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!

Ming,

Sounds like you need to troubleshoot this by doing a few tests. That is why I recommended Kuwata's suggestion.

My sense is that something is different in your sample solution as compared to the controls (could be salts,carbs, lipids, glycerol,detergents  etc).

I would suggest making a mock solution of extract using everything but the cells. Then add some of the standard peptides to this solution and see if it alters the profile of elution. If it does at least you know the problem resides in your sample preparation solution.

I would also suggest diluting the peptide HELA solution 1:10 into starting buffer and run that. If you get the same profile I would agree with Degterev in that your sample is just different in that it contains this type of composition of peptides and there is no real problem. If so you may need to alter chrome conditions to get good separation of these hydrophilic peptides.

Good luck.

Amended on 18 May 2018

I must say that ODS silica gel should be used in acidic phosphate buffer (pH 2.0) conditions (please see files; Monch Protein separation and Lysozyme by RP-HPLC). Silica gel is labile to alkaline eluents.

Please, handle the silica gel without using TFA. Acidic TFA solution generally gives low recovery of peptide or protein as compared to acidic phosphate buffer (pH 2.0) (my unpublished observation at The University of Tokyo, Yayoi-cho, Bunkyou-ku, Tokyo, Japan). Since MS is not a quantitative detector at all (personal communication from the Analtical Chemical Center (Bunseki Center), Shimadzu Co., Kyoto, Japan), the acidic phosphate buffer seems not to reduce the signals (my personal and unpublished observation). Therefore, desalting before MS analysis is not necessay at all (my unpublished observation at The University of Tokyo, Bukyou-ku, Tokyo, Japan).

I would like to recommend that fractionation of peptide by the above RP-HPLC method (please see again file; Lysozyme by RP-HPLC), and fractioned peak should better be quantitatively identified by Edman degradation method (please see file about PDMD (protein/peptide-direct-microsequencing-deciphering) method; HepG2 fucoidan).

I also would like to say that "slow but steady wins the race".