Can't tell what species it is peptide level data? Enter PEPTONIZER 2000!

 

Given the sheer multiplicity of peptide level data, we're probably approaching a point where just trying to do metagenomics classification seems silly compared to the metaproteomics stuff, right? 

The way I think of it - 

Yo, is this species A or species B?  I've got 20 bases of relatively fragile oligonucleotides, so  4 bases ^20 is what I'm working off of to make that species ID. That's a big number. Something in the e12s according to some extremely sleepy Excel thing that took 11 seconds and might not be right at all. 

Or. I've got some collagen which can have a half life of over 70 years....and I've got 20 amino acids from that. so...it's 20^20-ish. Change the 4 to 20 in that same excel spreadsheet and now you've got something that could occure by chance in the e26 times. 

Okay, but I know how to get good sequencing data of peptides....what's the next step?

PEPTONIZER 2000! 

Here come the LLM Search Engines, introducing ChatDIA!

 

Okay, so everyone else should be really embarrassed they didn't put together a preprint and get credit for "ChatDIA". I just submitted a one paragraph "paper" to Biorxiv on my lunch break, so ChatDDA is mine in the next 72 hours or so.

That's not to say that this doesn't have merit. This thing appears to fare well against DIA-NN! 

SAINTexpress portal- Good pull-down proteomic data analysis in your web browser!

 

You know what proteomics needs more of? Accessible data interrogation and interpretation tools, like the new SAINTexpress portal!  For some people they just really need tools online because it takes them 6 months to get permission to install things. Not me, I hang out with the IT Director whenever I can. 

SAINT (Significance Analysis of INTeractome) (whoa. my font went all funny, that's what I get for copy/pasting. Come on, Ben you can absolutely spell Sigifnivcance on the first try. Wait. Maybe not.) is a great way to take your immunoaffinity, affinity enrichment, pull-down, IP-MS, AE-MS (wait, maybe thats what one of the longer things stands for) data and make sense of it. And now you've got a webportal! 

Infinite multiplexing by attaching ...giant peptides....to...your...peptides...?

 

Okay, so I went back and forth on posting this one for a while, but I might honestly be missing something. 

I'm certainly missing the RAW data files, because those weren't deposited... but the initial idea seems clever...until you try to design something that can multiplex more than 3 samples at a time....

The basic idea is something like "why swap isotopes around when you could SWAP AMINO ACIDS AROUND?"

As shown in figure 1 - if you had INFINITELY LARGE PEPTIDE LINKED TAGS! 

...attached to your tryptic peptides.... 

(wait. what?)

 ...you could have INFINITE MULTIPLEXING! 

Y'all don't line up immediately to add a 2,000 Da multiplexing tag to your all of your peptides so you can have the equivalent of a TMT 16 plex experiment. I think you should definitely contact the authors first to ask to see these .raw files because there aren't enough details in the method section ....of a paper accepted in ...Analytical Chemistry ... to reproduce the experiment. You can guess some of the settings, though. TopN? Guess. Ion injection time? Guess again! Can you employ normal dynamic exclusion?  What is the mass of the tags that were searched in MaxQuant? If you want to know, you need to do the math yourself. Really, just a disappointment to see something slide by an editor and reviewers, particularly when something seems - on the surface  - unlikely to work...? 

The OpenLC project! Build an autosampler out of an old OpenTrons?

 

Our library doesn't subscribe to this journal, and I don't have $50 to spend on one paper, so it sure was handy for the authors to put links in the abstract to the -

OpenLC Github! 

There is a repository with .STL files you'll need to print adapters and the code you can use to over-write the original OpenTrons one.

There are a couple of related papers that I can access as well, such as this one that can probably provide a starting point on useful details like this.

What's the point of these super fast LCMS methods if your HPLC only has room for 1 or 3 plates? You can't even go on a weekend trip. But if you have an OT2 autosampler could you have room for 12 plates???

Sure looks like it to me! Totally worth thinking about....

JASMS pulled together an amazing piece on the passing of Amina Woods

 

I didn't know Dr. Amina Woods as well as you'd guess considering the 20+ years we were doing mass spectometry stuff in the same city. We'd spoken a few times, most recently maybe two or three years ago and I was always a fan of her work and presentations of it. 

