Thursday, June 14, 2018

The Lazy Phospho Normalizer!

Okay -- so there are easily a million smarter ways to do this. I know it. However -- I doubt there is a lazier way.

Here is the scenario

You did a global phospho TMT study.
Because this is a time course long enough that trancriptional/translational regulation isn't something you can rule out you also kept the flowthrough (or part of the sample that you didn't enrich) and you also ran it -- ala SL-TMT or something similar.

How do you combine that data?

Like I said -- 100 smarter ways to do it, but last night around 8pm I would have given just about anything for an Excel sheet that said this --

Around 11pm I started channeling that frustration into watching some tutorial videos on the Microsoft Office website and early this morning I woke up and finished this tool that does EXACTLY what I needed last night

You can download it from my Google TeamDrive here.

As it says in the instructions -- if you make something easier and better (or already have one), please let me know. As always, happy to see comments regarding what could be fixed or improved. Can't guarantee I'll actually do it -- as I said -- this does what I want it to do, I'm just putting it out there in case it would help anyone else.

Also, if you're just writing me to make fun of the fact I could do this in R in like 3 seconds, I'll have you know that I've installed 6 copies or R and R studio on this PC and I don't even know which icon on the desktop is the most current one. Keep that in mind.  You could do it in 3 seconds, I'd spend an hour -- easy -- clicking the wrong desktop icon. "Where is the one that links to that cran thing..?"

Wednesday, June 13, 2018

MaxQuant goes Linux!

It's been possible for quite some time to run MaxQuant on Linux in different kinds of "virtual environments" and things. I know people who have been doing this for a while. These, unfortunately, have loads of overhead and sap your processing power.

MaxQuant having a true Linux version?  That's a big deal. Nature Methods level big deal? Sure... why not!

Monday, June 11, 2018

Tired of trying to find your PARPs? Cleave 'em off!

Do people still look for PARP inhibitors? I haven't heard of any in a while.

PARP is obnoxious because it's a polymer PTM. (This is a good review on it.) Unlike more friendly PTMs like ubiquitin, there isn't a friendly cleavage site coupled with a nice mass shift.

This new note at JPR shows a great way to get to the proteins that are PARP'ed (PARPyPARPylated?) by getting down to the proteins and knocking it off.

Much better idea!

Edit: I didn't try hard on the nomenclature at all. This link at Ribon pharmaceuticals explains the different types of these things.

Sunday, June 10, 2018

ProteomeTools takes on 21 different PTMs!!

Spectral libraries are coming back with a vengeance. Check out this great new study in press at MCP!

What were their limitations again?

1) The libraries weren't big enough?

2) Integrating library search into normal workflows wasn't straight forward?

3) There aren't enough PTM libraries?

1) ProteomeTools has already dropped 400,000 synthetic human peptides through their site and through collaborations with institutions like NIST. A LOT more are coming. Couple this with the MASSIVE's new libraries and the treasure trove at NIST?

2) More on this later, I think. But more and more of our normal software workflows are becoming spectral library compatible. Mascot supports them now (right?). I've seen two mentions of spectral libraries in MaxQuant in the literature, so that's coming and all the DIA software is ready to go for libraries of all kinds.

3) NIST has had a huge human phospho library for years, sounds like MASSIVE has a ton now as well -- but ---

This team synthesized a ton of peptides with weird PTMs on them!! I'm sure they have or will release the libraries -- but what might be more important right now is that the RAW files are available via ProteomeXchange now (PXD009449, here)

Is that really a crotonylated lysine you're looking at? Ever seen one before? (I'd never even heard of it until recently...) sure would help if you could download a couple RAW files with real ones in them and find out they look like this in HCD, right?

Better image from the RAW file itself (yeah -- that 152.107 is in virtually every MS/MS spectra -- you know, just checking...):

This paper is an absolute treasure -- if you're European you probably know that Andromeda can make use of these diagnostic ions in the scoring algorithm. So...if 92% of all crotonylated lysines made a 152.1070 fragment ion, Andromeda can take that into account and help you weed out the false cool is that?!?  I just went through PD and even in the text editor interface for modifications in MSAmanda 2.0 I don't seem to have the ability to do that at all...(you can, however, get MSAmanda to preferentially score you new neutral loss masses, but it requires some finagling to get it right. Can't guarantee I've got it, but you edit those here in the Administration).

