Hi Tomasz, no, SDS has nothing to do with that, even precast gels will contain SDS, because that's the only way you will be able to separate your proteins by size in a one-dimesional gel, unless you are running non-denaturing gels to study protein complexes. Some facilities don't like silver staining of your proteins, which is more sensitive than Coomassie, but today you can cut large pieces of the gel containing multiple proteins and still have a quite good identification of all the proteins in there, just ask in advance to your facility.

What you have to worry about when you prepare samples for MS in general is keratine. This is in your skin and hair, so it is very easy to have it everywhere, even if you think you don't. This means that extreme care is necessary not only for preparing the gels, but also all the solutions and the equipment used. We wash thoroughly everything in 0.5M NaOH and then distilled water (bench and equipment - glasses, combs, running chambers, bottles for solutions, etc). We wear fresh double gloves (because the first pair of gloves you will touch with your hands, although you will be careful to touch them not on the fingers' side!), hair and mouth protection and of course a disposable lab coat (your regular lab coat is full of keratine from your body and everything you touch) to do anything related to MS, including preparing the lysate (from the lysis solutions to the tubes, the mouse tissue, and the loading buffer: if you work with cells you are already half way through... no hair from the mouse around!). In any case, expect lots of keratine hits in your MS results. Normally, since most of them are human, you can just rule them out if you are not working on human tissue. But it is very important to minimize their amount in order to increase the representation of the peptides from your sample.

Good luck.

HPLC-ESI-MS usually works fine with proteins separated and digested in self-made gels. 

Cheers, Christian

Hi Tomasz, no, SDS has nothing to do with that, even precast gels will contain SDS, because that's the only way you will be able to separate your proteins by size in a one-dimesional gel, unless you are running non-denaturing gels to study protein complexes. Some facilities don't like silver staining of your proteins, which is more sensitive than Coomassie, but today you can cut large pieces of the gel containing multiple proteins and still have a quite good identification of all the proteins in there, just ask in advance to your facility.

What you have to worry about when you prepare samples for MS in general is keratine. This is in your skin and hair, so it is very easy to have it everywhere, even if you think you don't. This means that extreme care is necessary not only for preparing the gels, but also all the solutions and the equipment used. We wash thoroughly everything in 0.5M NaOH and then distilled water (bench and equipment - glasses, combs, running chambers, bottles for solutions, etc). We wear fresh double gloves (because the first pair of gloves you will touch with your hands, although you will be careful to touch them not on the fingers' side!), hair and mouth protection and of course a disposable lab coat (your regular lab coat is full of keratine from your body and everything you touch) to do anything related to MS, including preparing the lysate (from the lysis solutions to the tubes, the mouse tissue, and the loading buffer: if you work with cells you are already half way through... no hair from the mouse around!). In any case, expect lots of keratine hits in your MS results. Normally, since most of them are human, you can just rule them out if you are not working on human tissue. But it is very important to minimize their amount in order to increase the representation of the peptides from your sample.

Good luck.

Thank you both for your answers, I'll be sure to keep in mind the precautions against keratin.

I can confirm the above answers. I think the use of self-casted gels is not a problem.

The procedure is similar to the 2D-PAGE analysis but instead of a spot you will have a slice. In 2D-PAGE the second dimension is in fact an SDS-PAGE.

I've carried out a procedure similar to your one here:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131519

Take care of environmental dust (mainly keratin) and usually the MS facility will require a blank slice of the SDS-PAGE that does not contain band that should be trypsin-treated as the other band slices. I've used only NOT-recycled Coomassie.

Thank you also for more info, If I can ask for one more thing. Could you please tell me, where are you sending your samples? Especially I would appreciate this info from Christian Q. Scheckhuber and Pietro Pilo Boyl.

Hi Tomasz,

I usually send my samples for protein identification to either Alphalyse (http://www.alphalyse.com/) or Proteome Factory (http://www.proteomefactory.com/).

Cheers, Christian

I'm used to carry out the work in collaboration with MS people who take part in the project. See the link above for address: Alberto Vitali and Valeria Marzano

Hi,

The problem with mass spectrometry a part keratins are DETERGENTS. But for sure it is not a problem when you work with gels (because the gel step is done to clean up your sample: It exists also in soluton digestion but then if you have detergents there you will through away your sample). So don't be worry. MS spec could be done with both home made and precast gels (depends from the complexity of your sample and from how much you wish to separate your mixture).  Make all solutions clean, put always glowes and labcoat.Actually we have also a facility so we can keep in touch.

Ciao

Simone