What a person she must have been to have inspired such a touching piece from the nitpicky chemistry nerds at JASMS. It's a legitimately nice read and maybe being good and interesting enough to inspire such a thing is something we should all aspire to. 

Somebody went all out on diaPASEF peptidomics exploration - analyzing baby formula!

 

I was initially a little appalled by the amount of optimization of diaPASEF windows in this paper, but I read more of it while walking a mean little dog in the rain and there is a lot here. 

Food science is very different that your run-of-the-mill proteomics samples. Often the thing you're worried about is the allergenic peptidome, and those sure don't look like tryptic peptides. It's likely that this team ran some baby formula using the default methods on the instrument and they didn't see a single thing of interest.

They had to go back to the drawing board do wide IMS window DDA and build their windows for their targets iteratively until they had a good method.

Even in this context, I have to admit there are just too many purple DIA windows to make this more than a useful reference for the next time we have to look at something singly charged and 10-50 amino acids long. I certainly don't want to read the whole thing until that happens, but I'm glad to know it's out there! 

You can slow an Astral way down and do TMT on it? I thought you needed MS3?

 

Wow. Okay, so am I ever confused. Since 2012 or so there has been a constant irrefutable message from one of the world's largest science companies. And that message has been something like "the only way to do good multiplexed quantification is by using an exclusive, incredibly slow, and over all low sensitivity method which can only be performed on our most expensive and complex instrumentation." You've seen that, right? This method relies on doing MS2 fragmentation in an ion trap and then (in later iterations on the Fusion instruments) selecting multiple large fragment ions for MS3 based quan in the Orbitrap at high collision energies. So...yeah...slow....

There were some surprising outliers, however. CPTAC - the largest proteomics initiative ever attempted up to that/those times purchased Fusion 2 "Luminati" devices for all participating labs. After a thorough analysis they chose not to use the MS3 based method for heavily fractionated multiplexed samples. Weird. Those data are amazing. Reanalyzing those data with new algorithms has been the backbone of at least 3 companies, and probably a whole lot more. In my mind they're the high water mark of the quality of data you can get with LCMS proteomics and I hope to someday do something as good. (But at a tiny fraction of the cost! The goal in CPTAC was irrefutable quality and that's expensive). 

There were a lot of confused conversations when said gigantic company started showing MS2 based quan for these experiments on their extremely expensive new quadrupole Orbitrap TOF device (Asstral). Some betrayal? Maybe. I know at least one lab where this turnabout made them start to investigate alternative vendors for the first time in the lab's storied history. There are a lot of fast TOFs out there now and some cost like 3x more than the others. 

So...there are places where this new preprint might not go over super well. 

It's a short and easy read and does pack some surprises in it. For one, though, the MS2 based reporter ions look really really good. For real, there isn't much to pick apart in these data aside from the following fine method details. 

We see two different methods employed here. The first is with a 1.2 m/z isolation window. Which probably sounds like an absolutely huge window for an MS2 based multiplexed quan experiment - because it is. That's letting in your peptide of interest and a whole lot of it's friends. It's approaching the size of some DIA windows today. 

To run the TMT HR method you can go down to a much more typical 0.5 m/z window and then use a 50 millisecond accumulation time to get the signal you want. 50 milliseconds? Ouch. Wait. 

I don't know how fast a 120,000 resolution MS1 scan is on this instrument. I'll assume 2x faster than an HF-X so let's put that at 60-ish milliseconds. If you have zero overhead across the board for everything else (which you don't, but we'll pretend) that is almost 19 scans/second on low samples. Right? 1,000 ms - 64ms divided by 50ms? If it perfectly parallelizes MS1 and MS2, which it might, it's impressively smart hardware, maybe the Orbitrap scan doesn't count against you. Even if that's as slow as you can go but most of the time you're not using the whole 50ms fill time that's not the amazing 200 Hz or whatever this thing can get by cutting the ion beam into tiny little windows. So there might be some serious sacrifices here. 19 Hz puts you in "wait. why not use the Orbitrap?" scan speeds. 

