If not all , you can characterize most of them by mass spectrometry based proteomics. But quantifying them with reasonable accuracy is a tough ask. If you have very specific answers that you are seeking, one can design cell based experiments and quantify a subset that is amiable to quantitation. You can look up techniques like SILAC, iTRAQ and ICAT to see how they work and their limitations.

As it was told it is hard to quantify unless you have a particular protein overexpressed and you are looking for it. Before doing the mass you can also fine tune the purification steps using Ammonium sulfate ppt at different concentration and then go for detection; or run a monoQ gradient etc.

Well, Sujay and Debajyoti are very right. In fact, mass spectrometry is a qualitative technique, it can help you to characterize or identify you proteins however, it cannot help you to quantify your proteins. I think that the best way is to extract your proteins, whereby you can use ammonium sulfate precipitation as mentioned by Debajyoti. Then you can use HPLC or SDS PAGE to quantify them. Even though these techniques does not give an accurate quantification but they will at least give a close estimate of your proteins in your mixture.

I hope this will help

Thanksalot for putting IN answers,..

what i get from the answers is: Quantification of proteins is a tough job and if even its done still you may not guarantee accuracy,..

now i wonder if only qualtative techniques are used, then may i roughly tell about up and down regulation of any specific protein or enzyme??

and how SDS may help in protein quantification?? is it via the route of 2D SDS>> Recovery though HPLC >> LC-MS ???

Dear Maliha

It is better to run a 2 DE for qualifying of the proteins in your samples.

For activity of proteases, you can use zymography technique.

For other proteins, you must use special techniques.

Please don't hesitate to contact me, if you have any questions about 2-DE and zymography

Are you talking about quantification or about qualifying proteins? Maliha's last comment refers to quantification but Masood comment is focused on protein identification.

All the techniques mentioned above are reaaly good to characterise proteins but they are not good if you try to quantify proteins. For protein quantification there are several colormietric methods: Lowry, Biuret, Bradford, etc.

The comment by Dr. Rosa María Martínez-Espinosa is compeletely true. The protein studies are two parts:

1. Qualitative: SDS-PAGE, IEF, WB, IHC, ...

2. Quantitative: Bradford, BCA, Lowry and ...

You must identify your aims and proposal.

Dear Maliha,

All of the above answers have merits.  You can use enzyme assays only if you have a specific substrate.  Also if you substrate or product does not interfere with eachother's assay then mass spec is a very good option to assay more than one enzyme at a time.  But you will need to clean up your samples before injecting them on the mass spec. This will allow you to measure if your enzymes is up or down regulated.  Using Western blotting for quantification is NOT trivial, but it will allow you to detect changes in total protein levels (dead or active enzyme).

If you need more elaborate protein characterization then you will need to purify your proteins.  For this I would like to suggest ion exchange chromatography which can be performed without HPLC in a batch mode approach.

@Rosa Maria,

I want to do both i.e. qualification and quantification, like what proteins/enzymes are present and in what ratio or quantity they are present. Bradford and other methods would give me idea about total protein content, but what part is individually contributed by each protein may not be calculated/assayed.

Thanks @ Masood Sepehrimanesh & Marc Ouellet 

Actually i have a mixture of enzymes,.. both intracellular and extracellular,..

and i want to characterize and quantify major classes of enzymes involved in the reaction,..

Gel filtration chromatography may be used for the fractional separation/purification of proteins??

If you have a mixture of enzymes, it's much better to start by using other chromatographic resins such as ion exchange. Gel filtration works really well as final step in purfication processes but not as the first step.

Dear Maliha,

An easy way to separate intra from extracellular protein is to simply spin down the cells.  Indeed, gel filtration chromatography can be used to fractionate proteins but I would only use this if you do not know anything about the isoelectric point of the proteins that you want to separate.  Ion exchange chromatography is a much more powerful purification tool than GFC.

I assume that you know what enzymes that you are trying to separate, than you should be able to find or calculate their isoelectric point.  I still feel that mass spectrometry may be the way to go to assay many enzymes at the same time.  As I mentioned before you must know if any substrate or product will interfere with any of the reactions you want to study.

Marc