Wow -- as soon as I think this paper has stopped giving -- there is more stuff. If you're interested in any sort of weird PTMs -- this study should be on your desk. It'll be handy when it's formatted, but it's worth cutting 45 pages out of a tree right now --

HOLY COW --- The very last figure of the supplemental info casually shows you how to resolve one of the single hardest things I could ever think of trying to work with -- there are different neutral loss patterns whether a peptide is symmetrically or asymmetrically dimethylated....the more I think about that the more I'm certain I probably couldn't manually sequence that without this information....but -- could I go back into the settings above and feed MSAmanda this information and get it and PtmRS to use this information to resolve these correctly?

I can't believe how great this study is.  I don't use this .gif lightly...

Saturday, June 9, 2018

Trypsin and urea? Keep it at room temp, y'all!

This makes a lot of sense -- and makes me really happy since I only recently found out where our incubator is (room temp overnight digestion, FTW!)

Friday, June 8, 2018

ASMS 2018 takeaways!

I didn't get to see much the final day of ASMS as I traveled back, but the two of us from our group who got to go are working on a wrapup for those who couldn't. It'll honestly take months for me to sort through all the notes and for the Google Scholar alerts to stop coming in for all the cool stuff I don't feel I can't talk about yet.

My biggest takeaway --

Our field is still beset with difficulties -- but the instruments don't seem to be the problem right now. How can it be with so many people achieving near theoretical proteomic coverage? With thousands of PTMs of any type seeming like something that even I could get?

This ASMS felt like -- time to confront our biggest shortcomings, like:

1) The fact proteins are really hard to get out of cells easily and consistently

There were sample prep robots everywhere! And high tech new sample prep kits, like S-Trap, the awesome thing Pitt is working on that I can't find note of yet, some new stuff coming from vendors themselves (finally?) that all seem tilted toward being automation compatible

2) The big one -- How immature our informatics are -- and how we fix it!

I think I'd been lulled into some sort of a complacency finally that our data processing pipelines are just fine. A major emphasis of ASMS on the proteomics end is that they aren't. They're better than they have ever been, but this explosion in scan speed and data complexity is showing that some of our core early 2000s data processing assumptions are in serious need of updates -- but really really smart people are working on it. We probably don't ever need to get as sophisticated as the genomics people -- our raw instrument output is easily 1,000 times less noisy and more accurate than theirs (have you looked at raw output from these next gen things?!?) but we've got some ground to make up.

(Images like that do make me feel better. I can always manually sequence my peptide if I have to, best of luck with those 4e7 transcript reads)

(Another way to make myself feel better, I went to some metabolomics talks -- they're trying hard and making up ground, but they are way behind where we are, partially due to the smaller size of their field, some really poor assumptions that were made in the past, and --most importantly -- some really unique problems they face. "Oh-- cis- and trans- makes a huge difference here? Great! Best of luck with that! I'll...umm...check..back...later...")

3) Glycomics and glycoproteomics are coming -- and is about to become a primary thing we hear about. I'd have to stop and check the signs while walking around "yup -- I'm still surrounded by posters about sugars..." Everything is glycosylated -- and glycans totally suck to work with -- but it wouldn't be crazy to suggest that they are more phenotypically important than unmodified proteins.

New reagents, new columns, new methods, new software. It's all going to help when that scientist knocks on your door and has no intention of letting you cleave the sugars off and throw them into the waste bin. "Oh -- d- and l- makes a huge difference here? I'll...umm....")

4) Proteogenomics may finally be something that I can do! Surely, out of all of these new tools there has got to be at least one -- at least one -- that I'm smart enough to figure out how to use, right? I hope more details to be on the way soon!

Thursday, June 7, 2018

ASMS2018....Day 3....

ASMS 2018 described in too many words, what about this --- Amazing, dedicated, super nice and scary brilliant people everywhere who are all unified by some strange desire to use vacuum chambers and magnets and electric fields and stuff to somehow make the world a better place.

ASMS described in <140 characters?

Day 3 was awesome. My day started with Michael Shortreed describing his team's MetaMorpheus software. If you haven't used it, I strongly recommend you check it out. If you were awake at the cursed hour before 9am (whatever it's called -- I don't even know or care to find out) and got to see it, I'm sure you know why.

I have a problem (or two, I guess...) -- but the one I'm thinking of is something about a processed protein that gets stuck in something weird and I need to know how much of that protein is stuck in that weird thing (like -- up to what amino acid is it stuck in this weird thing, does that make sense? -- this is often about the level I understand around work) and the talk on DiPS presented the solution I've been looking for! You can read about it here. DiPs relies on multiply microwave assisted acid protein cleavage and de novo sequencing to fully sequence proteins. So -- if you're protein is stuck in something weird up to an unknown point, maybe you can figure out exactly where the sequence ends!!  This digestion isn't new, of course, but the strategy for overlaying the fragments from different runs is EXACTLY the right twist to make this work (I think!)