Again - these data look great, but don't get it mixed up. You can do MS2 based quan on the Astral, but given the apparent sacrifices you might be better off running it like an Exploris with MS1 and MS2 in the Orbitrap. If you are looking at this thing and you see you can do TMT 32-plex quan and you can do 200 Hz or 300 Hz (whatever it is now) based MS2 with the TOF in parallel, it's important to remember these statements and experiments don't appear to be happening at the same time. 

At the end of the day it's super cool to know that when you get an instrument and your research shifts that you can do a pile of different experiments, and this at least shows you really can do TMT based quan on this box, and those of us doing MS2 based quan should get a little less flack from reviewers in the future for it. (Lolz)

Tandem mass tag (TMT) label on a chip for high throughput labeling!!

Ummm....if this is real it could be enormous for a field or two!

Yesterday I was reading another paper in this issue of Analytical Chemistry that I was excited (and very skeptical about) and I got to the end before I realized there were no files to support some very lofty claims. I was already really mad about some dumb shit my country was up to and I guess the reviewers and editors were just like - meh - whatever, this is going to be super controversial, so we'll just let you push it through without showing any work. That paper will not feature here.

This one will because I've totally got all their RAW files now! (There's only 14! But there is proof they did something!) 

What was it? They built microchannels so they could TMT label single cell digests in a high throughput manner! Does it look like a pain in the butt? Totally. OMG. It totally looks like no fun at all. But have you seen how crazy DropSeq is (how they label cells for single cell RNASeq)? It's crazy. You eventually get flow rate A and flow rate B under a microscope to line up and the bubbles join and then you can do like 10 million of them while you're at lunch or whatever. 

This is a step in the right direction. Now....because I'm a jerk and I do have these files, they do look a little weird. 

This should be 1 cell, 1 cell, 1 cell, 1 cell, 1 cell, 1 cell, 1 cell, 1 cell, blank, 1 cell, 50 cells of each cell type (100 cells). 

I've scanned through few thousand MS/MS spectra and while they look pretty consistent the ratios appear to be off. Good example. 

It's very rare that I land on a spectrum that I expect... one cell is 2e4 and 100 cells is 2e6, by rare, I'm seriously talking about less than 1%. I'll probably queue them up for reprocessing in a second, of course, so I'll have real numbers. There is some level of error intrinsic in just scanning with your finger on the forward arrow key. 

I'm sure the reviewers for such a nice journal looked at this already and were satisfied with it, so I bet I'm missing something. Right now I'm missing the last bit of sunshine to take my trash out to the curb, so I'm can't spend more time on it. Pittsburgh stairs can be steep and dangerous in the dark. Mine are no exception. 

Again, if this is real, it could be really really cool and that's how we should think about every paper we read, right? I'm just personally invested in processes to speed up my single cell labeling and I don't want to get financially invested in something without being super duper skeptical. Please interpret my words and effort implied in this post in that light, because I wouldn't post anything about this paper if I didn't like it. 

Proteomic analysis of postbiotic inhibition of Salmonella typhimurium!

 

Borrowed this picture from the MicroChem website while I was looking up what a Kirby-Bauer Inhibition assay was. Its a microbial zone of inhibition assay! 

Paper link!

What's a postbiotic? It's a metabolic secondary product of "good" bacteria or other microorganisms. So the question was something like "what would inhibit Salmonella typhimurium" and when we see that inhibition, what is causing that?

And since Salmonella is not a fun thing for humans to get, the goal is to find compounds that will inhibit it to limit infections. 

Interestingly the proteomics from these researchers was performed by sending the samples to the University of Copenhagen Chemistry department where they digested them and did peptide mass fingerprinting by MALDI-TOF. I suspect microbiologists are so used to doing everything with lasers that this is due to the contacts they generally have. The authors come up with a solid table of both small molecules identified by GCMS and proteins identified by these methods that may be next line inhibition chemicals for this nasty bacterium.

As an aside I sincerely hope that these authors are safe and continue to remain so in the wake of the blatantly illegal actions of an 800 year old narcoleptic dictator that began a few hours ago.