Hi,

Which output did you obtain after LC-MS/MS? Few years ago I performed a knock in model overexpressing a small protein, but it did not appear in our proteomic results (we performed SILAC). In that case, that small protein did not display any unique peptide, so we were unable to check it by MS. If you received only a list of the differentially regulated proteins, you may have a similar issue.

Otherwise, if you have access to all the proteome data, did you performed your WB using some internal control? You may normalize the LC-MS/MS data using the same control protein.

Hope some of this info may it help.

Hi Saurabh Tiwari,

since the both methods are very different in the way to make a comparative quantification of a protein, there can be several reasons for the results to be different. What do you mean with beeing confident with the WB results? Have you performed it multiple times and also with different normalisation standards? Do the protein of interest exists in different proteoforms and maybe your WB antibody is specific for one proteoform only, whereas the LC-MS analysis has been done with summing up all together? Again, there can be several reasons for different results when comparing WB and LC-MS results. It is hard to find any possible explanation without more info on how both experiments were performed and analysed.

Best,

Murat

Hi,

In addition to Murat Eravci, There could be several reasons why the protein was not upregulated with label-free LC-MS/MS despite seeing an increase in target protein concentration through Western blotting. Some possible reasons could be:

1. The protein may be present at low levels in the sample, making it difficult to detect with label-free LC-MS/MS.

2. The protein may not be differentially expressed at the level of the proteome, but rather at the level of post-translational modifications or protein-protein interactions.

3. There could be technical issues with the label-free LC-MS/MS experiment, such as poor sample preparation or data analysis.

It may be helpful to investigate these possibilities further to determine the cause of the discrepancy between the Western blotting and label-free LC-MS/MS results.

Hi Pere Llinas

Thank you for your comments.

I have received the proteome data, and the target protein was identified and Validated by Scaffold 5 software. However, it was not differentially expressed.

As I previously mentioned, we performed Western blotting first (we found an increased target protein concentration during treatment) and then MS. It will be a good idea to check the results after normalizing the LC-MS/MS data using the control protein.

Hi Murat Eravci and Rajan Singh

Thank you for your valuable input.

Our target protein is an isoform of Na+ - K+ ATPase (alpha 4 isoforms) and I expect that it is present at low levels in the sample (Based on normalized emPAI values in MS data). It's interesting to see that MS data does not have an alpha 1 isoform of Na+ - K+ ATPase, which I expected to be present. Although, several beta isoforms of this protein were present.

Western blotting was performed in five biological replicates with a reference standard for normalization using an antibody specific to Na+ - K+ ATPase alpha 4 isoforms.

• I believe I should look into the differential expression at the level of post-translational modifications or protein-protein interactions.
• As Pere suggested, I will try to normalize the LC-MS/MS data using the reference protein and see how results change and if meaningful interpretations could be made.

Thanks, again for the discussion. I will keep the updates posted.

Hi Saurabh Tiwari,

in your initial question you have stated that you "did not see this protein upregulated with label-free LC-MS/MS" which suggests that your protein has been identified but is not significantly different from your control. From the provided info it seems as your isoform of interest is not assessed with your LC-MS analysis because of its low abundance. Maybe you can improve the sensitivity of your setup by increasing your amount of sample and run a longer gardient and if this is not successful maybe think about doing a prefractionation with an increased amount of your sample and analyse the sample fractions by LC-MS.

Can I ask what LC-MS platform you ave used for this experiment? and what software and protein DB you have used?

Best,

Murat