Monday, March 7, 2016

Natural genetic variation measured in nematodes shows mRNA levels are a small part of the picture!

...darned nemotodes...

Wait...natural genetic variation? This is a proteomics blog (most of the time..) don't you need PCR or some super sensitive genetic technique to measure genetic variation? NOPE!  You can do it with mass spec, and you should be doing it with mass spec, and we need to get that into people's heads out there!

This brand new study from Polina Kamkina et al., is a great example. In this study they work with some hungry hungry nemotodes using SILAC for protein abundance and microarrays for transcript abundance. What did they find? That "...only a fraction of the changes in protein abundance can be explained by changes in mRNA abundance."

This isn't particularly novel. We've seen this before. In this nice model organism, they come up with a higher correlation between the transcript abundance and the protein abundance than we've seen in some other systems, but its still just a fraction.

Which begs the least a little...why does it appear that transcript abundance is still king over lowly protein abundance? Which may be another rant for another time. Maybe...

From a transcript level we might argue here that microarrays aren't exactly the cutting edge, so its a somewhat unfair comparison of technologies, but this organism is pretty simple and extremely well characterized.

Anyway, this group used SILAC and a couple of very divergent nematodes. They did some top-notch proteomics here with sample pre-fractionation with high-pH reverse phase into 47 fractions. They pooled the samples and normalized the fraction pooling content by UV absorbance at 214nm and use their own in house peptide mixture to QC their separation (nice extra steps!)  They quantify using a combination of MaxQuant and Progenesis IQ and do their enrichment analyses and statistical analyses using some R packages. To top it off they go back in on their quantifiable targets and verify them with Skyline. This group doesn't mess around. If they say its differentially expressed, its differentially f'racking expressed!

All the data at the end was uploaded into the WORMQTL (which I assume is something well-known in the nematode world. And the RAW data was put into PRIDE.

Absolutely a solid analysis that is another great example that you need to look at the proteome level if you really want to know what is going on in your organism. Again, stuff we're all to aware of, but that the biologists out there really need to get on board with!

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