Youli, do you have the option to perform both as they are complementary. If you run a SDS-PAGE 2D gel and excise the band(s) of interest, you can then perform in-gel protease digestion (e.g. trypsin) and run the peptides by LC-MS for identification of the protein (and potential post-translational modifications).

Good luck.

A very good question - For protein identification in my point of view you should opt for MS as you have the possibility to cover the whole protein sequence, however, if you want to target the question of sample purity first, then you might get more information out of a 1D or 2D gel...

Youli, do you have the option to perform both as they are complementary. If you run a SDS-PAGE 2D gel and excise the band(s) of interest, you can then perform in-gel protease digestion (e.g. trypsin) and run the peptides by LC-MS for identification of the protein (and potential post-translational modifications).

Good luck.

hi youli, have you heard of CE-MS? Capillary electrophoresis -Mass spectrometry is better than 2DGE, its easy to automate, fast (30-45mins run) high sensitivity and accuracy and at the same time it has high resolution capacity. 

If you actually want to IDENTIFY your protein, then 2D PAGE will not do that, you'll need to use MS. I assume you are talking about comparing two samples and want to see what changes occur. Once you've done that, you want to identify the changed proteins?

From your question, it sounds as though you haven't had much hands on experience with 2D gels. They can be a pain, but if you are in a lab that runs them regularly, they can be very reproducible. They also have the advantage that you can easily visualise thousands of proteins at once making comparison relatively easy.

In short, the answer to your question is not so much which technique is better, but what is available to you and what do you have experience with? You are always better off sticking with your strong points.

Dear Youli, the answer to your question depends a lot on your "induction condition", the amount of unknown protein you expect (is it highly abundant or not), and as Peter said, the techniques you have available in your lab or department.

If you suspect the unknown protein is highly abundant between control and your induction condition (and you don't have 2DGE in place or available as service), you'd better start with SDS-PAGE, excise your band of interest and send it to LC-MS/MS for identification. Second step would be to to small 2DGE to check the heterogeneity of your protein of interest (PTMs) if desirable. But even small 2DGE is not so straightforward, and requires some experience to obtain nice and reproducible images.

If there is no major difference in abundance between your control and "induction condition" (or at least if it is a minor protein whithin the whole proteome), then you need a real proteomic approach, and dedicated tools. If relative quantification between control and induced condition is your major issue, and your protein of interest is fairly soluble, and it is not extremely acidic or alkaline, then it is worth envisaging the large 2DGE approach (or even DIGE). But I would not recommend trying that if you do not have any experience or support in your lab or department with 2DGE (it can take monthes to reach the experience necessary to obtain nice images). If you can perform large 2DGE, comparative image analysis will "identify" spots differing in abundance between your conditions; you will then have to run a "preparative" 2DGE to excise the spots of interests, and send them to mass spectrometry for identification. In our practice (with well-established 2DGE techniques and very-well trained personnel), it take one month to run all the necessary gels for image analysis and then for MS identification.

On the other hand, if you have proteomic facilities available, you can investigate wether they would perform a comparative shotgun approach by LC-MS/MS; there are numerous alternatives (label-free, or various labeling techniques), but sample prep or pre-separation can be an issue depending on your sample, and can rapidly make the experimental design quite complex and expensive...

Hope this helps... and I agree with Peter: you are always better off sticking to your strong points, or find a lab with a strong capacitiy in the techniques you need and establish a collaboration....

Niels

Hi Youli

You have not given much detail to go on.

Is the source from cultured or ex vivo samples? How much material do you have? How easily can you extract the protein of interest? Do you have an idea of where the protein is located within the tissue or cells? Is the protein a membrane bound or transmembrane type protein? Are you wanting only the ID of the protein or do you want relative quantitation data as well.

From your skills it would seem you do have 2D gel expertise. This should already give you an idea of what you can or cannot do. If the protein you are looking for is a membrane protein or a low abundance protein you are probably wasting your time with 2D gels. DIGE could give you some answers as to the relative changes of protein abundance but again you need the soluble and high abundance proteins for this to be successful. The use of SDS PAGE followed by in-gel trypsinisation and mass spectrometric analysis can at least give the protein identity but you will have mixed proteins of the same approximate mass so the problem would be to determine which protein is the one of interest.

If you have access to mass spec facilities then mass spec can give added information about possible PTMs and relative concentrations without major added costs.

The use of CE-MSMS as mentioned by Lothy Casim is a good idea but this is relatively unavailable at present and uses top down protein ID technology unless the sample is pre-digested with trypsin.

Good luck 

I'm totally agree with Niels . ..good luck in finding proper MS based tools for the unknown protein you are searching for ..Good Luck !

Hi Youli, I agree with Peter and recommend doing both. The MS can identify the sequence, as most people have pointed out but doing a gel will also let you determine specific linkages such as disulfide bonding which otherwise may be missed by MS. The MS exclusively requires the disulfide bonds to be reduced otherwise the estimation of molecular weights could be difficult. Indeed the in-gel trypsin digestion followed by MS/MS seems to be the best method but its efficiency depends on how well you are able to extract the peptides from the gel matrix. Good luck!