Wednesday, August 29, 2018

mPOP --> Streamline single cell proteomics!!

Have you taken a swing at ScoPE-MS?

Are you also really bad at pipetting?

It doesn't take too much distraction before you will lose that single freaking cell that is in the bottom of your stupid plate. ("Did I add the 0.25uL of label to this tube? I'll get the microscope to see. Hopefully it won't evaporate before I get back")

Okay -- this HAS to help!

I'm aware there is some concern regarding ScoPE-MS, such as why it hasn't been peer-reviewed yet, but I know of at least one independent lab that is pulling in results from this approach (not counting me -- yet -- pipetting issues....) so the data is coming! If you use Tweeter, or whatever, I recommend you follow @SlavovLab. This group is kicking ass and appear characteristic of the growing community of research labs that are looking to alternatives to the classic peer review model for rapidly pushing science forward.

Tuesday, August 28, 2018

You can add Ion Mobility to your Fusion!

*Cue someone to email me and tell me FAIMS and Ion mobility are different.*

high-Field Asymmetric waveform Ion Mobility Spectrometry is back in the form of the FAIMSpro device you can add to any Tribrid mass spec!

What's it do? It allows gas phase selection of peptides directly inline on your instrument.

I took this from the manufacturer's website here -- 24% increase gain when using FAIMS on a Lumos, vs not?  This is ion trap MS/MS, btw, but that is an amazing boost!

Some details I've found between the spec sheet and the press release.

The data can be processing in Proteome Discoverer 2.2 (no digging through GoogleGroups to find the patch to process your data like some other ion mobility medium resolution system we hear a lot about)

FreeStyle can also process the data (might mean you finally have to ditch QualBrowser -- which, honestly, you really should. Yeah, I'm still opening everything in Xcalibur too -- but FreeStyle is so so so much better.)

FAIMs Fusion operational software templates are also ready to go (new Fusion tune upgrade coming?!?)

Skyline can also deal with the data automatically!

If the manufacturer plans to put this device on it's fastest and most sensitive mass spectrometers, I don't see it mentioned anywhere, but it provides an enormous boost for the ones made here in the U.S.!

Sunday, August 26, 2018

BoxCar update -- MaxQuant live page is up -- stay tuned

MaxQuant live is coming. There is even a page up where you can sign up for updates! You can find it here or by typing MaxQuant live into the Googler.

Thursday, August 23, 2018

MS2Go -- Export all your Proteome Discoverer results in END USER Friendly sheets AUTOMATICALLY!

How are you going to send that data to your collaborator from your PD output? Are you gonna go through manually and cut a bunch of Excel sheets and spend the next hour combining them?

What if you could take that 12GB output report and automatically create the exact output you want?!?

MS2Go just went live at!!

Unzip and run MS2Go.

Point it toward your .pdresult file.

Give it your parameters (there are instructions at the link as well).

Kick out beautiful automatically generated spreadsheets!!

Here are some examples of how it looks!


Okay -- so MS2Go is looking for particular formats so you might need to tinker just a bit to get it to kick out what you want. By default it is going to be looking for the other IMP PD nodes. Works great for me, but if you aren't running MSAmanda as your go to search engine, you may need to read the instructions. I do, so I haven't. 

Heck -- if you haven't switched over to MSAmanda 2.0 as your default search engine for PD, maybe this is the impetus you need to finally do it! Or you could probably read the instructions....

Wednesday, August 22, 2018

Chemical crosslinking in vivo in plants!!!

Somebody seriously just asked me about this recently and I came up with nothing. And here it is!!

Plant proteomics sucks. Give it a run sometime!

Annnnd....Another peptide from...{{drumrolllll}}.....RUBISCO!
(I can't find the originator of this Rubisco gif, just random repostings for karma. Happy to credit appropriately)

Okay -- so how on earth would you use chemical crosslinking to do interactomics? You stop thinking about it and just do it this way.

