Cause no one ever asked for it! My favorite papers of 2018!

It's already time to break out my sequin jacket and end the year at the Laser Disco?!??  How did that happen??  I didn't get a fraction of the stuff I planned to do in the last 12 months done...logically, instead of working on those things I'm going to do my biennial review of my favorite papers of the last 12 months?  Meh. Whatever.

In no particular order!!

BOXCAR

You're tired of hearing about it. So am I (not!). This is maybe gamechanger of the year, y'all. When you can boost the S/N of your low abundance ions by an order of magnitude or two, VERY good things happen. I swear I've got an 80% written draft waiting for some feedback so I can get it out the door to be summarily and rapidly rejected by my peers like every other thing I've submitted recently. Hint: BoxCar is great for proteomics, but it's waaaaaaaay better for other things. Where do you need dynamic range the most? BoxCar it!

SCoPE-MS

We can't amplify stuff like those weird DNA people down the hall can with their PZR machines, or whatever they're called. With SCoPE we can -- sortof. You can amplify tiny amounts of DNA into loads of it and then you can use whatever super expensive, low quality, error prone sequencing technique you want to. SCoPE lets you leverage TMT reagents to the increase in interscan dynamic range to quantify peptides that are below the dynamic range you would get with MS/MS sequencing. SCoPE is a revolutionary idea and I don't think we've even started to explore the ramifications that it can have for us. Perhaps more important than the original SCoPE paper is mPOP that makes it a lot easier to do SCoPE (trust me, it's not a great project if you're rusty on sample prep. I screwed it up, I think, and that's why it's a robot's job now.)

MetaMorpheus

If you haven't downloaded this marvelous piece of software from the Smith lab you are missing out. We use this every day -- and every week or two it somehow gets better.

Since MetaMorpheus came out this year, upgrades have landed like
FlashLFQ (crazy fast label free quan)
Crosslinking analysis!
And MetaDRAW (which hasn't been published yet, but I'm using right now). Once MetaMorpheus finds that PTM you didn't know was there, didn't think to look for, and totally didn't have to do anything to find (cause that's what it does -- BOOM! PTMs!) you can use MetaDraw to make sure it's real.

Yo. That looks like MetaMorpheus found me a darned acetylation! The team is super responsive, and loves to get feedback. I wrote them about a feature that would be really helpful for my work -- and the next time I updated MetaMorpheus (it sends you little popups that new builds are available!) the feature I needed was there. If you don't have it, you can get it here. 

FDR for Spectral Libraries!

Spectral libraries sure seem like the future of mass spectrometry again, right? While DIA methods are pushing the development, shotgun proteomics needs them as well and we're seeing great new stuff! MS-Ana is a great new engine (wait. where is the MS-Ana post?), FDR for MSPepSearch added in the NIST stand-alone and in development in other software where it's used, and we can keep on going on. Now that FDR is in hand and there are amazing new libraries like ProteomeTools -- I just need to get off my butt and start using them again. So should you!

More Proof that NanoLC is mostly dumb!

Okay -- sometimes I say something like "I need 10ug of protein to work with" and the person I'm talking to stares at me like I'm wearing an awesome sequin jacket and strobe lights on my head, but most of the time, people have MILLIGRAMS of protein to work with.  I LOVE this paper. NanoLC is likely the weakest link in your pipeline. There are alternatives. Let's not get caught up in dealing with these engineering atrocities as if they're something we can never do without. Evidence is mounting that they may cost us more in productivity than they provide us in sensitivity and this paper is the best one yet.

Biological Application of a Phase Constrained Orbitrap!

I hope hope hope hope the application of the phase constraint algorithms for Orbitraps is just around the corner. Remember the jump in speed we got when eFT was added? This looks like it could be bigger. Do I want 60,000 resolution in the same amount of time it takes my QE HF to do a 15,000 resolution scan? Yes. Please. Now. Thanks.

ProteomeTools PTMs!

Yup, I already mentioned ProteomeTools once. This project is too awesome. This study on the fragmentation of 20+ PTMs from synthesized standards is an absolute gold-mine. There is more to be learned about PTMs fragmentation in this one study than in any book on the topic in the world. No. I haven't read every PTM book in the world, but I'd go to this paper first.

On the topic of PTMs -- what about Chemical Proteomics?

Big, far reaching implications here! Multiple reasons this study is in Cell. You don't have to take the experiment as far as these authors here. Just considering the unmodified peptides that drop in concentration when they're drug treated as something of importance might be the thing to blow the doors open on your project.

I admit it. I'm obsessed with the idea of what we could do with real Clinical Proteomics.

And this is the study I carry around if I want to prove to someone that this isn't 10 or 20 years down the road. We can help now if we're given a chance to.

A re-evaluation of FDR demonstrates some scary stuff about it -- but also how to fix it!

I've noticed a famous software program has some new feature that looks like they took this study seriously. I haven't investigated.

Wow. This could go on all day. Unfortunately it's late, I didn't sleep a ton last night and I have to cut this here. It was an AMAZING YEAR for Proteomics. And I expect nothing but bigger and better stuff from you awesome teams out there revolutionizing all the things. 2019!!!! Let's go!