Random favorite stuff:

Hong Wang's (pretty sure he's in Junmin Peng's lab) from St. Jude's -- comprehensive proteogenomic study on how to deal with rare (low n) diseases was a serious WOW. Had to text colleagues to come see it (including an infamous MD I work with, so that I could verify that it was as smart as I thought it was), but it hasn't been published yet. Can't wait to talk about it, though. Google Scholar Alert set!

Mark Chance showed CrossTalker -- it isn't out yet, either?!?!  I've had the fortune of getting to spend some time with Dani Schlatzer in the phenomenal Case Western Proteomics Center. They have amazing in house tools -- and it looks like some are coming to the outside world. CrossTalker is an open network and pathway analysis tool that has some really cool twists. I hope to use it soon.

Amber Moseley (who was too flooded with interested people for me to catch up with) showed an application of thermal profiling for studying mutations!!  I swear, I always think about this tool as one of those things that is amazingly powerful -- but is looking for an application. Amber's team at IUPUI found one and it's scary smart.

Pierce is releasing a TMT QC reagent!!  It isn't on their site, but -- how useful will that be?

I got an inside perspective of what we'll see out of CPTAC 3 from David Clark at JHU -- just the controls they are using is worth a serious publication. This going to be an amazing dataset to mine forever. (Hurry up and get it done, y'all!!)

The middle of my day was software demos and looking at sample prep robots. Paul Stemmer just upgraded his robots at Wayne State to the Agilent AssayMap and had nothing but good things to say -- given the throughput and quality of work that comes out of that lab, that says something. However -- that robot is VERY sophisticated and that translates to pricy in robotics. I know a number of people who are loving the cheaper (though write your own python scripts to use it...) OpenTron route.

Okay -- these are clearly some of my favorite people to talk about (do other proteomics people have real life PIRATE STORIES?!?!) but Mak Saito's team at WHOI has been doing amazing stuff profiling the metaproteomics of the sea. Jacyln from that group (probably doesn't need a last name if you spell her first correctly) detailed the study of huge ocean zones where there is virtually no dissolved oxygen. Huge areas completely full of some of this planet's most ancient (pre-oxygenic catastrophe! life) and how she's studying it with proteomics. Wait on that paper -- I suspect there is a reason she's getting NASA funding, for real.

(Probably joking!!)

However -- they've got loads and loads of data from the ocean and they've been working to develop standards for oceanography data collection and dissemination. OceanProtein Portal is the first attempt at the dissemination part.

Check this out -- you put in a peptide sequence (partial or intact, or identifier) and you can see if they've collected information on it and it's abundance -- at different spots on their global explorations.  (P.S. I've provided feedback that the word "cruise" is not a good PR decision for what are extremely long and dangerous sounding deep see mission)

This obviously isn't my field, so I can't really do anything smart with it except punch in peptides, but for people who are in metagenomics/metaproteomics how lucky are they to look at such a cool resource?!?!?

Woooooo!!!! ASMS, yo!!

Wednesday, June 6, 2018

ASMS2018 -- Ben's even more useless recap of Day 2

Hey! Are you looking for a postdoc who can make great proteomics informatics posters? Is quality of handwriting not the highest priority? 😋  You can reach Donxue (she's in Kuster's lab! I hear they're pretty good there.) here.

With that out of the way (clever idea, Dx!) time to dig out the ASMS 2018 day 2 notes. Besides crack 20,000 steps again today...umm...what did I do again? 

Wow -- Ben Garcia's acceptance speech of the Biemann Medal -- wow -- informative, funny, sincere

--- included a very detailed SpiderMan reference (there is a histone thingy on that chalk board in the movie)-- what more could you ask for? Seriously... one of the best talks I've EVER seen about anything.  I still don't quite understand what a histone is or does, but it is clearly a problem that nothing currently exists that is better suited to conquer this problem than our technologies.

This was PROTEOGENOMICS day! How do we finally merge all this transcriptomics and proteomics data together? And there are crazy powerful tools out and more coming.

Some highlights!

PepQuery is live here and can build off of VCF, BED GTF or WTF files (one of those is made up, the rest are things your genetics people talk about -- and is peptide centric comparisons off of those file types.

