Monday, February 6, 2023

Finally! A complete kit for offline fractionation in plates!

 


Disclaimers in addition to the ones over there somewhere --->

1) I haven't tried these yet, but I'm adding them to our ordering sheet today.

2) I don't know these people, I don't think.



HOWEVER, when they stack their spin elution high pH fractionation columns up against those that shall not be named, the data look comparable.  

AND they come in 96-well plate formats. If I can get them in house (always sketchy here, I still can't order IonOpticks columns or pay my ACS open access fees since neither are approved vendors at my university), you bet I'll be showing data from them.

You can check them out here

Saturday, February 4, 2023

Another US HUPO 2023 guest on the podcast! This week Jenny van Eyk!

 


The next episode of The Road to Chicago podcast series just posted, featuring the inspirational Dr. Jenny Van Eyk

If you were on the fence about sunny Chicago in about 30 days, let Jenny tell you not only about her work, but about the speakers and projects in real translational clinical proteomics that you'll get to see and hear about in Chicago.

Thursday, February 2, 2023

What the developers don't want you to know about MSstatsShiny! (Download an annotation template here!)

 


In advance I want to apologize if this post comes off soundy a little sarcastic. That isn't my intention. We all know that sometimes people who do great science are not good at communicating said science. One example might be the MSStats thing that most mass spectrometrists have heard of but is only accessible to people that commonly compile code. 

MSstatsShiny has a pretty web interface but you can only use it if you have a small input file and only if you know the absolutely top secret - you can spend 3 days looking for it and you won't find it anywhere - annotation file format! 

If you want to run it locally, all the steps are at the bottom of this post and you still need that annotation file format. 

I broke out the big guns here and had one of the best real bioinformaticians on the planet wade through pages and pages of stuff that looks like this


to find that top secret format. 

Want it? Too bad, I didn't spend 3 days looking for it AND cash in a huge favor just to give it away for free.

J/K, but y'all owe me. Download it here

With that out of the way, let's go through THE MASS SPECTROMETRISTS GUIDE TO INSTALLING AND RUNNING MSstatsShiny!

First you're going to need a pain in the butt called RStudio, and you'll need the newest one. You can get it here

Once you install it, you'll want to open it and go to the console tab.

Now you're going to need bioconductor. You can get that here: https://www.bioconductor.org/install/

Bioconductor is a big package that is necessary for installing all sorts of other things. It's like how you have to install Steam to get games from the Steam store.

Anytime you see something in a grey box on Bioconductor like this, you can just copy and paste this thing and then you put it in the R console by the "greater than" sign. In this context it is probably called a carrot. 


Protip for R. If your console has been doing stuff and you don't see a greater than sign, go to the top and hit the little stop sign. Chances are it is running something in the background. 

Copy and paste the thing in the grey box to the lowest greater than sign on your screen and hit enter. 

Now, I don't know why, but when you are installing stuff IT WILL LOOK LIKE YOU JUST TRIGGERED A NUCLEAR WAR FROM YOUR COMPUTER. You'll see what I mean. The angriest red scrolling text that you've ever seen, generally means things are going okay. Some nerd in Australia found it funny and no one has ever changed it. 


Example of what it looks like. I've also circled console. I have not idea what the other tabs do, but the one looks similar and is completely useless. 😉


Protip for R #2: What I've found with the catastrophe that Windows has become is that my program files will sometimes be locked as read only. You may have to follow the directions in the R console to the file it can't write to then right click, get the little box to pop up, go to properties and unclick "read only".

Once the stress of what a terrible thing you've obviously done is over. It's time to do it again! 

Go get MSStatsShiny here! https://bioconductor.org/packages/release/bioc/html/MSstatsShiny.html

Copy the stuff in the grey box to your greater than sign and avert your eyes from the terror on your screen! 

It might ask you questions about things you don't know about while you're installing. Just say yes. It will probably be fine. 

