2 hours in and complete brain overload here at HUPO! So much good science out there in the field!
One important side note that I think you guys will like, though. The first free OpenMS nodes for Proteome Discoverer 2.0 are now available for download. The first is LFQ for label free quan! The second is a workflow in OpenMS I'm unfamiliar with, but will read up on ASAP.
About a year ago a paper came out that introduced me to the concept of Thermal Proteome Profiling. While this concept will likely have several different applications, it is definitely really good at figuring out what proteins a drug is interacting!
Being a Nature Protocols paper, the methodology is set out completely so that any of us can go right out and replicate it. This team also developed software for Python and R that can be used to process the data and they make this all available.
Are you stuck on what that stupid drug's mechanism of action is? You should probably check out this paper!
This summer one of my biggest workshops was a PD 2.0 training workshop Alison Wiedergreen set up at the NIH Bethesda campus. It was really well attended and there were tons of great questions.
The crowd requested a follow-up workshop after they got going with the software and we finally made the schedule work! I'm happy to announce PD 2.0 workshop 2.0!
In the AM the brilliant and charismatic Dr. Talamantes will be going over the basic functions of PD 2.0, including getting you going if you are new to PD completely or if you have used PD 1.x in the past.
In the afternoon I'll stop watching and we'll go over some more specific workflows. I think we'll look at some real big datasets and how to organize them as well as maybe how to combine quantitative analysis of global proteins with phosphoproteomics or something similar. If you have a suggestion for what you specifically want to take a look at, shoot me a suggestion and/or a dataset and we'll try to make time for it! We'll also look at what is coming in Proteome Discoverer 2.1 (which is just a bunch of improvements to the PD 2.0 interface. It works and looks just about the same, I promise!)
Now, I know I've rambled on about NeuCode in this blog a bunch, but if you are unfamiliar there is a good description in this GenomeWeb article here. In a nutshell, its very much like SILAC except by NEUtron endCODE(ing) the mass discrepancies between the various channels are very very small. You are limited by the number of channels you can use by the maximum resolution of your instrument. More resolution = more NeuCode channels.
(I stole the figure above from this open access paper here.) P.S., the technology has been progressing significantly since the original study. I saw a slide a while back that suggested 40-plex is theoretically possible.
In this study, the nematodes are studied with an Orbitrap Elite that is running 480,000 resolution at the MS1 and 30k resolution at the MS/MS (which they refer to as "medium" resolution! man, I love this field!!!)
Now, you might think 480,000 resolution? That's so slow, they'll never identify anything that way! What did they see? The top 50 most abundant proteins? Well, they did a little better than that. This might be the single most extensive proteome of the nemotode out there. Along the way they did phosphoproteomics and also worked out some of the key regulators or stress response in this important model organism.
If you are still using the FACE technique or a series of different enrichment strategies leading up to FACE you might want to take a step back and think about what you want to get out of your samples. Is a generic anti-phosphotyrosine antibody still the best for what you want out of your analysis? If you are interested in pathways, for example, that preferentially use phospho-Ser, maybe there is a better option now than we had 5 years ago!
If you are looking for information on top down proteomics via Prosight or info on the Prosight nodes for Proteome Discoverer, you need to check out Proteinaceous.net.
Here is a direct link in case you have as much trouble spelling it right as I did...
Shoutout to David Kusel for the link for this one! Is there anything about the history of mass spectrometry that you've ever wanted to know? This Nature Milestones project probably has it covered. This was compiled by a huge list of authors who all seem to know at least a little about this field and is written to be accessible to everyone. It would make a great reference for our customers or collaborators who aren't really sure what magic we're doing in our noisy rooms!