ABRF San Antonio -- Making a Proteomics Core Nimble & Efficient!

Alrighty Buckaroos. Time to throw another shrimp on the barbie(?) and mosey on down to San Antonio! for --

The "at a glance" is now up and it looks like another ridiculously awesome and action packed meeting for us core lab folk.

What I want to direct you to if you're making plans to attend is one of Sunday's workshops:

Making a Proteomics Core Lab Nimble & Efficient!! 

Let's all sit down and try and tackle the biggest challenges that face getting the turn-around and sample quality up!

Speakers include:

Brett Phinney ....who, while new to this core lab thing...wait...

...nevermind ---who will be talking about how to handle diverse (weird) complex samples within a core environment. (I copied that Tweet months ago and figured I'd use it sometime!)

Birgit Schilling  -- who will focus on implementing that DIA stuff in a core environment. (Oh -- if you haven't seen this new paper yet, I highly recommend it!)

and this weird blogger (Me!) -- who will try to say something coherent about addressing the key bottlenecks in a core environment! How do we wrangle up the biggest problems....and...then...um.. do whatever it is you do after you've wrangled something up....(giddyap? is that a thing?)

Giddyap, it is! Get that thing that arrived proteomerized or metabolomerized and get that data out the door!

Edit...misspelled "proteomics" in post title...

HLA Splicing, Yes/No/How much? -- Part 17!

Alllllllllllrrrrrrrrrrrriiiiiiiiiiggggggggggghhhhhhhhhhhttttttttttt......

Are you working on endogenous peptides?  Are the awful fragmentation patterns of peptides lacking basic residues things you see for just a second when you first close your eyes? Would you LOVE to see another entry into the "are these things spliced or do they ionize and fragment so poorly we have no idea what we're looking at" arena?

You're in luck!

HLA peptides are BIG right now (for great reasons). And -- honestly -- what we know about these endogenous peptides seems to border right on nothing -- despite the fact Don Hunt's lab showed us we can study these things by mass spectrometry -- 25? almost 30? Years ago?  I swear the first paper is 1989, but Google Scholar thinks I'm making that up.

Biostars...always helpful...

Top comment on a question I was going to ask Biostars, but someone else already did....

...for some reason I had this weird idea that this was an okay place to ask questions...maybe cause this is where Bioinformatics is Explained...

I'm only miffed, because that was the only comment that wasn't an insult....

Washington Post Editorial if you haven't seen it already.....

Washington Post direct article is available here.

I'm sure they weren't trying to imply anything with the image. It was probably the only science picture they could find.....

Hey Galaxy People! Want easy proteo-transcriptomics!??!

Edit: Forgot paper link. Here!

Galaxy is one of the biggest players in the genomics/transcriptomics playground. As I understand it (from listening to smart friends in these fields):

1) Loads and loads of tools
2) You don't have to be a super coding expert
3) You don't get access to super cool new code until someone integrates it, but the community is big and active enough that the only people that get access to new tools faster -- are the super coding experts

Galaxy for proteomics (often referred to as GalaxyP) hasn't been as ubiquitous in our realm -- we've got our camps like TPP, MaxQuant, PD, CompOmics, OpenMS, etc.,.

GalaxyP shouldn't be ignored, though, it's an amazing tool for the (seemingly much more common) bioinformaticians coming from the genomics realm to jump right into ours.

QuanTP is a great new gap bridging tool! You can statistically correlate the transcript IDs with the proteomics data within this! What else can do this? Perseus -- I think, but you'd have to work all the data up into the right formats which would require loads of preprocessing from both sides. (I could be wrong here, of course.)  In Galaxy you'd already have all the transcript processing tools available and the proteomics tools.

Therefore....

....Yes. I felt very stupid while making this. And while posting it. But it's done now!

Q4SRM -- Fast check of all heavy labeled standards in SRM experiments!

To avoid my post Xmas cheer rambling -- you should just check out this cool new QC software for heavy labeled standards in SRM experiments.

You can get the Q4SRM software here!

It has a cool logo!

Rambling you should skip:

I have a tendency to give triple quadrupole (QQQ) instruments a hard time. It's tough to go back when you've seen the power of 3 decimal places at MS1 and MS/MS. SPECIFICITY!!

I've done a couple of head-to-heads and I'm 100% convinced that even a Q Exactive Classic can still achieve higher sensitivity than any QQQ on the planet (note: in complex matrices! if you're diluting resperine in water, the QQQ wins every time. If you're studying pure reserpine go with the QQQ!!! 😇), but you need at least 100 ms of fill time to beat something like an Altis or a 6500+. That's -- at best -- 10 scans/second?.... our Altis can do over 600 scans/second... so....there are some definite advantages.