NOW IF YOU GET THROUGH THIS HERE IS THE NEXT SECRET. 

To run the program you'll need this top secret code: (I read somewhere that I might not need all these words, but this is what works for me) 

launch_MSstatsShiny(
    launch_app = TRUE,
    port = getOption("shiny.port"),
    host = getOption("shiny.host", "127.0.0.1")
)

(Copy and paste that to the lowest greater than sign on the screen. Hit enter!)

If it worked, R will open another window. I find that window a little buggy, but there is a button that says "open in browser" and if you hit that your default browser will open and you have a locally running copy of MSStatsShiny that can take a 2GB SpectroNaut output sheet! 

It will look just like the online MSStatsShiny in your web browser, so if you get confused by it turning down your big file, you might just be on the wrong one.  

Wednesday, February 1, 2023

3D print an LCMS tool kit for those Thermo tools you lose all the time!

 


How freaking great is this?!? 

Do you know where all of those things are right this second? I can definitely find them all...eventually...

You can check it out here and download the free plans

Tuesday, January 31, 2023

Almost forgot! SKYLINE NEEDS YOUR HELP TO STAY FUNDED AND FREE!

 


Every couple of years this goes out and I nearly forgot! This takes 90 9 seconds or so (unless you write a personalized letter, which helps) and helps keep Skyline developing and provided as a great unifier for the research community.

Please click here and follow the instructions, cause Skyline is waaaay too annoying to pay for! 


Cyclic ion mobility can resolve closely related phosphopeptides of TAU in real brain samples!

 


Cyclic ion mobility? That sounds weird, right? I hardly need to use my ion mobility that goes in a straight line and often forget that it is even there.

But what if there are critical phosphorylation sites out there where a phospho on the 8th residue turns the protein on and the 10th and 13th residues all do something completely different?

Maybe it's time to crank up the spin cycle with this thing? 


That's what this group did


This is one of those pieces of hardware I know are out there and it seems like it could have some really cool applications, but I couldn't personally think of one. I could probably use one of my instruments and eventually resolve the three phosphopeptides they did here. Eventually. In a freaking brain lysate? Wait. Does this stupid thing have 4 prolines in it? With enough work you can do anything, but - ouch - now I know I could suggest annoying peptides like this to people like Matt Padula who have invested in this new technology cause here is a real reason to spin it up. 




Monday, January 30, 2023

The need for new biomarkers in ALS!

 


I've had a scholar alert up for quite a while for ALS proteomics and this nice review puts into perspective where proteomics can have a major impact in at terrifying human disease.


I also have a poster up in my office at Hopkins so it is the first thing I see when I walk in because it is the poster from the long long ago ALS community challenge. I'm going to leave the poster up until this paper is written. When the senior author on the planned paper (and the only person who knew anything about the disease) was lost to it, the study lost all momentum, but the data is really really cool and we had a draft going. 

HEY YOU. Yes, you. Would you like to help finish it? Contact me! I've got tons of notes and an outline and amazing data from like 10 different groups who all found really interesting things in it.

My favorite is a glycopeptide that almost every group identified that, as best I can tell, has never been seen before (from the ASMS Halloween poster): 


Now, there is some disagreement between Byonic, ANN-SoLo, MetaMorpheus, Byonic and MaxQuant as to the sugar monomers (since, you know, the whole sugar monomers having the same stupid mass in a lot of cases and the relative libraries/approaches they used) but the MSFragger team found that after batch corrections this thing is certainly differential. A rotation student in our lab helped me pull out the original spectra and we hand checked them. Definitely real (though I can't say the monomers either). Some groups found a second glycan length at the same place. 

Seriously, check out that awesome review to get motivated and if you want to help those authors reach their goals of finding some new proteomic biomarkers for the disease, a huge dataset is sitting right here. I'll send it right over. 

Sunday, January 29, 2023

THE Proteomics Show Road to Chicago is finally back with Ying Ge!

