Yesterday I made a short post on this new study in Open Reports. I'll be honest, I thought it was a seriously impressive sample set, but I was a little skeptical about the results. Not that I didn't believe the authors could find the bacterial proteins, but I was skeptical that the results could be that clean cut. I've seen a few "identification of digested bacterial proteins in body fluid" studies and even with high resolution data the matrix is complex enough that there is noise there. These results just seemed a little too good to be true.
Shoutout to ProteomeXchange/PRIDE and these authors for making this data extremely easy to obtain, sort and reprocess overnight!!
For my re-analysis, I threw all the files into PD 2.2 and used the default processing workflow for LTQ-Orbitrap LFQ and LFQ concensus with enhanced annotation. I made a 77MB FASTA in PD by parsing a UniProt TrembL I downloaded in July on "Steptococcus pneumoniae" and used my normal Uniprot SwissProt human + cRAP databases.
I can confidently say, with no reservations at all, that I believe this group did exactly what they said they did. This readout is the simplest one to display what I'm looking at. Green means protein found in that sample.
I have to go into really low scoring stuff before I see a peptide or two from the bacteria flagged as present in the controls -- and the first 2 match cRAP hits as well.
Okay...so this study wasn't published for almost a year from the time it was submitted. I'm willing to bet $10 that part of the holdup was that a reviewer (or two) didn't believe the data could be THIS clean. It is.
I don't know how long it takes to culture CSF in a classical microbiology assay to test for these bacteria -- or how trustworthy results from those classic assays are. What I do know --this team has shown clearcut evidence that our technology can make determinations of CSF infection with 2 hours of instrument time per patient (on a mass spec first released 7 years ago!)
Thanks for this reanalysis and the recognition of the team's work. A 2h run is not suitable for point of care in resource-poor environments, but at least we have candidates. Nice cross-verification using capillary western blotting too.
ReplyDeleteThanks for the great paper! Shotgun proteomics applied to microbiology has had some successes here and there, and this sure looks like one of them. I didn't even get into how nice the cap.westerns looked -- was this "Wes"?
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