On that same tilt -- ProteomeGenerator is also on BiorXIV here. More tools to make integrating all this data together, please!

Side note --
RawMeat may be coming back finally! Should be investigated. You might LOL when you find out what it's called (pdf download of poster is here)

Something ABSOLUTELY WORTH CHECKING OUT -- PeakStrainer --> it reads directly from RAW and does some amazing stuff in terms of discerning peptides from noise based -- not on intensity -- but on the frequency. Super smart....

Back to the proteogenomics -- besides the tools there were amazing examples of large integrations from tumor samples to patient cohorts to using the two to tell between human and mouse in xenograft models. With better tools maybe all of this is going to become easier (it still seems tough and scary to me...but there are some great templates coming)

Just to double back to the very top -- Donxue's poster was on healthy tissues, something I feel like we sometimes forget about the need for profiling. If she did that work (it doesn't appear to be out yet) and you've got a position somewhere on earth that isn't too cold, it would be my recommendation you shoot her an email -- if only to learn about what she's working on. Suuuper cool.

Okay --coffee -- more informatics --go!!

Tuesday, June 5, 2018

ASMS2018 -- Ben's totally useless recap of Day 1!

Cause I need something mindless to do while staring at this coffee -- here is some useless rambling about ASMS2018 day 1

Actually, I'm going to start with this awesome Tweet first.

Great thinking points, right?!?  This should be hung up somewhere....

A random list of observations:

1) Be sneaky if you have your kids with you and you want to walk around posters. No children are allowed in the poster area. My best guess is that there is a fear they will die of boredom and the San Diego convention center will be held liable.
-I'm no expert on being sneaky, but the poster room has 4 entry ways with doors that only lock from the outside. There is only convention center staff at the single main entrance. Have someone (me, if you see me) walk to a side door and let you and your kids in. Your kid gets a paper cut and that's on you, though.

2) Shimadzu has the best food -- their TOF still only has 100 resolution in high mass mode, but you can't have it all. (I can make this joke -- my work is getting a second one)

3) I learned that despite all of our differences, all mass spectrometrists seem to be unified in their hatred of Budweiser (whatever that is) -- definitely the most common topic of the last 3 hours of the conference.


Informatics is king! We're continuing to realize our limitations on this end and more tools to improve everything is coming.  One thing I can't wait to post -- JudgePRED (best name ever?) isn't out yet -- watch out -- it's cool. Triqler is a much smarter way of doing FDR (integrating peptide and quan FDR) and the python code is all at that link.

Then Bill Noble came on stage and gave one of the scariest talks I've ever seen. I think I can talk about it because he presented it at RECOMB last year and the notes are in Cell here.

So....if you randomly shuffle your decoy database every time you process the same set of global proteomics data --- you...umm...can get terrifyingly different results.

He showed a couple extreme examples just to see if anyone would run out screaming using both large datasets and large and small FASTAs. If he used a FASTA with less than 1,000 protein entries and then reshuffled the decoys (changing NO other parameters) the peptide IDs could differ by 20%(!!!) from experiment to experiment. (This is real footage of what happened next)

Did you know MCP doesn't allow you to use global data searched against FASTA's <1k...? This is why.  His team has a solution coming and it might even be available on Crux now.  For now -- umm -- let's all ignore it and not let the collaborators know.

I've scanned the other talks and -- I'm seeing a lot of stuff that isn't out there yet.

There was a great PACOM talk that really showed what it can do -- and was delivered with much more confidence that I could have done with Jurgen Cox sitting in the front row. 😅. We all REALLY LOVE MaxQuant and Perseus and the fact we're trying to build things above this lofty bar is a testament to that. PACOM makes some reeeeeaaaallly pretty plots, tables and graphs.

Instrumentation-wise -- I'm a little behind. I got a personal demo on the Q Exactive UHMR last night -- and I totally want one -- but its gonna be a tough sell for me. This is the top down instrument that the field has been needing. Its an Orbitrap that scans up to 80,000 m/z (not m+H+ --- m/z!!) can do 280,000 resolution, has magical front end trapping (essentially making MS3 feasible in a Q Exactive!) and doesn't require the double calibration stuff that the EMR does. Native top down and they have data for mega-dalton top down work.  Is it the most powerful top down, intact mass characterization instrument ever? Yeah -- without a doubt. Can I find a biological problem that would mean we absolutely needed something this awesome? Working on it....I'd love ideas....

