Saturday, January 17, 2015
DigestIF - A QC standard for tryptic digestion!
This falls under the: Why the heck didn't I think of this!?!?!? category.
One of the reasons the genomics people sometimes get to eat our lunch during grant competitions is their reproducibility. It's high. Crazy high. From lab to lab and from sample to sample. A big reason for this is the fact that every person using a certain sequencer uses the reagent kit provided by the manufacturer. They use that kit and follow the directions to the letter. People that make variations to those instructions are treated as outcasts and forced to fend for themselves in the desert alone.
I probably exaggerated a little. But it seems that way. In our field, however, no one preps their samples the same way. Sometime in the next 4-6 weeks I will be visiting my 100th proteomics lab. And I can tell you for certain that no one preps their samples the same way. Even in the same lab you'll find one guy who preps with FASP and another person who only does liquid digestion. And even if two people are using FASP, you'll find different concentrations of ammonium bicarb or iodoacetamide between people. We've been used to tinkering with things. We find better methods that way. But when you're up for a clinical grant and the money goes to the guy with the hi-Seq and not you, this has a lot to do with it.
<End pre-coffee grumpy tirade>
So, what is this DigestIF? Its a QC standard for determining the efficiency of your tryptic digestion! Its a synthetic protein that gives off peptides of varying hydrophobicities when digested. So you have not only a read out of your digestion efficiency, but you have a way of troubleshooting your LC and MS once the peptides are produced.
A LOTof work went into designing the protein to make it realistic, with regions that are easy for trypsin to get to and also regions that are difficult to digest. Lets go back to the first sentence in the blog. Okay...maybe I would have thought of having a digestion control, but I wouldn't have thought to (or had the necessary knowledge to...) design a protein in that way.
So this probably falls under the: Why the heck didn't I think of this idea and then tell someone with the skills to pull this off?!?! Fortunately, I didn't have to. These guys came up with this and its brilliant!
Eventually, I think we'll see a lot of standardization in our field, particularly for when proteomics really takes off in the clinic. Until then maybe we can use something like this and be able to control to make sure we're at least all getting the same digestion efficiency. Even better, maybe this is the control we use in the future that will help us get to the best standardized method.
I think everyone should read this paper from Dorothee Lebert et al.,. There is some guy named Aebersold listed as an author on it as well.
TL/DR: You can spike this protein into your lysate and it will make an amazing QC standard for your whole system, including digestion. Ben needs to get this puppy off his lap and get some coffee...
Subscribe to: Post Comments (Atom)
Post a Comment