isotopic profile of heavy labelled peptides

[Gautam Saxena]

I have an "amateurish" question possibly: should the isotopic profile of heavy-labelled peptides be any different than the normal, non-labelled peptides or should it be identical? (I'm trying to understand some spectra that I'm seeing where what I *think* is the monoisotopic mass has a much smaller peak than what *appears* to be the +1 peak for peptides with precursor mz of ~600Th at charge state 2....But, I could be interpreting the data incorrectly or it could be simply a peak from another peptide which is causing the interference etc, so I'm trying to shore up my theoretical understanding of "what ought to happen")

[Vladimir Gorshkov]

Hi Gautam,

The isotopic pattern for normal peptides is corresponding to natural isotopic abundance of constituing elements (i.e. ~1% of carbon is carbon-13). The monoisotopic mass (and peak corresponding to it) will be sum of the most abundant natural isotopes of elements, i.e. C12, N14, H1 etc. In heavy peptide, one or few amino acids is built including "unnatural" isotopic abundance (typically enrichment is about 95 - 99%, i.e. 95% of carbons are carbon-13, but the rest are carbon-12 and other isotopes). That to said, some of the molecules will be built with the only most abundant natural isotopes, so you can expect to see smaller peak of "non-heavy" peptide.
There are many tools, both offline and online to predict the isotopic pattern by elemental composition, some of them allow defining non-natural isotopic abundancies. for example http://yanjunhua.tripod.com/pattern1.htm You can try to play with them, to get the feeling how isotopic pattern changes.

Best regards,
Vladimir

[David Bouyssié]

Hi Gautam,

This is a typical problem when using 15N labeling, the shape of the isotopic pattern of the heavy form is often slightly different than the one of the light form.

However this is less the case when dealing with SILAC labeling.

Be aware that the correlation between theoretical and experimental isotope patterns is very often far from being perfect for multiple reasons.

If you compute the theoretical pattern by using an averagine formula then you may have a wrong estimation (over/under) of the number of atoms contained in the considered peptide. This can be more or less problematic in function of the nature of peptide (i.e. presence of specific PTMs).

However, more often the main problem comes from the experimental data.

For low abundance species, the isotope pattern abundance distribution is not always accurate/reliable.

You may increase the accuracy by averaging the patterns observed in multiple spectra, if you can do it of course.

Best,

David