Would I have thought to introduce a crosslinker under vacuum? Nope! 

Would I have used inexpensive and powerful dimethyl labeling? Nope! (I only just found out what a nice reagent this is. Another scientist in my group generates beautiful data with it) 

How do you get rid of the infuriating rubisco? 

Extensive fractionation
Gradient centrifugation
And enrichment (...cause you use a crosslinker that can be enriched!)

It takes a lot for me to post a paper about that Scumbag Arabidopsis, but this is useful enough that it could definitely be applied to real plants and things that matter! 

Monday, August 20, 2018

ClueGO! Another promising downstream analysis tool.

Shoutout to @PastelBio for bringing attention to another great resource in this amazing catalog of stuff we can use!

We've been making some serious progress in the area of software to use once you've got that quantitative protein list.

ClueGO is another nice entry in the category. It is a plug in for CytoScape that allows you to directly use data you're already using within that interface, or to pull in formatted text files from other programs. The visualization interface is focused on user-customization and allows you to display your findings in ways that help you to find the patterns hidden in that big list.

Sunday, August 19, 2018

Promising technology? Mixed mode spin columns?

This is a reminder for me to read this when I get to a computer that pays for the ElfSeverer journals.

What it looks like is a fast and promising way to digest and fractionate samples on a spin column. After some recent dabbling in metabolomics and small molecule ID, I'm interested in any technologies we can borrow from the chemists that could supplement our boring ol' reserved phase C-18 separations.

Saturday, August 18, 2018

New MCP format!!

If you haven't already -- you should check out the new format for MCP!

It's 🔥🔥🔥🔥🔥🔥🔥!!

Friday, August 17, 2018

Finding the missing proteins in the 5+ year old NCI-60 proteomes!

This study makes me so happy!

I've been digging through the NCI-60 proteomes from Gholami et al., since they came out 5 years ago. I use this study as my favorite example of how valuable a proteomics dataset can be when done right -- you can mine them for new stuff for years and despite the fact many people have been through this one -- there is still new stuff to find.

In this new JPR study, this team does an amazing job of driving home this point. Yo -- most of these files are from an Orbi XL (select samples are also repeated with Orbitrap Elite OT/OT). They pull out all the data, intelligently combine the results of search engines search engines, incorporate RNA-Seq, and by being really smart with the data analysis find evidence of almost 200 "missing proteins".

5 of these proteins meet the guidelines the Chromosome Centric Human Proteome Project (C-HPP) has for proving presence. This is a big deal. These are proteins that genetics suggests should be present -- but you can't find mass spec data of so it's thought the genetics expression model is wrong. But -- here they are-- hiding in Orbitrap XL data that y'all can download here and look at anytime you want.

Thursday, August 16, 2018

Want to learn DIA by participating in a free study (and get free samples)?!?


Tired of hearing about DIA and want to give it a shot? If you participate in the free PRG DIA study you'll get free standards AND protocols and you just have to send them your (anonymous) data in return!


Are you thinking your a DIA hotshot? Want to prove it? Same study! Get this stuff, send it in, and then revel in your mastery over your everyone else who gave it a shot, because you know those anonymous files (you get to name them) are yours. LORD IT OVER EVERYONE IF YOU WANT!

Side note -- there was a single global proteomics participant in one of the ABRF studies last who blew EVERYONE away. Same instrument. Same sample. Someone out there (who didn't lord it over everyone, thankfully, cause I was right there in the middle) IS WAY BETTER than everyone else. That might be you in DIA!  Wouldn't that be great to know?

You can sign up for the PRG DIA Study here (I think they even give you access to software to process your data, but don't hold me to it.)

I'm gunning for that upper outlier, so y'all better bring your A game. a team...

Wednesday, August 15, 2018

NextGen NanoHPLC is here -- its EvoSeP time!

Once upon a time, HPLC companies focused on making really good HPLCs. They didn't waste everyone's time making terrible mass spectrometers.