 


At long last THE Proteomics Show is back! Wherever you get podcasts! 

What better way to kick off 2023 and the final leg of the Road to Chicago than with the one and only Dr Ying Ge

I think my first question was if she had 50 postdocs given the productivity of that group. She doesn't. How are they so incredibly successful? You'll have to listen to find out. 


Glycoproteomics patterns in alzheimer's disease -- deep analysis of 30 human brains!

 


30 human brains? 

Multi-enrichment to get to the glycoprotein/peptide patterns? Check this out!


They used "healthy" brains, brains from people who had alzheimer's but were asymptomatic, and some from those who had symptoms at point of death?

Would you be surprised by differing patters of glycan distribution? Me either, but I'd have no idea what to do with those data.

What really shines in this paper, besides the obvious biological importance and loads of work that went into it is how they visualize the patterns of their observations.

If you're wondering what to do with glycoproteomics data this is definitely worth a look. Frustrating things about these PTMs are (among many) the fact that your biology might be that one sugar may be over/under represented in the glycan chains observed. How on earth do you display that?? Like they did. 


Saturday, January 28, 2023

BIRCH -- Identify those batch effects and fix them!

 


Well....that was a rabbit hole that ended at some old scifi horror show about tree monster. An ad that said the "root of all evil" made me think they probably gave it the correct level of seriousness.

HEY! Remember when we did like 2 condition proteomics? That was cool, because we could run our control, then our treated and spectral count some stuff. No fancy stats, just crap data! (J/k) 

Now, however, we have to do the grown up science with lots and lots of samples. BATCHES of samples, you might say, and now we need to start thinking hard about BATCH EFFECTS.

What's that? Well, that's the differences that aren't your phenotype. Those are the ones that are due to the fact you had a lot more dog dander on yourself when you processed the samples on Monday than the next batch of samples on Friday. Or, if you're in an old dungeon where the HVAC swings your internal temp from 45-95F between those dates....those changes.....

And that brings me back to BIRCH

You can get it here

You need the things above! Your non-normalized file. Your normalized file. A stable internet connection (not sure why) and the software at that Github.

It'll try to sort out what effects were caused by that dog dander when it was 95F in your lab and what was actually due to your CRISPR knockout! 


US-HUPO LFQ Battle Royale -- Sunday, March 5th at 4:30pm!


We have TOO MANY WAYS to do label free proteomics quantification today. 

What we should do is hunt down the most die hard specialists of each one together -- with some folding chairs -- and leave Chicago with at least one less than we flew in with! 

I don't actually know if that is what we're doing, but it is a funny thought. 

I have my opinions. I bet everyone who shows up will have theirs. Maybe we still need more than one method for doing LFQ based proteomics. Do we still need 45? Maybe? But I bet a bunch of us in a room with some folding chairs can sort it out! 


As an aside, it just came to my attention that when I use the "line through text" button on this blog it DOES NOT DO THE SAME THING AS MICROSOFT TRACK CHANGES. 

Friday, January 27, 2023

MS-Ana -- Big DDA spectral library searches with really cool statistics!

 


I got MS Ana in Vienna at when I was lucky enough to speak at the Proteomics Summer School thing about 1 pandemic ago and have mentioned it on the blog a couple of times. 

And the paper is finally out! 


In some regards, it might not matter to you how much great MS Ana is, particularly if you are using the free versions of Proteome Discoverererer. Starting in the 2.5 (I think?) PD viewer, MSPepSearch requires a commercial license from Thermo. 

BOOM -- MS Ana restores your ability to do spectral library searches! 

Even better? MS Ana gives you cool abilities like choosing how your decoy spectral library is generated. And it generated column after column of statistics on your peptide spectral matches that you can use to evaluate your forward and decoy match data! 


(Mirror plots are awesome) 

Also, are you one of those weirdos who hates Proteome Discoverererer? Guess what, weirdo, there is a rocket fast stand alone version! 

