Thursday, August 18, 2016

Nanospray single run analysis of peptidoglycan!


I'd wondered how long it would be till someone pulled this off!  Bacterial cell walls are sugar chains connected by peptide crossbridges. To get to it you use enzymes that destroy all the DNA and protein and you're left with the cell wall. You chop what is left up with lysozyme, reduce the sugars so they don't all elute in a single peak and then use LC-LC-MS to quantify the compounds and figure out what they are to reassemble a picture of the cell wall.

Marshall Bern (wait, I know him!) et al., show in this new study that they can ditch the first LC part. Just run the digested reduced peptidoglycan out on a single dimensional LC gradient and use ETD and HCD and BOOM!  you're done. (That sentence is too long).

By simplifying it they get to see some new, low-abundance muropeptides (glycopeptides) in C. difficile that they haven't seen before.

The paper points out that the traditional method is slooow. You use a phosphate buffer system to really get very clean separation of your compounds and fraction collect them. The phosphate buffer system is flushed out and then the remainder is analyzed with reverse phase. Yeah. Its slow. But this is the reason they've been doing it this way:

(From a Dave Popham paper. They all look like this.)

Using the phosphate buffer system you can quantify it like its a NIST standard.  Can you get a 4% CV? Heck yeah, you can.

Just shooting muropeptides on reverse phase alone as they did in this study?

Great for finding new muropeptides!  Ummm....maybe not so great for quantifying them and ultimately elucidating the peptidoglycan 3D structure? Hmmm....if only there was a quantification technique that didn't care about peak shape...then you'd have the whole package.

But for straight-up discovery of new muropeptides? Single shot 1D ETD/HCD with Byonic processing looks like the solution!


  1. Thank you, Ben, for the great blog post! Just to add a word on quant -- we used XICs rather than the blobby TIC for quantification. And we quantified PG components over four "logs" that way. We didn't have technical replicates in this study, but I don't think CV would be a problem.

    And in something of a surprise to me -- ETD is great for PG analysis !

    1. Its a nice work. Could you isobarically tag it and then multiplex the quan? There are some glycan tagging reagents...but reducing the terminal sugar might complicate it?

  2. HI there. Could you please direct me to the source of your peptidoglycan image? If it is yourself, could I please request use of it in a research project proposal of mine for my honours work?

    1. I got it from Google Images. It appears to have been on a test someone posted online from Georgia University.,d.eWE&psig=AFQjCNHOIZoYAydVWjB0Jk4kfLZ5L0V5Vg&ust=1489504826150982