Hi, Reza -

Can you give us some additional details?  It's difficult to answer your questions without a better idea of what might be happening.  There are many possible reasons for your problems, but it's not really clear what the problems are.  For example, are you using a QTOF, an ESi-TOF, or a triple-quadrupole for your MS experiments (i,e., are you getting exact masses for your peptide ions or only nominal masses)?   Have you done enough runs with known standards to be familiar with the systems (i.e., do you get good MS/MS spectra with, say, a well-defined commercial digest, but only bad spectra with your actual samples)?  How long between the MS experiments and the MS/MS experiments?  If you re-run MS experiments with the samples after storing them and redissolving them, do you get the same  results that you got the first time?   Are you just beginning to do proteomics or are you experienced in the area and this is the first time you've had problems?  Fill us in a bit.

Best regards.

Pete                                  

Hi, John-

Thank you for your answer and your attention.

I am using MALDI- TOF/TOF ( Bruker ) and I am getting nominal masses. I have done enough runs with standard and I got good MS/MS spectra with standard but bad spectra with my actual samples. It is almost five months between MS experiments and MS/MS experiments. When I rerun MS experiments, I didn't get the same results that I got the first time. I am beginning to do proteomics and I don't have more experience.

Best regards,

Reza

Is it your actual sample has volatile in nature or stable?

Hi Reza, 

Can you also comment on the complexity of your sample?  If it is whole proteome digestion and there are interfering compounds (e.g. buffers, denaturants), or even if your matrix is old and degrading, MALDI could be very irreproducible.  MALDI-TOF/TOF is best for very simple samples containing only a few proteins.  You might want to do some control digestions with a standard protein such as BSA to understand your technical variation.  Also try to get nLC-ESI-MS/MS data if possible because the separation provides valuable interference reduction and chromatographic information.

Best,

Jesse

Storing peptide samples for 5 months at 4 degrees celcius is not advisable. They can be kept at 4 degrees for several weeks or a month at most. If you intend to keep them longer than a week, it is best to freeze them at -20 degrees.

For now, I think it is best to redo the experiment and do both MS and MS/MS measurements in one go.

Few additions to what others already said.

- storing:  a complex sample does not have a "specific" storage time at 4C. There are peptides that are gone within hours at that temperature. It is advisable to organize your experiment so that the analytical part (all of it) happens at the same time (or close to).

- MALDI ToF-ToF: this is not the best instrument to do MS/MS. You do it only if it is strictly required. Having results as quick a a heartbeat is fascinating, but you are not applying any other separation step aside the mass separation. As Jesse pointed out, a complex mixture can give you extremely irreproducible results.

Some examples: if you have an expressed protein which was highly purified, MALDI is the perfect instrument (time-sparing).

If you have more than one protein, you may try to run a MALDI experiment (with nr. of proteins < 10 and a very pure sample). Or you may directly switch to an LC-MS/MS technique. Alternatively, perform packed-tip fractionation.

- MS/MS on a MALDI ToF/ToF: remember to modify the parent ion selection window. Bruker instruments select wide ranges (e.g. for a 2kDa peptides the range is about +- 12 Da). If you don't, you might include neighboring peaks and get a very messy MS/MS spectrum.