If you have PEG signal, it is due to a too low amount of protein. The analysis software show you PEG polymer when the can not find another peak. Increase greatly the amount of material.

A mi nor point becareful of the plastic used: No lowbind plastic, thy have more additive. No color tube or tips as well.

Antonio, there are lots of variables in your system that we'd like to know to give more specific answers.  But here are some ideas:

1- Maybe you don't have enough protein.  Depending on your machine, Western blots are much more sensitive than MS.  Can you "see" your protein by some kind of stain? (Coomassie or silver?).  

2- Maybe your protein aggregates.  Westerns would fix this because of the SDS and boiling, but for MS, big aggregates may not properly fly through the machine.  

3-  I think the PEG-polymer is a key clue.  Where is this used in your setup? Filters? Concentrators? It is very common to lose protein during the "cleanup" steps.  The more steps you have, the less protein you'll end up with so check to see if you still have any protein in the end.

4- Some proteins just don't fly well through an MS.  Trypsin digestion would help.

You need to work on sample preparation ..try a microcon tube with a cut-off depending on the Mw you expect for the protein  and redesolve the protein in a proper buffer for ESI-MS ...or try bottom up or top down MS approach.

What about cutting your protein after coomassie staing of a SDS Page? If you see the band you have enough protein for Mass Spec. And you know that the size ist right and you don't have additional proteins

ok, thanks, #Sven Reister but how can I elute it from coomassie?

Hi,

the elution for us is done by our mass spec facility. But your can do things like electroelution in a suitable buffer ( like blotting also moves your proteins). Have a look onine there are noumerous protocols.

Poor input = poor output for mass spec, so just concentrate on getting a)enough protein in b)the right buffers and then everything will work. Point 1 of the above is very true (except that MS is far more sensitive and wonderful a technique than westerns!). Elute in the 'best' buffer, coomassie/silver stain (dilution series, estimate conc?), Find That Protein, repeat to make sure this buffer is really reliable. Then move on to b). Can you use different buffers that are more mass-spec compatible? Unless you're doing top-down mass spec, solubilize 8M urea, reduce, alkylate, dilute to 1M urea, digest with 1:10 trypsin. Clean up using Harvard apparatus ultra micro C18 spin colums (clean-up loss is not a problem here). I'm guessing it's all to do with a), though. 

if you know the size of the protein then cut the band and go for in-gel digest protocol for Mass spec,,it should work ..if it does not then start with more sample ,,, For pull down experiment we normally start with 750 ug to 1 mg sample...

here is a protocol for in-gel digest...

http://www.nature.com/nprot/journal/v1/n6/abs/nprot.2006.468.html

What about the size of your protein?...you can try passive elution.  Good Luck !

Isn't there a bit of controversy in your experimental set up?

>>>a DNA Binding protein having a particular conformation, so I'm interested in isolate it and analyze it as intact by mass spectrometry. 

and then you boil it...

>>I've tried to elute the protein with a buffer having 100mM of Glycine at pH 2 and giving a brief boiling (a couple of minutes).

If you are not that really concerned about its conformation that you boil it, go to bottom-up and be done with it (there was a link to the protocol of inGel digestion above, or you can just make it in solution to avoid additional step of PAAG-EP (a lot of IS digestion protocols are available). 

If you want to see your protein intact, are you really sure that you are able to do it in your MS setup - not all MS systems are able to show you proteins (mass-range and resolution might be inadequate) and not all proteins are keen to show themselves in MS (again it was mentioned above).

Could you please provide  more information concerning your isolation protocol (the more details the better, please; otherwise how can one tell you where your PEG might come from)? 

Actually, you can get PEG from any source (any plastic (e.g. your sampler tips), any chemicals, and I am afraid that uncoloured plastic might give you PEG almost as easily as coloured one). And it can easily mask your protein signal. It is strictly necessary to locate the PEG source and eliminate it. Do you make LC-MS or syringe injection for your protein? If you see PEG during the gradient it can easily come from LC setup - in this case you would hardly be able to get rid of it, but it might interfere less with your protein signal provided it does not coelute.  You can try (just as a control) to take some other protein that you can see in your MS setup in a syringe injection mode in appropriate concentration and run it through as many steps of your protocol as you can and check whether you can see it in your final ESI-MS analysis. Your protocol might greatly reduce your initial material... And if you see it by immuno-blotting it does not really tell that you would be able to see it by MS. IB might easily be much more sensitive than MS especially in top-down proteomics.

By the way, you do not see any protein signal at all, or you have some expectations about mz and you just do not see this particular mz? 

What is the purpose to do mass spectrometry after pulldown? To prove that it is actually the correct protein you are looking at? Why does it need to be analyzed intact? There are many problems with intact protein MS, both with the size and number of PTMs you protein can have.

Large proteins would have a wide charge and isotope distribution and analyzing proteins above 30-50 kDa can be difficult (independent of mass resolution). If you protein can have PTMs (or endogenous cleavage products) it will also be distributed between several masses.

If you want to identify your protein after IP i would do as Svein Reister suggested and do coomassie after gel separation. Elution can be done by tryptic digestion and then analyze the tryptic peptides by lc-ms/ms. The you overcome problems with intact protein mass, and not all the peptides would have PTMs. Most of the detergents would also not be extracted from the gel. There are many in-gel digestion protocols online.