Hello Sven,

I think you are confusing DNA and protein here. If the protein is enzymatically cleaved, the gene encoding for it remains intact. The strategy you suggest would thus not give you information about the cleavage site. In general, I think that mass spectrometry would be the method of choice for identifying the cleavage site. The amount of protein you need for a MALDI-TOF experiment is not very high in comparison with other analytical techniques. But if you know which enzyme is used for the digestion, you can perhaps search for cleavage sites in the amino acid sequence of the expressed protein.

Hello Sven,

I think you are confusing DNA and protein here. If the protein is enzymatically cleaved, the gene encoding for it remains intact. The strategy you suggest would thus not give you information about the cleavage site. In general, I think that mass spectrometry would be the method of choice for identifying the cleavage site. The amount of protein you need for a MALDI-TOF experiment is not very high in comparison with other analytical techniques. But if you know which enzyme is used for the digestion, you can perhaps search for cleavage sites in the amino acid sequence of the expressed protein.

For search/predict cleavage sites in you protein you could you several online tools.

1. PeptideCutter

http://web.expasy.org/peptide_cutter/

2. PROSPER

http://lightning.med.monash.edu.au/PROSPER/

Hello Sven,

I totally agree with Guus, intact mass measurement of the cleaved protein is the most obvious thing to do. You could use either MALDI or ESI to measure the intact mass of the cleaved product and if your protein contains no PTM's the mass measurement will be accurate enough to go down to the amino acid level.

Thanks for your first answer. Because of the reason that the MALDI-TOF people say the concentration my colleage get is not high enough for MALDI-TOF the idea was to search for an other method, and PCR we think maybe can work.

Again, like Guus told you: You´re mixing DNA- and Protein-analysis. MS is for the analysis of Proteins. PCR is for the analysis of DNA! When there is a cleavage of your protein, than there is no way to use PCR.

Yes Oliver, you are totally right and Guus for sure also. It was more an idea that htese maybe work in which way we don't know. Sometimes methods exist you don't know - this was we hope for because as I say, MALDI-TOF not work.

Hi Sven. I read the comments of colleagues and I agree with them. The expressed protein is a protease, OK? Therefore, it is possible to attempt a panel of synthetic peptides to know the preference of hydrolysis of peptide binding. The kinetic values ​​indicate the preferred type of peptide bond and after sequence analysis of the target protein will be possible to know the cleavage site of the enzyme.

Hello Sven ,

Like the others I think that you are mixing DNA and protein. And I don't know about this shedding experiment but usually the process called 'shedding' is a kind of secretion (very often unconventional) which protein or vesicles undergo from cells.

Attached is an article where the authors use mass spec to analyze the cleavage site of a protein.. youcan check out whether you can perfom this expt too.

Hope these words help.

Article Determination of the Proteolytic Cleavage Sites of the Amylo...

Hello Carlos,

the expressed protein is a extracellular protein, no protease. So the enzymes do shedding in the way of extracellular domain shedding.

Vilmont, I know about the mixing, it was just an idea because maybe there is anywhere in the world a method I don't know (think there are many of these methods) which can help for the problem without using mass spectrometry. The shedding I mean is extracellular domain shedding, sometimes a little bit confusing because of the meaning of shedding you mentioned. Because of these problems I also write cleavage.

Dear Sven,

I think knowledge of amino acid sequence of her protein and the enzyme that she used to cleave her protein may help to solve the problem. Becase proteolytic enzymes have specificity of action and each enzyme has a specific clevage site. Trypsin specifically cleaves peptide bonds next to lysine or arginine. Chymotrypsin – next to aromatic amino acids. Elastase – peptide bonds formed by glycine, alanine and serine.

It's a matrix metallo protease

Matrix metalloproteases are a family of proteins. If you know which particullar metalloprotease is studied you can find its amino acid sequence and ID in UniProtKB/SwissProt knowledge base it is possible to try web services offered by Andriy Kubarenko (see above). Also you can use methods of identification of N- and C-terminal amino acids in the peptides obtained after digestion. These amino acids participate in formation of peptide bonds in site of cleavage.

Hello,

The most accurate way to find a cleavage site is to purify the cleaved protein in the polyacrylamide gel, digest the band by sutable protease (like trypsin), extract/purify peptides and analyze them by mass spec. When performing database search enzyme specificity should be "semitrypsin" because on the C-terinal side it could be any residue. I attached our recent paper as example.

If you want to know the cleavage sites of a given protein, assumed the aa sequence of the protein is known, then you can use peptide array to pinpoint them. On a typical protease substrate peptide array, numerous synthesis peptides derived from substrate protein will be spotted and are subjected to proteolysis by a given protease, and the cleavage peptides can then be identified. Pepmatrix Technology (http://www.pepmatrix.com ) can make custom proteolysis substrate arrays. thanks,

Thanks again for the answers.

if you are looking for cleavage sites, add a bunch of proteolytic enzymes, run the fragments on a gel, do a western transfer and send the samples for N-term degradation upto 10 AA. also send some sample for intact Mass. these should give sequence information as well as mass of the fragments. you can also try some online tood where it will predict the protease sites based on your AA sequence like peptide cutter. link attached

cheers

http://web.expasy.org/peptide_cutter/

I would focus on either increasing the yield of the target protein so that you can get enough cleavage product for MALDI-TOF, or using a different type of mass spec that is more sensitive. The sensitivity of a Q-TOF can easily be in the femto mole range, so you really would not need much target protein with it.

Oftentimes, people add a HA tag or other tags to the substrate that is to be cleaved, and then the fragments are purified, by affinity resin or immunoprecipitation. The protein/beads are placed right on the holder that goes into the MS machine and subject to Formic acid treatment or ammonium bicarbonate before the sample is analyzed to remove the bound protein fragment from the resin.

This has been used by Hinkle and Lee et al to determine the cleavage site of TGFalpha by TACE. Also, you can see Brennaman, Moss, and Maness, et al 2013, to determine the cleavage site of NCAM by ADAM10.

And for further instruction, you can contact my colleague, Leann Brennaman on Researchgate who has done all the work.

If it is a known MMP, then the sequence may be predicted to some extent by inspection. Most MMPs cleave two residues after proline... Pxx Phage display has been done with many of the MMPs. See Deng et al for MMP13, Smith et al, for MMP2 and 9 and Navre et al for MMP7 and 3.

So you can make peptides surrounding these areas and also mutate the sequence, by replacing proline with alanine, for example.

In the Brennaman paper, this is what was done, ie the suspected cleavage site was muta ted, and this prevented shedding.

Hi

The best and most affordable option Can be clone into a vector and sequence this with primers of the vector. then the site could be help to you http://www.cbs.dtu.dk/services/

If you have a good proteomics LC/MS/MS setup at hand, working on the peptide level might help as it is around 2-3 orders of magnitude more sensitive than intact protein MALDI-TOF. If you can isolate your cleaved protein (Tagging and Fishing; or simple SDS-PAGE) you could digest with trypsin, and then perform a ragged end/semitryptic search. With some luck you might find peptides involving the unknown cleavage site, or from the sequence coverage map at least be able to narrow it down to a region. Simone König in your faculty should be able to help you here.

I tend to disagree with a peptide level approach. It is a matter of chance whether the terminal peptide contain g the cleavage site is 'visible' in the instrument - too large, too small, 'flies' poorly. I would be much more inclined to recover the pure protein (folded or not) and simply measure the mass, using a good ESI-QTOF and using software such as maximum entropy 9Waters) to deconvolute the charge envelope. With a good instrument the mass should be accurate to at least 1Da in 20,000Da which is more than enough to define the cleavage site.