Nazhan,

can you see the LDL-R in Western Blots? Might be an extraction/solubility issue. Is it situated in lipid rafts?

Hello Nazhan Ilias

Can you tell us more about the gene product (protein) of low-density lipoprotein. What is the UniProt accession number? From that, you could predict the m/z value for the protein (or peptides) you may anticipate.

Next, what steps were performed to produce the LC-MS proteomics results? As LDR contains fat and protein molecules, was it confirmed after extraction from the precipitated material? And were the extracted proteins digested with trypsin to produce peptides for LC-MS?

If you can answer the questions above, maybe we can move forward a little more.

Good luck.

Best,

Peter

First of all, thank you for the responses Peter Allen Faull and Wolfgang Schechinger

Peter Allen Faull

1) Since our data doesn't have the desired protein, so I've checked the accession number from UniProt which is supposed to be P01130. I haven't screened the peptides part yet though. This protein is a receptor on the cell membrane. Anyhow, several proteins related to the pathway that we're looking into exists (but somehow the proteins involved are incomplete).

2) As for the method, cells were treated accordingly and extracted by mechanical detachment using a cell scraper. Cells were homogenized in buffer containing protease inhibitor and protein concentration was determined by BCA assay (concentration was fair enough). From there, we proceeded with protein precipitation adapting the methanol-chloroform method. Next in-solution protein digestion was done using LysC/Trypsin. Finally, separation using EASY-nLC with C18 RP column. MS 1= 310-1800m/z and MS2= 110-1800m/z.

Question: You did mention that if I have the accession number, I could predict the m/z value. May I know how? Would it be from screening our data?

Wolfgang Schechinger

1) Another problem is that our CCD cam is broken so less likely to proceed with WB. Although it can be done elsewhere, access is quite restricted due to the pandemic.

2) Yes the protein is located in the lipid raft. Based on the method I briefly explained above, does it interfere the current results?

3) As for the extraction/solubility you mentioned, we highly suspected that the problem could be due to low abundance...

Nazhan,

I think to confirm that the receptor is present and that it has been solubilized is mission critical. For the Western, instead of using ECL, you could e.g. try a visual staining procedure, based on the precipitation of a dye. There are ready to use kits on the market.

Then, you can start to establish a procedure (i.e. a suitable detergent mix) that will dissolve the receptor from the chloroform-methanol (HCl?) precipitate. As you're removing ~all lipids during this procedure, dissolving membrane proteins for the mas spec analysis could become quite difficult.

Alternatively, you could try to dissolve the cells on the dish with up to 1% Tween 20 or TX100 in PBS. This will leave the nuclei and the cytoskeleton on the plate but should get you all other proteins. This also may be down after scraping the cells of in a tube, mixing well and centrifuging the insoluble material down. If you can't see your receptor yet in the mass spec, try adding other detergents as you should find with a web search for "solubilisation of lipid rafts".