You can get both at www.pd-nodes.org or directly at the MS-Ana page here

I do want to send a special shoutout to this team for helping me with this tool and sharing this manuscript while it was in review. I couldn't have got my first ever paper in JPR without it. (I guess I'm like 14th author on something I honestly did less than 1/14ths of). I also couldn't have gotten that paper without the help of a bunch of other people. The acknowledgement section might have been longer than the paper itself if I hadn't just thanked #MassSpecTwitter as a group.  

Thursday, January 26, 2023

Label free single (big) cell proteomics in 11.5 minutes per sample??

 


This is a preprint, so all the preprint disclaimers apply here, but Simion Kreimer is just about the baddest mass spectrometrist on this planet so I'm cool with wagering just about anything that this will be in press soon. 



Recently Dr. Kreimer (who might have the coolest job title ever) et al., demonstrated a really fast valve switching double loading method for proteomic sample analysis. It recently showed up in ACS here. And if you've been at any conference in the last 2 years you've probably heard the amazing Dr. Van Eyk talk about how important single cell proteomics is to understanding cardiac diseases.

100 single cells per day! LABEL FREE! 

The ability to discern between cell types! 

Yes, cardiac cells are sort of big, but cardiomyocytes are miserable to work with (95% of all protein is 4-10  proteins!) and this group is hitting 1,000 proteins per cell.

AMAZING WORK!

Wednesday, January 25, 2023

Prep hundreds (or thousands) of single cell proteomics samples in a single day!

 


Want to get going in single cell proteomics? Here is even more resources! 

Check out how unbelievably cool this JOVE protocol is from the Slavov lab!  The video protocol for SCOPE2 is freaking amazing. 

No joke. There is like an 11 minute video explained by the scientists at NorthEastern university -- step by step, that would allow you to prep hundreds and hundreds of single cell proteomics samples per day. 

How did I find the amazing NorthEastern video? I was considering doing a JOVE video and then I felt like a dumb person for not looking this up first. 

Conclusions? 

Single cell proteomics doesn't have to be terrifying. It can be approachable today and there are great resources to get you going. 

Tuesday, January 24, 2023

Deep proteomics on 875!!! different drugs! DeepCoverMOA!

 


You know...sometimes I see papers with huge titles like this and think....oh no....what are we overselling today....

This is a study that deserves a big big big title. 

This relatively small group of authors did proteomics on 875 drugs. 

Eight hundred and seventy five. 

acht­hundert­fünf­und­siebzig

八百七十五

שמונה מאות שבעים וחמש

åtta­hundra­sjuttio­fem

otte­hundrede og fem­og­halvfjerds

walóng daán at pitóng pû’t limá

ثمانية مائة وخمسة وسبعون

ochocientos setenta y cinco

How'd they do it? This figure pretty much covers it! 

Okay, so who cares, right? I'm not going to download 11 million spectra and neither is anyone in the Pharmacology department I work in.

To make it useful, they'd have to have a ridiculously easy web interface that allows you to look at any drug you want and how it affects around 9,000 different proteins. 

Welcome to 

Check this out. I know someone who studies/develops compounds similar to Olaparib. I know every drug has like 12 different synonyms, but -- I'll type the first 3 letters into the compounds box and BOOOM!


Not only can I pick a specific protein to see if it is perturbed by Olaparib treatment, but this resource goes way further than that. It generates correlation plots between it and the other drugs in the library based on the protein level effects! 

This is so so so so so so so so so good. I'm just floored by how much thought went into this. There are, of course, similar things for drug + mRNA levels that everyone uses (most of the biggest ones are old microarray data...blech....) but people use those all the time. Protein level??? Nothing I've ever seen has ever come close. 



Monday, January 23, 2023

Save 19 hours per multiomics prep with BAMM!!

 


Multiomics sample prep is totally doable, but it takes a long time and can have a load of steps!