However -- SRM specificity still sucks. Wait! I read this review to help me sleep on a plane, but it ended up being awesome. I think it was this one (sorry -- its ElfSeverer.....) and the author brought up this great point -- the increase in sensitivity of QQQ devices has actually made the specificity problem worse. That when you can see lower and lower levels the chances that you will find something with an MS1 +/- 0.7 Da that also has an ion with an MS/MS fragment (or coeluting compound has an MS/MS fragment) within +/-0.7 Da gets increasingly higher! Interesting, right? Don't surplus that Quantum or 4500 just yet! 

Okay -- what was I rambling about (sorry...I should put a warning at the top...).

Q4SRM!  Okay -- the smart people at PNNL do a lot of specific targeted assays.

They do it this way:

1) Relying heavily on chromatography (what?? Since when does PNNL have an emphasis on chromatography being a critical component of proteomics? Where were they when we were all filling up TRANCHE with the worst chromatography of the last half century? Wait....oh...right...)
2) Putting in stable heavy labels for every peptide they quantify
3) Developing awesome software that can rapidly run QC on their stable heavy peptides as soon as the instrument has finished acquiring the spectra!

If you're going to do SRMs and you do those 3 things, I'm going to be super impressed. You've got an assay that is awesome!!

Get an SOP and get that thing out there helping people!

Is there other software you can make do what Q4SRM does? Probably! But we're all using different software packages and this is the first GUI I've ever downloaded that is specifically designed with this in mind. As light and fast as this is, you could stick to your normal data processing pipeline for your targeted quan and just keep Q4SRM on the acquisition PC to verify that everything is going according to plan.

BOOM. Massive point of data acquisition Quality Control upgrade!!  And that is NEVER a bad thing.

Happy Holidays!!

Happy holidays y’all!

This is the end of a scarf my badass sister knitted for me around her easy schedule of medical school at some famous university in NYC....wait...she also made this?!?!

DIWA -- Dual Isolation Width Acquisition for Combating Ratio Suppression!

We're not done innovating, people! Smarter instrument methods keep coming in all the time, and DIWA is an awesome and elegant new example that you can read about here!

Also, an earlier pre-print version is available at biorXiV here. I'm not sure how I missed it. It would have been really helpful...stupid job getting in the way of me reading every paper that comes out, I guess....

Here is the idea -- if you are doing MS2 based isobaric labeling quan (TMT/iTRAQ) you have 2 choices:

1) A really narrow isolation window that will help you get less coeluting ions. (Obviously of varying efficacy depending on your complexity, actual m/z, etc.,)

2) A wider window that will allow you to catch your more of your peptide isotopes.

-- here is a cartoon I have for something we're working on --

If you use a 0.4Da isolation width on any isotope of these two coeluting peptide species, you're going to miss a TON of signal. Maybe the majority of it! Are you going to get enough signal to get an MS/MS that you can match a peptide out of? Maybe....?

Dual Isolation Width Acquisition (DIWA) says --

(In case you're wondering why you remember this -- we did the research -- this was a commercial for a grocery kit that contained both hard and soft shell tacos. --@ProteomicsNews -- not afraid to ask the hard questions)

Two MS2 scans were acquired for each ion selected for fragmentation on an Orbitrap Velos system. In order to sort the scans out, they had slightly different HCD energies. A wider isolation was used to get the best signal for identification and super narrow isolation was used for quan.

How'd it do? Impressively well.

You don't have to believe the authors.  You can get the files at PRIDE here when the full print of the paper goes live. 

Things to note -- this was TMT 6 plex. I'll always hold the great Orbi Velos I once had in high regard, but it's slooow for TMT 10/11-plex. 6-plex? No problem!

This can totally be applied to the other instruments, like the 2x faster Elite or Q Exactives! I'm not sure how to write it for the QEs, though.... I'll think on it and if I figure it out, I'll add it. (Definitely ping me if you have the method!)

There are other important points here such as the ability the narrow isolation spectra give you to do regression analysis to assess your isolation interference. And -- this sounds totally smart -- the authors mention the potential application of ion mobility as something they might be exploring?!?!

Prep for metabolomics AND proteomics out of the same sample!!

YES. THIS. This is exactly what I've been looking for.

Check out this ASMS poster from the great people at Cold Spring Harbor Labs! 

Sometimes proteomics will solve that tough biological question that smart biologist just brought you and trusted you to solve.

As metabolomics tools continue to mature, I'm finding just as many problems that can be solved with (much easier to set up from the instrument side!) getting a view of the global metabolome.

The problem has been that the classic sample prep methods force me to choose. Do I do proteomics? Or do I gear up for metabolomics? What if  I didn't have to choose at all and could get both samples SIMULTANEOUSLY?!?!?

Honestly, metabolomics run in triplicate sets me back about 45 minutes. It almost makes sense to do the metabolomics -- and if that doesn't seem to unlock the key to the puzzle then I've probably got digested peptides dried and ready to load. (This probably isn't the way I would do it since I don't have a dedicated metabolomics unit -- I steal borrow one that is meant for small molecule structural stuff when I need metabolics or top-down capabilities)

Either way, though -- the possibility that I could explore both avenues with one single sample?!?? That's a big deal for me and my collaborators.