I think MS-based proteomic techniques can identify 35S-labeled peptides from tryptic digests given a database of peptide sequences in which the isotope replacement is specified. The MS facility may object to being given radioactive samples, so you should check with them first.

The latest generation of FT-ICR instruments have what they are calling isotopic fine structure, basically just really really high mass resolution.  One of the specs for installation at the moment is 10,000,000 resolving power at I think m/z 609 in a narrow band mode.  It depends on what m/z your ion will appear at, and what the mass difference will be, but in many cases what you are describing is entirely possible.

Adam and Mark,

Thank you for your answers.

So even if the mass of a labelled peptide is only 3 Mw units (35 - 32) larger than its unlabelled counterpart, MS can still differentiate between them (I think the charge will be the same)? And it seems the only problem left to me is to find an available MS machine with high enough resolution?

In our department, we only have something like AB SCIEX TripleTOF® 5600+. Is it good enough?

Thx again.

HY

It comes down to the size of your molecules whether the resolution of your instrument is good enough, but since you said you want to measure peptides (instead of, say, large protein complexes in native conditions), I'm quite sure your TOF instrument will be able to resolve the difference of 3 Da. I think with your instrument you could probably even go ahead and sequence the peptides (with MS/MS) and find your individual methionines, which at least should verify their isotopic labeling.

And as Adam said, there is always the concern of whether you will be allowed to measure radioactive samples, even though the radioactivity of 35S is relatively low and for such an MS experiment I'd also expect minute sample amounts.

Thanks Arne. I'll consult our MS facility to know if they receive 35S samples.

But what about the quantity of each nascent peptide in my samples. For example if I have the two samples, from WT and mutant yeast, both pulse-labelled with 35S (so they cannot be sent into the machine at the same time), can I quantify the (relative) abundance of a nascent peptide by certain means (e.g. spectra count)?

For quantitative comparison between samples, consider using the iTRAQ method:

https://en.wikipedia.org/wiki/Isobaric_tag_for_relative_and_absolute_quantitation

@Adam. Thank you for reminding me this. So I pulse label newly synthesized peptides in both WT and mutant with 35S in vivo; but iTRAQ in vitro only the sample from mutant? If I can do this it'll be great!

Dear Hy, isobaric tagging (TMT or iTRAQ) are probably not appropriate here as the 35S labelling renders your labelled peptides non-isobaric. You are also unlikely to find someone willing to contaminate their mass spec with a 'hot' label. Do you really need the 35S. Depending on your objective, you can do pulse/chase experiments with stable isotopes:

http://journals.plos.org/ plosone/article?id=10.1371/ journal.pone.0080423

If you want to go the mass spec way, you should consider this approach instead 

Dear Andrew,

Thank you. Great suggestion!

I figured this out the other day when I was told by our MS facility that only stable radioisotope is allowed there, and decided to use SILAC Methionine.

As for isobaric labeling, since I only care about SILAC-Met-labeled peptides (and the ratio of labeled peps in WT to mutant), is it OK I still use this method? For example:

Peptide X contains 3 Met sites. After pulse-labeling with 14C-Met, newly synthesized X will have 8 different labeling states: 3/3 labeled (1 state), 2/3 labeled (3 states), 1/3 labeled (3 states) and unlabeled (1 states). With different isobaric tags for WT and mutant sample, I can still compare the quantity between WT and mutant for each labeling state (e.g. 3/3 labeled with 3/3 labeled, still isobaric)?

Thanks a lot for the help. I really should know more about MS, really a useful tech.

HY :)

Hy, 14C is radioactive too. You mean 13C. Usually with SILAC, you aim to generate a 'heavy' cell line by growth in the appropriate medium for long enough to more or less replace all the undoped Met. Obviously for pulse/chase, you will get a more complex mix and resolving incomplete incorporation is a bit more challenging but I think you can do this with the MaxQuant software package from Matthias Mann's group. I am not an expert on MaxQuant so I would try and find someone who is.

If you want to compare two separate pulse/chase experiments than labelling on top with an isobaric tag will work. I would use TMT rather than iTRAQ not just because we make it ;-) but because TMT is fully isobaric (all the tags have exactly the same isotope composition) so this will not complicate your spectra even further in the MS-mode. iTRAQ tags don't all have exactly the same composition due to their use of 18O in the 4-plex and since they don't publish the full 8-plex structure, you just don't know. You can also obtain the TMTduplex reagent which is a little cheaper than the 6/10-plex tag as it is only doped with a single 13C in both tags.

Oh, yes, 13C, or 15N :)

I'll take a look on MaxQuant.

Hah. In the past we do TMT when we had a former student of my boss as our MS collaborator. But now we can only do MS in our facility, and only iTRAQ. Maybe I can advertise TMT for you to our MS facility? :)