ESI LC/MS of gelatine hydrolysates

[jrussell]

Hi all,

I hope someone can help me. I apologize upfront for being long-winded, but I think some background will help to explain my situation.

I am a 1st year graduate student working on my master's thesis, mainly pertaining to the characterization of different gelatine hydrolysates (I also work for a gelatine manufacturer). I am interested in determining the distribution of peptides in different enzymatically digested gelatines to see if an enzyme or a combination of enzymes produce different ratios of peptides and/or free amino acids. There is probably a better way to do it, but I would like to use ESI LC/MS, and from what I have gathered, it is a fairly novel technique in the gelatine industry, and hopefully I can convince my boss to buy a system if results are good!

Before I started doing this research myself, I had a local university run a gelatine hydrolysate sample for me. They used a Shimadzu LCMS 2010 mass spec using ESI in the positive ion mode. I have serious doubts about the validity of the test, here is my reasoning. Gelatine is basically 33% glycine. After harsh processing of collagenous tissue to make gelatine (bovine bone in this case), and then enzymatically digesting the gelatine to an average molecular weight of 2000 Da, I would expect to see some free glycine. However, there was not one peak that corresponded to free glycine, or any free amino acid for that matter. This makes me very suspicious. Maybe there are not any appreciable amounts of free amino acids, but I don't think this is true.

Does anyone have any suggestions on how I can proceed? Does anyone know of anyone who has done work similar to this? Is this a bad idea?

Please be gentle, I still don't know a lot about this subject, but am very excited to learn everything I can. I just found this forum, I think it will be a valuable resource. My boss told me this research may be a dead-end leading me to become a career student, but I am stubborn, and at least I will know it is a dead-end instead of only wondering if it is so.

Thanks for your help,


JRussell

[MG]

I am new to proteomics, and my background is in small molecules. So keep that in mind when reading this. I say that because what you are doing sounds similar to what my colleages do to look for proteins. That is, they digest a protein sample into constituent peptides. Whether they see free amino acids, I don't know, but to my knowledge they aren't usually looking for them. Rather, they are looking for larger peptides.

You said gelatine is "33% glycine". Do you mean that it contains this much free glycine already, or is that glycine bound up in proteins or peptides.

If the glycine is bound up in larger molecules, then there must be a biochemical reason why the enzymes would yield free glycine rather than larger peptides. You will have to answer this for yourself, because I don't know. You stated that the average m.w. of the digest was 2000. Given that, is there a reason why glycine (m.w. = 75) would be present?

OK, let us assume now that free glycine should be present. One reason you aren't seeing it could have to do with the conditions used for the MS. First, the operator must have been scanning low enough. Make sure the scan range was down to ~ 70 m/z. (It is common to use 100m/z or higher as the lower scan limit, due to low mass noise present in LC/MS). Second, if this instrument is an ion trap, be aware that they don't do as well with low masses. You should be able to see the m/z 76 ion that would be observed in ESI(+) for glycine, but the "low mass cutoff" or "trap drive", corresponding to the R.F. amplitude on the ring electrode, must be set properly.

Finally, be aware that under typical reverse-phase LC conditions, glycine would probably be unretained by the column, and may coelute with any salts or other unretained polar compounds. If it coeluted with any sodium in the sample, for example, then you might see sodium adducts (M+23) rather than proton adducts (M+1).

[jrussell]

MG,

Thanks for your reply. Gelatine is about 33% glycine as measured by the proportion of amino acid residues in a given chain. In gelatine that gels, most of the glycine is bound. However, when I made the gelatine hydrolysate (non-gelling) I used a series of enzymes, including some that are strictly exopeptidases. It makes sense to me that if an enzyme were to clip off an amino acid one-by-one from the C or N-terminus of the peptide chain, some free amino acids would be present, with the largest proportion of those being glycine. Some Brazilian colleagues performed a free amino acid analysis some years ago and confirmed that free amino acids are evolved through enzymatic hydrolysis, however, I have not personally confirmed their data. As for the average Mw, SEC shows a mass spreading from roughly 100 Da to 4000 Da. This should also indicate the presence of free amino acids, but once again, this may be hand-waving on my part.

I will consult with the university that ran the sample for me and bring up the points you addressed. I appreciate your help.

JRussell