Likewise, mass spectrometry companies kind of stuck to what they were good at, maybe they'd make an HPLC, but we'd all laugh at it, buy a real one from an HPLC company and everything was just fine.

At some point these businesses all got really greedy and tried to do everything and they all failed. Now you've got a choice, buy a great HPLC system with a turd of a mass spec attached to it, or buy the best mass spec in the world and get the unsupported virtually nonfunctional atrocity that the Walmart of Science has turned the once-great EasyNLC into. And you really don't have another option, since the vendors -- now in direct competition somehow -- have no incentive to develop compatibility. No other option --

Until now.

EvoSep!!  And this paper shows that some of our European friends are looking at this awesome little company as a way to ditch the HPLC monopolies and get crazy fast comprehensive proteomes in rocket fast time.

EvoSep is it's own company, is working to be compatible with everyone and has the following advantages over everyone:

1) FAST LOADING (what do you mean, 40 minutes to load a sample is a problem?!?)
2) No carryover (what? how is that possible in 2018?)
3) Some new technological advances!  (shirking the last decade's pattern of HPLC advances -- namely lowering the performance by subbing in cheaper components and focusing on profits over customer support staff --  but -- oh my gosh -- they CHANGED THE COLOR OF THE BOX! And instituted telemarketing?!? Science!)

I've kind of rampaged about how amazing the EvoSeP is here before. And that was before all these awesome people DID PROTEOMICS WITH IT.

If you read the HF-X paper out of Jesper Olsen's lab there is this really sad/funny part in it -- they can't shorten the gradients any more -- because the poor SleasyNano requires 22 minutes to wash it's needle (or something, I haven't read it in a while), so you might as well run a 22 minute gradient since you can't inject another sample anyway.

What if your autosampler was faster than your separation gradient? What if you could PRE-FORM your gradients so they're ready to go? What if your next sample was pulled up and waiting for the instant the last run was about to end?

Well -- then you could actually make use of the HF-X's 42.4 Hz acquisition speed. Maybe you could knock out a complete proteome (>10,000 proteins) in a few hours.

OR in the 20 minutes it takes your current system TO WASH IT'S NEEDLE you could ID 5,000 proteins. For real. 20 minutes.

Time to take my NanoLCs out in a field....

Tuesday, August 14, 2018

Ever wonder how the metabolomes of sled dogs change during races?

Okay -- I'm going to guess you haven't ever wondered....honestly....neither have I and the time I got to go out with some sled dogs in the Rockies several years ago is at the top of my "best f'ing days ever" list. However... someone did wonder... and now we all can know thanks to this great open access paper!

It looks like all the metabolomics was done with RP and HILIC on a Dionex U3000 coupled to a Q Exactive. Metaboanalyst was used for all downstream processing (and presumably the pretty heat maps!)

Monday, August 13, 2018

Conserved phosphorylation "hot spots" in Eukaryotes!!

What happens when you bulk analyze over a HALF-MILLION phosphosites across 40 species of eukaryotes? Maybe you end up with a big picture of how phosphorylation dynamics have evolved.

BOOM! You can find these awesome brand new results here!

This study is big. Like really big, and is just a joy to flip through. The RAS proteins, btw, are really interesting in this regard -- particularly when aligned against random structural features. There is something critical to learn here, but I can tell I'm too dense to get it. I guarantee I'm going to be thinking about this paper on my commute today!

Tuesday, August 7, 2018

Protein ID with no MS/MS using complementary enzymes!!

Ummm....I don't hate this idea at all...I had to think about it on a long humid run and I decided that I might love the idea, or I'm out of brain electrolytes -- are those things? I don't even know.  I do know I strongly suggest you check out this brand new JPR paper. 

There are an awful lot of people out there identifying and quantifying peptides with triple quads. We know that a high enough resolution MS1 scan can match or outperform a QQQ SRM in complex mixtures -- and the magic number from independent studies is around 60,000 resolution.