There is another contender in the DIA software space -- with Scaffold coming in with a really nice interface. I'm really in love with Pinnacle from OptysTech -- and it can do a lot of other things I really really need (untargeted Discovery! -- I.e., great SIEVE replacement), but it's always great to have options. Both are doing live demos here if you're looking at getting into this field.

Coffee down -- time to hit Day 2!

Sunday, June 3, 2018

The Skyline User meeting recap!

The Skyline User Meeting was 🔥🔥🔥🔥🔥🔥 (I'm not sure what that means, but context clues from Twitter and Reddit suggest to me that I'm using it right).

How do you have a successful user's meeting for awesome open source software?  Maybe you have it in an amazing old public library -- you get support from vendor sponsors to provide really good food, and -- most importantly (of course) -- maybe you stack a ton of talks on things that we didn't know this great software on our desktops could even do!!  Even though a vendor had offered a free cruise of the harbor for lunch -- enthusiastic Skyline users filled this less flashy event.

Talk 1: Chris Ashwood of Medical College of Wisconsin (and Head Editor of Glycomics Methods for

Glycan analysis and characterization with Skyline! The first paper came out recently, but he showed that this might be the tip of the iceberg. Even better maybe? What about a fully developed internal/QC standard? For glycomics? Yeah!

Talk 2: Paul Auger at Genentech

Automated QC with Panorama for peptides AND small molecules!! Okay. I knew Panorama was out there. I knew that Mike Bereman's work with sProCoP had been integrated entirely into Panorama. What we didn't know? That you can set up a local Panorama server (in case your instruments aren't accessible to the outside world!) and get all the benefits of Panorama inside your network!

Benefits like: real time QC/QA on ALL of your instruments.
Know that ion transfer tube is gonna need a new one dropped in --- before you're 2SDs low on sensitivity!   This talk required an email to our director (we need him to buy us a server so we can put Panorama on it)

Talk 3: Robert Ahrens -- LipidCreator/LipidExplorer + Skyline!!

Have you even heard of this?  This open(?) lipid software looks ridiculously powerful and the developers got in touch and now its a Skyline powered quantitative lipidomics platform!  Is there anything better than adding new applications to a piece of software you already know really well? Sure, you'll need to learn something new -- but you aren't starting from scratch and that gives you a massive lead on getting that application going in your lab!

Talk 4: Don Davis of Vanderbilt and developing Clinical Assays in Skyline!!!

This was a great lightning talk where the real consequences of the implications of the work this team at Vanderbilt is doing didn't really hit me until about 3 minutes into the next talk. For a lot of us, our end goal is new biomarkers or diagnostics -- and Don showed how feasible moving what you've developed in the lab could really be -- without the added complication (again0 of having to learn something new. With Skyline piling on new Auditing features (which Brendan assures us you can actually see -- just not on a projector 😜) why not? If all the necessary security protocols are in place -- why couldn't we develop an assay and just port it right into the clinic -- without having to re-optimize with new software??

Talk 5? Yao Chen from Catalent -- what about antibodies?!?  

Heck, why not. Why not use Skyline as a (digested) antibody characterization system?! Apparently it can do everything else. There are some really important reasons to use more targeted approaches for mAB characterization, but I've been trying to pull his references and I'm not sure what has been published, so I'm gonna stop that one here.

Talk 6? Kristen Geddes from Merck -- Panorama QC of a diverse portfolio of instruments doing all sorts of things.

If Conor and I weren't already sold on Panorama -- seeing this great talk -- Merck isn't a single vendor kind of place. Their LC and MS systems aren't even single vendor -- but QC with was shown to be powerful and well automated with a local instance of Panorama handling the whole thing! Yeah -- we need to get this set up.

Talk 7? Buyun Chen from Genentech -- taking a deep look at peptide and protein quan. 

Again -- Buyun gave a great talk, but I'm not sure how much has been published. She raised some really important questions (with some solid data) about peptide and protein quan -- and how Skyline can really help you find those peptides that TRULY reflect the amount of the proteins present. Some basic fundamental questions are being explored and if I see that this work is published, it's going to end up here for sure.

Talk 8? Lindsay Pino  SIGNAL calibration in quantitative proteomics. 

I think this is being written up (maybe I'm just being lazy) so I'm not going to go into it much -- but -- what if you could do a PRM on your Orbitrap Elite AND compare it to PRM on your Orbitrap Fusion (I'm using this because it's an extreme example -- I often get areas >1e11 on the Elite -- I don't think numbers that big can be displayed on the Fusion -- Signal calibration is something we're going to be talking about in the future -- Lindsay demonstrated with some amazingly diverse datasets that 1) Signal calibration is critical and 2) She can show us how to do it.

