Just as showed in attached picture, in a 120min LC-MS run, there is a high and wide peak group in about 15-30min and in the rest of the elution time, the peak intensity is low which means that most of the peptides are eluted very early.
But, the HeLa Cell Lysates Trypsin Digestion Sample used for the standard checking the mass spec's state has a very well-distributed TIC or Base Peak shape. It means that it is not the problem of the instrument.
Then I re-prepared all the buffer,solution and reagents to insure no contaminants was introduced in the process of cell lysing and FASP reduction and alkylation,digestion.
At last I use new prepared buffer to do the C18 tip desalting.
But the problem still happened.
I am not a green hand in proteomics sample preparation but now I am in a new place to do the experiment and this strange problem happened.
I am sure that the instrument is no problem as other people's sample didn't have this kind of problem. But I have re-prepared all the solution in the experiment and it should be no contamination and I desalted the peptides very carefully using C18 tips. But the strange phenomenon still exists. Why?
Anyone has any ideas for this very very strange phenomenon? Thank you very much.
Dear Ming Yang
Withouth information regarding the type of LC system (nano, capillary, standard flow?) and of mass spectrometer used (it seem Orbitrap TIC) it is difficult to understand the problem. However, it seems to be a gradient or dwell volume problem. Check for LC connections, void volume, mixer unit and gradient slope. In nano LC, small leakage can be detrimental, and it has consequences on gradient, peak shapes, spray stability and signal intensity
Best regards
As Eduardo already mentioned, it is diffcult to find the problem without knowing your system in detail. In addition to the dwell time, there might be more problems such as:
* Solvent content at the start of the gradient is too high (-> start with more water in the gradient)
* Solvent content in the sample is too high (-> Evaporate & redissolve in water, or dilute sample with water, or inject less volume)
Thank you Eduardo Sommella and Michael G. Weller very much. As other's people's sample is normal and only my sample has this kind problem so I think it is not the fault of the instrument. There must be somethings wrong in my experiment. But as I have re-prepared all the solurtions to ensure no pollution in the reagents and solution so I can not understand what pollution can cause the peptides binding weakly with the C18 material. If not the reason of contaminants, and the start solution conditions are the same as others...so, where is wrong?
May be I need to use other guys' solutionn and materilas to test again....
Other's people's sample is normal and only your sample has problem........ Please digest and clean up your sample (sample 1) and other sample (sample 2) at the same time (using same reagents). If samples 1 and 2 have same problem, it is clear that reagent or something is wrong.
Dear Ming
First of all, you did not provide any chromatographic and MS-conditions of your analysis. It is very hard to propose what could be wrong when you don't know what is right. Nevertheless, I have several thoughts about your situation:
- Does it a real problem? Maybe the nature of your sample is very different from the standard ant it really contains mostly hydrophilic peptides. Does the results of your experiment are really bad?
- Did you perform a compound search across the whole chromatogram? Usually it is not very robust to estimate a compounds content based only on a TIC intensity. Also, more hydrophobic peptides may have less well chromatographic properties, so the tailing of their peaks could cause a MS signal intensity fall.
- Did you repeat a runs for the standard sample? Maybe the problem appeared after HPLC-MS system suitability check
- Try to use a different standard (some type of the commercial peptide mix containing different hydrophobicity compounds)
- Make your m/z range a slightly wider and perform a test run to check a sample for the too short or too long peptides
- Check your protease specificity (by perfoming a previous step, for example) or use an enzyme from another lot
Good luck!
- Badges
- Science topic
- Similar topics
- Chemistry
- General Biochemistry
- Proteomics