However -- if you try to pull out a peptide from a complex mixture by just SIM at 60,000 resolution -- you're likely going to find a bunch of things that match -- maybe even some with nanoLC level retention time alignment values (+/-20 minutes or so -- which is possibly an exaggeration).

What if you were smarter about it and used complementary enzymes and extracted MS1s? Could you get away with lower resolution? Could you use MS1 scans and then get stupid high levels of confidence?  It would probably suck to compile that data -- unless -- some extremely productive software lab in Moscow was already churning out the scripts!  Ben, Put the link in before posting!!

KeyStone Proteomics Symposium in Stockholm in April!'m not sure what a Keystone is, but holy cheese, this is one heck of a meeting in April

....Ummm...important people all talking about the most important area of science...?!?!?

Confirmed speakers appear to include a lot of people that have done enough stuff they only need one name:


and the speaker list doesn't drop in prominence after that -- I just got to Steen and I realized I had to change my template or stop -- and you can look up the list of presenters yourself. Why am I typing it all?

My wife and I were already planning to go to Sweden this year!!  The plan was to see what caused the people in the country to create the greatest musicians in the history of the universe... Something special is going on if you invent both Melodic Death Metal and ABBA in the same century.  The hope was to see see some of greatest metal bands in this entire plane of reality on their home turf. I don't know if these events can line up, but if luck doesn't them maybe we'll get creative

umm.....that doesn't get less weird on the 100th repeat....

Monday, August 6, 2018

Analysis of adaptive evolution at the amino acid level!

EDIT: Fixed multiple errors about 10 minutes after posting -- I misplaced my espresso....

Okay -- I admit it -- this sounds really boring. And, yes, it's on Drosophila... and it's full of maths and Greek letters and things that it assumes the reader knows what they are and what they mean... 

...what was I doing before I fell asleep...? oh yeah! This new bioRXiV thing! 

Maybe I'm tired of reading pure mass spec papers cause of this grant thing we've got to do this week that I worked on all weekend -- or maybe it always seems like all the evolutionary stuff is done at the nucleotide level, which seems lazy and somewhat naive. 

Natural selection acts on phenotypes. And phenotypes are controlled at the protein level. Sure, genetic info is where everything is passed along, but if the genetic changes don't change an amino acid, then unless there is a stop codon or something like a silencing event -- there is no change in phenotype and therefore no selective advantage is acquired. Seems like you could get a much more accurate measurement of evolutionary pressure by focusing on the protein sequences. These authors point out that people have been doing amino acid evolution stuff since the 70s and point out a big study from last year, so people do realize it. I bet it's 1 out of 100, though, and it's always good to see another contribution!  

What size steps, though? 

Sunday, August 5, 2018

PHOTON -- Is there finally a solution to phoshoproteomics signaling?

I made this statement twice in one week recently "what we'll give you from this experiment is a very large list of phosphopeptides and their relative abundances, and if you figure out what to do with it, please let us know"


Sure, I can go to PhosphoSitePlus and I can see that my phosphopeptide has been found before and when (yay....)

Or I could pretend these are genes and put them into IPA and generate...well...something....

As a field, I think we've done a great job of ignoring this. Fixing it? That would be really hard. But -- I think that Perseus is sneaking in a solution.

Check this video out (did I post this before?)

There aren't loads of details on PHOTON outside of the video -- and here is where you can get the software to load into Perseus ( or newer, though, I think this might be the newest). I presume there is a ground-breaking paper on the way!!

Saturday, August 4, 2018

InDigestion --> Find tryptic peptides from UniProt on your iPhone thing!

I was scrolling through PastelBio's enormous list of proteomics tools and databases and -- since I just switched back to Apple's yay $1 TRILLION dollars? Maybe there's finally a little bit left to improve the quality of life of the people who make the  shiny friendly phone interface -- there is a new tool I can try!

