Tuesday, December 12, 2017
Trypsin may be a limiting factor in alternative splicing event detection in proteomics!
Thanks WikiPedia Alternative Splicing page!
Alternative splicing is a big deal in eukaryotic systems. One of the first big revelations in "next gen" transcript sequencing is that these aren't rare events.
However, there hasn't been a huge amount of data from the proteomics side to back up the numbers that the transcriptomics people have been finding.
This new study in press at MCP suggests it might be our fault -- primarily our reliance on good 'ol trypsin.
Earlier this year we had this bummer study on cysteine alkylation reagents -- and now another weakness of trypsin...? Isn't anything perfect?
Really -- this is only going to be a problem if you're specifically focused on alternative splicing -- it turns out that lysines and arginines are involved more often than other amino acids in these junctions. The trypsin cuts them up to things far too small to sequence -- or cuts them at just the right point that there is no useful information about the alternative splicing. (The espresso must have kicked in just now, there isn't a single period in that paragraph. Maybe I should go back later and add one).
These authors in silico digest some human proteomes simulating different enzyme activities. Trypsin doesn't lose all the information, but it appears that chymotrypsin and AspN provide better coverage of these sites -- however, as in all things it looks like using all 3 (separately, of course) will provide the greatest amount of coverage