Hi Marieluise,

1. Trypsin is very adequate. where the C-term will have Arg, your N-term will be still labeled.

2. Hard to tell - depends on your instrument parameters, but I would use the manuscript as a start (assuming you don't have the method already on the instrument).

3. the idea of this method is cleaning up co-isolated peaks that may skew your quantification. makes the quantification better, but the throughput slower. 

Cheers,

David.

Dear Marieluise

the orbitrap fusion parameters for the synchronous precursor selection are described in detail in the following publication of the Gygi group (PMID: 25521595).

Hope this will enable you to perform nice SPS MS3 Fusion experiments.

In our hands, trypsin digestion was sufficient as each N-terminus is still labeled and b-ions are prominent after TMT-labelling.

So for cells in culture MS3 ID rate for fractionated samples showed to be nearly as good as for MS in HCD mode.

 Kind regards, Stephan

Hi Marieluise

The MS3 vs MS2 question is one that you will have to make a choice for yourself depending on your application. MS3 will remove more interference and will give you more accurate data and will reduce ratio compression but one of the advantages of TMT is that it has relatively high precision in both MS2 and MS3, i.e. the data is quite reproducible. If you really need to see a 5-fold change accurately then use MS3, but if you only care about seeing the fold-change and are not too concerned about the exact fold-change, then you should be okay with MS2.

For early stage screening you may prefer the higher id rate of MS2. We also find for phosphopeptide work that MS2 gives better coverage and sensitivity. In general for PTMs, when you want to find as many modified peptides as possible, MS2 would probably be recommended. Interference is also typically lower in samples that are highly enriched for PTMs such as phosphopeptides as you are getting rid of a lot of the potentially interfering peptides so again MS2 is adequate.

As my colleague Stephan mentioned above, the overall protein ID rate (particularly in a 2D LC study) will not be reduced in MS3 by as much as you think due to the redundancy of ids at the peptide level but obviously you will get a more substantially reduced number of peptide ids. For a more standard proteomics experiment where you are only looking at protein IDs, the quantitative accuracy from MS3 may be preferred.

The choice and tradeoffs thus depend on what your application is.

Kind regards, Andrew