InDigestion is a fun little app that can give you instant access to UniProt and peptides from proteins there!  It's listed under "commercial" so my guess is they charge something for it, but I don't know what.

I'll try it as soon as I remember what the password is for this bright shiny stupid little thing!  Why does it need so many passwords?!? Why did it fingerprint me?!?!?!?

Friday, August 3, 2018

Identification of glioblastoma tumor antigens IN PLASMA!

I've gotta move fast today, so I'm going to just leave this here. If you're looking at MHC/HLA petpidomics stuff -- this new study is an understated gold mine. 

This phenomenal amount of work was done on a Q Exactive Plus using 150 minute gradients. If you are doing this stuff, I'd suggest paying particular attention to the LC separation and the MaxQuant data processing parameters. I'm not 100% sold on the 95% confidence logic, but they make a solid argument.

This is still in press at MCP, so PRIDE/ProteomeXchange hasn't released the data yet. I did bug the corresponding author, extremely out of normal working hours on a weekend to see if they could release it early so I stare at all of it. He said it will go live next week! (Thank you!)  When it does it can be found here!

Thursday, August 2, 2018

Want to shape the future of DIA proteomics?!?! Your opinion is needed!!

Okay -- maybe I'm biased but I don't think it's out of line to say that MCP is our high water line. If you can get your data through the filters and the 100 annoying rules and don't find yourself thinking, for just a second, about keying the cars of your anonymous reviewers -- and then actual get your paper in - it's probably AWESOME!!!  (100% kidding...who even thinks about keying cars!??!)

Yes, you'll be rewarded with a lot of work to get yourself in a journal your collaborators have never heard of -- but WE will know what you went through. We'll appreciate the work you did. We'll know that you could probably have sent your paper to a biology journal with 4x the "impact factor". We'll probably also be sitting here reviewing papers that came in from said biology journal and will probably think, for just a second "yeah -- this would never have got through MCP or JPR review process...and are the RAW's Nature something something....they don't require it..."

Part of the reason MCP is so great is this stuff --- DIA is coming bigtime -- and if MCP is going to maintain these crazy rigorous standards on DIA studies, it's going to need to come up with 100 very strict rules -- and they want input from the field to come up with them!

You've got till the end of September to throw your thoughts in!   This is MCP. It's supposed to be strict. Is there a PDF that you have saved as "DIASucks.pdf"? I know you do. I've got like 10 of them! It's very confusing. Write MCP and tell them why and how to make it better. This is our chance to shape how an emerging field in proteomics develops and help make it something that will make our field and science in general, better!

Wednesday, August 1, 2018

What ratio of isobaric labeling reagent to peptide is critical?

(Shoutout to A.J.Bureta for assembling part of this image and posting it under the Creative Commons Share-A-Like 3 where "free to modify" is rule number 2 {Hopefully my modifications aren't too obvious}).

Okay -- so I've noticed a trend recently -- I first noticed it right before ASMS in this great new paper. While at ASMS I hunted down a couple of studies that used isobaric labeling technologies and spoke to them about the ratio of peptides to labeling reagent they are using. The final straw was last week when I got to talk to a somewhat famous researcher in isobaric labeling techniques who referred me to some earlier works like this one is the question. When I pull up a protocol for isobaric labeling reagents, I'll find a recommended ratio of 8x labeling reagent to every 1x of peptides. In a protocol for another I find 10x.

Do we need that much reagent? In the first paper above, they clearly use a 2x labeling reagent to 1x peptides. In conversations with other researchers it sounds like 2x or 4x is becoming more of the secret (hidden deep in the methods section) norm. I can imagine that 8x probably does label closer to 100% of the peptides -- but if you could get 95% labeled with 2x or 97% with 4x  or 99% labeled with 8x --- umm..... that last 4%...ummmm --

[insert unrelated gif here]

....totally unrelated...