Wrap up:

Brendan MacLean filled in some interesting history on the almost 10 years of Skyline -- where it is -- and where it is going next.

Worth noting, maybe -- due to some rearrangements in the grant process -- (NIGMS no longer has a separate category for software like Skyline. The grants supporting this software are going to need to be fully competed (against things that aren't software). User feedback to the grant review boards and the vendors who chip in to support the process will likely become more important as time goes by. Not to end on a downer -- this was an AWESOME workshop -- but I'm sure it would be easier for the developers if the 10,000 of us that have this software had each paid $15,000 for it. So it's worth keeping in mind we may have to do surveys once in a while to keep Skyline free, and open and improving.

Friday, June 1, 2018

Blog is on hiatus for ASMS 2018

I have a tendency to blog too much at ASMS -- which sometimes gets me in hot water, so the blog is on hiatus this week.

I've got to focus on learning everything I possibly can and not causing any trouble at all this year.

Even better? According to the yelp! reviews, I don't think people will feel safe enough in my neighborhood to wake me up by sliding marketing material under my door this year!

SEE YOU THERE!!!  I'm the guy who looks like he's been sleeping outside.

I am, of course, joking -- about being on hiatus, anyway!

I applied for official press credential for a reason.

Thursday, May 31, 2018

Why is there a crab on the cover of JASMS?!?

What's this about?!? I believe some people are in need of these instructions!

Actually -- the study that scored the cover is really cool, so I'll give them a pass this time.

Multifaceted is a very nice word for -- this team did a ton of work -- including neuropeptide imaging?!? Check out how cool that looks (actually, it looks better in the paper).

Sure -- it's crabs -- but what a promising use of technology I didn't know was possible. Begs the question of how far away is mammalian neuropeptide imaging?

Wednesday, May 30, 2018

Phase constrained deconvolution -- can resolve TMT11 plex in 32 milliseconds!

Could this be a hint of things to come?!?!

Last year some people at Thermo, including some Makarov guy, published a paper on a phase constrained deconvolution alternative to fast fourier transform (FT) (that you can find here). Honestly, it looked like kind of a thought experiment. The fourier transformation is a great math trick that allows you to go from frequency of orbitals to masses in these instruments (but has, no joke, about a thousand other uses behind the scenes in today's world).

The phase constrained deconvolution goes beyond what the fourier transform can do -- pushing into the limits of "fourier uncertainty" where our instruments currently don't, and improving both resolution and sensitivity.  Again -- cool paper, but ---

-- this is WAY COOLER ---

Keep in mind that this chart is biased (smaller m/z gets higher resolution in the Orbitrap -- even with phase constraint model) -- but look at these flippin' numbers for the TMT reporter region resolution (this is an ultra high field Orbitrap -- I think this is the D20 with 5kV on the central electrode, but don't quote me --if I'm right, this is what is in the Tribrids and HF/HF-X; D20 with 2.5(?)kV is Elite)

Only 32 ms --- that is 31 Hz (ignoring overhead) and they got 80,000 resolution at the 127 marker -- enough to revolve the 6mmu (0.0062 Da) separation between the TMT127 N and C reporters.


Okay -- so I have to throw this in, because I've got a meeting with the developer of this thing planned --

--Cause -- Yury's booster system appears to be right at this same level --

The box on the right isn't the clearest, but the red peaks show complete baseline resolutoin of the 127 and 128 TMTC/N isotopes using 15,000 resolution on a Fusion/Lumos. Given that we use 50,000 resolution to resolve these in our lab -- 15,000 translates to 3 times faster!!

Plus, I've been told that it's really easy to just put it out of sight in case your service engineer is all nosy. If he's coming to fix one system you can just take the FTMS Booster and plug it into another one and use it on that one. Whichever one needs a ton of resolution that day.

I'm not trying to stir anything up this morning -- the Phase constrained deconvolution looks amazing -- but the FTMS booster is something that I could buy today (if I had the budget for it) and it costs less than an HPLC that I really want.

EDIT: MassSpecPro posted this link yesterday -- understanding the Fast Fourier Transform. If you want to know more about all of this stuff, this is a great starting point!

Tuesday, May 29, 2018

Let's check out the new PASEF TOF thing!

Have y'all seen this thing yet? I just got to see a great talk on it and I'm ready to say that -- for bottom up proteomics -- this is the most powerful Time Of Flight instrument we've ever seen.