Some people in Wisconsin who do a lot of multiomics did the math and came up with about 19 hours to do a good multiomics prep. 

They asked Emeril and he said...that's too long for anything, you need....


...it might get worse...maybe...it depends on what you think of Emeril. (I've made this recipe about 20 times over the years. I sub out the dead bird for grilled eggplant and it is super legit.)

This is what they came up with!




Of course, "multiomics" means different things to different people. For genomics people it is often, "I looked at nucleotides in 3 different ways."

Some of the cleanest proteomics samples I've ever gotten in my life were left overs from where people had done lipidomics and metabolomics extraction and the proteins were sort of just left overs. CLEAN PROTEIN remains. So, this makes all sorts of sense to me.

Total prep time? 3 hours! 

Sunday, January 22, 2023

Overlay your proteomics data on a 3D protein structure with SCV!

 


Wow, did I ever wish I had this before! 

Have you ever tried to just figure out what peptides you found in a 3D protein context? It is totally possible, but it is a miserable experience (for me anway)!

This is the easiest thing ever! 


Put in your peptides, put in your FASTA! Choose the protein you think it is!

Don't feel like reading? Just run it here!

There is a great YouTube video that walks you through it here.

Saturday, January 21, 2023

Time to adjust that textbook! A whole new set of DNA Damage Repair Proteins (in 2022??)!

 

You know...I'd be more blown away by this if I wasn't grappling every day with the fact that we don't actually really understand how human cellular division works. (Everything we know has been measured at transcriptional rates! And they don't line up very well at all because not every process in cellular division waits till we build up a whole bunch of mRNA. A lot of it is depolymerizing and reusing what is actually there.

BUT

It's 20freaking22 or whatever it is! We obviously know every protein that is involved in DNA damage repair! 

This study makes a very good argument that...no...actually...no, we do not and here is a couple more! 


They do some solid proteomics and then go back and do some even more solid molecular biology. Knockout these proteins and try that experiment again? These things are important! 

Imagine you just got back from the printers the 3rd edition of your textbook you sent out for printing in 2019 and some bozos with a mass spectrometer totally screw up Chapter 4?!? 

Friday, January 20, 2023

You guessed it! Nature's Method of the Year is....long read sequencing...?

 


For those of us who are maybe occasionally still writing 2017 at the bottom of things when we sign them, you might also be heartened to know we aren't the only ones! 

2022's method of the year is what I HOPE that the students in our department totally think is the only way of doing sequencing, because I thought that was all anyone had been doing for at least the last 5 years.

I'm very confused. I'm glad for the long read sequencing people. Good for (what I hope is like literally all of you, why would you still be trying to reassemble little tiny oligostrings back into a complete story when you can have very long strings of oligos...?) Hey, good for genomics and really good for proteomics. It is MUCH easier to find peptides linked to long read sequencing data. 

I feel like a jerk, but last year's was pretty pertinent and up to date. This seems like one they forgot to get to in 2017, but that's okay, I have things I started in 2017 that I haven't finished. Most pressing is this huge tree I cut down, but it fell into another tree and has just been hanging right over my garage for years. I've had a professional out twice and they've said they'd come back, and then didn't. One day Imma wake up to no garage. I hope, because I'm typing this right now in the office I made in said garage and there is another alternative...


Thursday, January 19, 2023

DIA for PTMs? This group tests DIA-NN vs SpectroNaut vs MaxDIA (etc) for DIA phospho!

 


One of the last remaining arguments for the dDa crowd like me is PTMs! When that domino falls (if possible), we'll see a full paradigm shift.

A lot of time when people talk about PTMs in proteomics they just mean "phosphoproteomics" and a lot of tools let try to look for them.

How do they stack up? You don't have to do it yourself, this team tried them all out! 


Worth noting, after 3 months of waiting for my university to issue a PO, I just downloaded SpectroNaut 17 and this paper used 16. 

Bonus -- you get to see an HF-X vs a TIMSTOFPro for DIA!