There should be support for ECRs as well. There are also country specific programs, I know Norway, Sweden and Denmark have recently announced their own programs. Just type in country of interest and keep checking, I assume most of EU countries will announce similar programs this year.

Yes!

We will look at proteomics in pharma industry from multiple angles
🌐How it integrates into the pharma matrix environment.
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...Looks like I have to force myself finally write that paper on this 😅

I also just remembered there was another, scientific, reason. When enriching protein the molar binding capacity is actually lower than when enriching peptides - some binging sites will become inaccessible because they are spatially blocked by the protein size. This was advantageous in our settings.

Economic reasons 🙂 the biotynated proteins make up only ~2% of the sample. Digesting enriched fraction resulted in 50fold savings in trypsin cost I think we went from $25k to $500 for one study.

Happy to help🙂

If those look good and issues prevail, I fall back to some relatively accessible positive control samples to pinpoint the issue. Alternatively you can use antibody against biotin for pull down to bypass streptavidin and check that the sample quality is good. Good luck.

That's too difficult to say. I would try with fresh batch of beads and if that doesn't help look for other reasons for failure. IDs are the ultimate read out, it is important to have reliable check points along the way like the yield and protein concentration if the import sample.

I discard the non-biotinglated peptides. Too high likelihood that some come from non-specific binding. Unless you need to pool to get over the sensitivity threshold. Even in that case I would only consider hits where there is biotin tag/specific fragment detected on given peptide/protein.

Absolutely, just want to mention than in order to optimize the labeling steps it it essential to understand the performance of the pull down and it's reliability (hence the need for positive control like the biotinylated cell lysate or similar).

And for elution. We also did on-bead digestion which allows for elution with high acid+ACN steps -> dry down resuspend in LC-MS injection buffer. There are some publications on this. If you tune all steps you can get to >90% peptides biotinylated in the final sample.

Can be hard. I would make a rough estimate from the prevalence of the PTM. We have done similar thing where we specifically targeted small % of proteome. A good positive control sample was biotynating HeLa lysate and then mixing it unlabeled HeLa in expected ratios. Allows MD without precious sample

Also, I've heard there are some bad batches of streptavidin going around.

For us the main optimization factor was carefully matching binding capacity to expected amount of biotinylated protein. Or rather making sure there is more biotinylated protein in the sample then binding capacity. The unspecific binding is basically unwashable.