EDIT 6/9/2018: I've spent some time talking to smart people about this thing and I've -- overall -- felt kinda dumb about this post for a number of reasons
1) I got really annoyed about my inability to see the instrument data or process it and kind of went ranting
2) The rants on this page aren't based on real facts, just some grainy marketing info on this device. I should really withhold judgment on this thing until I can see some real data -- however -- I definitely don't want to seem like I'm endorsing it until I do. I've seen MadMen. Marketing people are scary.
3) When in doubt we should be encouraging competition in the marketplace. Just look at what the resurgence of AMD has done in the PC marketplace. Intel has real competition for the first time in years and we're back to exceeding Moore's law for microprocessor performance! Maybe the same thing can happen on the mass spectrometry front.
4) Now I'm afraid people think that I've actually ranked my dogs in the order in which I would eat them if I had to....

I'm going to leave this post in place as a reminder to myself to do think before I post. The walkthrough on how to look at Bruker .d data in MaxQuant does appear correct, if you use the new version. And I'll put up a new post later when I actually review some data.

There are a lot of bells and whistles in the instrument, but probably the central technology is PASEF which was described here in 2015. TOFs are FAST. Screaming fast. But -- sensitivity is a serious problem. You need to accumulate some ions before you shoot them down a tube toward a detector. -- especially one that is several feet away to get anywhere near the sensitivity of a quad, a trap, or an Orbitrap.  PASEF and the TIMSTOF allows accumulation of ions before shooting them and that can bring up the sensitivity a lot. It also allows parallel filling and that speeds everything way up.

Okay -- so -- let's not go into details, but someone sent me a Zip file with some data from one of these things. I'm normally not nervous about science stuff -- but, holy cow, it is really hard to find data from one of these things. I'm sure that with brand new technology it probably takes a while to get everything formatted right for public uploads and things, but when I do see some uploaded via ProteomeXchange partner, I'll feel less like this....

If you get into the file -- you'll find it is an SQLite format (interesting!). That is what the processed data from Proteome Discoverer is -- so if you want to open it you will need to go and get a free copy of SQLite -- you'll need 4.5 or newer -- and if you don't want to hunt for it, you can get it here for Windows 64-bit.

You'll definitely need to check first to make sure you have a .NET framework that is this new. Here is a guide for how to do that. This will require administrator access. When you get there you'll see something like this, probably. If you do, you'll need a .NET framework upgrade.

To get that, Google "Download .NET framework 4.5" and verify that you're on the legit https:/ site (lots of fakes out there). Close everything, upgrade, reboot and come back to this. If you're on Windows 10, this should already be preinstalled. No problem.

To process it you'll need a recent version of MaxQuant, the newest version of PEAKS (haven't checked this one) or -- it sounds like the most recent Mascot! Easy! I'll go with MaxQuant. We need to get reintroduced anyhow.


Okay -- let's assume operator error, put a note on the MaxQuant discussion board and try something else!  EDIT: See bottom of post for correct MaxQuant version to open this data.

Here's a cool video that talks about the instrument! Let's check it out!

This is a really solid video. Dr. Rather explains how the PASEF thing works, and how we have to think about TIMS data in a different way than we're used to seeing. Being a guy who had the first MS/MS spectrum he ever published tattooed on himself, there's obviously some things I'm interested in seeing. At 22min and 37 seconds you'll find a spectrum to check out.

I drew two question marks here. Let's look at the one on the left first!

If you go to Google and type in "proteomics red elephant" it will take you to a blog post I wrote about a tool I use every single day, the pHpMS webserver. I love this tool.

If I punch in the sequence for this peptide in the Fragment Predictor it tells me this instrument is getting the right doubly charged mass

888.94 compared to 888.9329 -- I'm not going to be a jerk here, for real. Yeah -- I like 4 decimal places. Honestly, in this range we're only accurate (without post-acquisition recalibration) to the 3rd decimal, even on a Fusion 1. Even if we assume the very worst, that the measured mass was 888.9449, this is only 13.49 ppm off, right? I'd rather eat my dog that I like than send a peptide to a collaborator where my MS1 was off by 13.49 ppm, but this is the very very maximum, it could easily be 888.9351 rounded up, which would only be 2ppm out. For reference sake, I wouldn't eat my other dog for less than 8ppm.

