create a bigger well.
Another thing you can do is to use a small volume of elution buffer (say 50ul if your proteinA or Protein G bead volume is 100ul) and elute in multiple tubes, it is labour intensive, but probably will give you the best results,
Another thing to remind you, ESI TOF is not a great platform for detection of phosphorylation, you may need to think of MALDI-TOF in this case with more success
Hi Tushar,
The SDS PAGE will not help as the amount of interacting protein is too low to be visualized on the gel. You may need to pool 10-15 IPs to get enough material for digestion. On-bead digestion as suggested above will be useful but you will have contaminant from the Antibody and bait protein. Since the sequence of these two proteins is known, you can develop the method and selectively exclude all possible peptides from these two proteins and selectively fragment only the peptides coming from your interacting proteins. The problem here is that since Antibody and Bait protein are in huge amount, they will use up all the enzyme and will prevent digestion of the other proteins.
Alternatively, you can Elute the Protein using your elution buffer and then precipitate using cold Acetone thereby removing all interfering(MS incompatible) compounds and proceed for digestion .Anyways all proteolytic digestion enzymes require pH of around 8 so no digestion will happen in glycine buffer.
Two very important things
1) Whatever you do , process all your controls and experimental samples in duplicates under same conditions.
2) Since you are targeting a protein mixture , you will need MS/MS data to identify protein. A high sensitive instrument like QTOF and/or Orbitrap will only generate this type of data. MALDI TOF or stand-alone ESI TOF will not work.
Hope this is useful.
You can try this. Elute IP protein with Rapigest (product of Waters). This is a cocktail of MS friendly surfactants. If you want to remove antibodies, load the elute to a antibody removing column (for eg. Capturem Protein A Miniprep by Takara Clontech). Elute bound protein with a minimum volume (say 50-75 microlitre) of Ammonium bicarbonate buffer (pH8). Go for trypsin digestion
- Badges
- Science method