How do the MS/MS fragments line up? It's tough to tell with it being all no numbers and stuff, but we can extrapolate. y10 should truly be 1203.5423 and y14 with z=2 (**in the figure) should be 853.4143. If we assume that gap between the scale markers at 1200 and 850 are the same --

---Its probably just the visualization tool and a low res image of a low res image, right? y14 sits precisely on the 850 marker and y10 does not. The visualization tool used here could be at fault for bad as my eyes are I could be mistaking what appears to be a 3 proton gap and a nonexistent 3 proton gap. I don't even know why I'm still talking about this. I'm sure it's fine.

Let's go somewhere else. What about phosphoproteomics!?! Wait. I didn't do the other question mark. Ummm...let's come back to why we fragmented the same unmodified peptide 3 times...later...or not ever....
(Edit: 6/9/18) Surely it can do monoisotopic averagine modeling, right? Q-TOFs 10 (15?) years ago could do that.... some vague answers when I asked. I'm a little hung up on this one. Monoisotopic precursor selection (called MIPS or PeptideMatch by some manufacturers) is a big deal. Chances are you don't know your instrument is even doing it. If you want to see whether it's just a dumb button or not, go in, turn it off and run that sample again.) Actually -- new topic. Go to this paper from Dave Muddiman.  Turning off MIPS cut the IDs in half, and that version of MIPS was not very good -- at least compared to today's algorithms. (That one would throw out SILAC 'cause it couldn't resolve the isotopic differences. Buzz me if you need a reference on that, my memory is problematic, but I'm seeing some hazy details -- something about a smart ultra-marathoner from somewhere in Texas who showed that conclusively and the manufacturer fixed it...MIPS is that important! Dr. Bob Swaim would know exactly who I'm talking about, so I'll email him if anyone asks me about it. If you know him, email him for faster turn-around)

There is this REALLY cool thing and it's called the PhosphoProteomics eBook for this device. You can look it up on the Google. There are two spectra in it --- of basically coeluting isobaric phosphopeptide species (there was an AMAZING talk at ABRF about this from [correction -- Brian Searle gave this talk -- consulted my notes] about how often biologically relevant isobaric phosphopeptides elute together, but I haven't verified he's published what I want to ramble about yet). The isobaric species in the eBook look like this --

Here's the pitch -- without the ion mobility, you'd never be able to tell these two isobaric peptides apart. They'd coelute, muddy the water and you'd never figure them out.

Back to my outdated obsession with accuracy in scientific instruments in the year 2018 (bear with me, please) we have two isomers and one has a mass of 546.2626 and the other isomer has a mass of 546.2536

PPM calculator says --- 16.47ppm....I really like my dog. For real....who wouldn't!?!? Check out this dumb Mufasa thing he does sometimes. That is a really dangerous place to stand.

...but if you think that I wouldn't choose to eat that big majestic monster before I'd tell someone I had two isomers at >15ppm apart and the only evidence I had to back it their identity up in a human digest was fragmentation data scaled to the nearest 500 Da and one of the isomers has no evidence of neutral losses whatsoever.... I'm joking, of course. I'd probably eat Bernie if it came down to it, but definitely not Gustopheles.

Edit 5/29/18: Crazy idea I need to leave here so I don't forget. Could we simulate the 2 phosphopeptide fragmentation patterns and then see if Ascore or phosphoRS could tell them apart?

Wow. That was SO MANY WORDs. Not sure why I wrote them all now. Let's sum it up.

At ASMS we're going to hear a lot about a really cool new parallelized ion mobility trapping TOF thing.

It's probably the best TOF for proteomics the world has ever seen.

Yeah --- it can get upwards of 100 scans/second -- but at that speed it can't do sophisticated calculations like monoisotopic precursor selection on the fly. It also may not be capable of dynamic exclusion the way we're used to seeing it. And -- the mass accuracy isn't what we're used to seeing these days from fourier transformation or even modern TOF based instruments.

I'm not saying it's a bad tool. We've discussed how it might fit into our workflows at my day job, but there is this inherent danger in the explosion in value in the mass spectrometry commercial space when business marketing teams get to run ahead of peer reviewed science. There's a lot of flash around this big new box, but -- man -- there sure isn't much on the evidence side yet...

Update 5/28/18: MaxQuant was released on 5/27/18. It has native PASEF TOF support!

Monday, May 28, 2018


You know what I could really use? A --

Super Lazy phosphoproteomics protocol!

[Super Laid-back was suggested this morning. I dig it.]

[Stream Lined? That also works 😺 ]

11 channels for quan? That sounds like a good start!

Spin column phosphopeptide enrichment and elution?

SPS-MS3? Okay -- I can work around that. Especially if the results are really this good....