SL-TMT!

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....

Even more features added to SearchGUI/PeptideShaker!

About time someone published an updated paper on what you can do with SearchGUI! I didn't even know about all the stuff on the right side of this picture, but you can read about it all in this new study at JPR here.

SearchGUI is an amazing central interface for a ton of different open search engines -- including the deNovoGUI (which now has DirecTag and the super crazy no-way-it's-that-fast Novor de novo engine)

Now this paper reveals all the work that is going on downstream -- links to Galaxy!??

BoxCar/BoxFahrt real data and new mysteries!!

Ummm....

So...I'm confused. So far I've had exactly zero luck with forcing BoxFahrt to work on our QE HF using Thermo's factory issued software. The great Dr. Antonius Koller (now of NorthEastern University if you can't reach him through his old CUMC account and want to bug him while he's getting set up) and I have been in touch a lot as he has been working on making use of the basic time saving logic behind BoxCar to improve his results. He came up with a work around this week (raising the default mass settings to match the width of the BoxCar!!) that I haven't been able to try yet, but so far...

While editing (I'm trying to do a 48 hours before posting rule now, so I seem less slightly less odd, and don't tell you things like "I'm writing this from a 4 day death metal festival". I already like the blog less. P.S. I'm an adult, I'm definitely not blogging from a tablet and waiting for the Ruins of Beverast) I came across a reader comment -- one major problem with the QE manufacturer software is that you have just one inclusion list. If you use it for your targeted SIM -- it's now problematic for your T-SIM dd-MS2 -- which might be the main misconception for why people (like me) have always thought that method doesn't work. It isn't doing dd-MS2 within your window, it is doing T-SIM and then only doing MS2 if it sees what you're looking for in your T-SIM.  Toni's work-around (essentially increasing the T-SIM inclusion mass accuracy cutoff to include the entire BoxCar helps over-ride this).

As a side note -- why hasn't a complete industry popped up of people selling software to alter instrument software? For real -- there are thousands of them out there that could be improved. There is only one company I know of -- and they might be closed now, I wrote them for quotes about a month ago... you can run the Q Exactive with Visual Basic for goth sake. In the back of a lot of our brains is Basic -- we had to use it in order to be able to play video games. Commodore 64, yo!

Back to the awesome Bill Murray meme!

I'm not kidding. And I'm not cheating. No MS1 or MS2 spectral libraries. No FASTA with 7e6 entries. Just Proteome Discoverer, UniProt Human (and cRAP) FASTA entries. And BoxFahrt.  Heck, the chromatography doesn't even look that great.

😼😼

I'll post the method iterations. There is a lot to learn here on the Fusion -- and lots of room to improve from where I am right now.

However -- the approach isn't without some mysteries and drawbacks right now.

Mystery  #1) I can't use Morpheus with these files. No idea why. I get loads of PSMs and Peptide groups, but I only get 2 (possibly the same 2) proteins past 1% FDR. If it is the same 2 proteins, for real, we need to figure out what is special about them. I bet they're full of ANGST.

Edit: 5/28/18: The development team (If you've never been up to Wisconsin to see why they're so great at mass spec -- you should try to go visit. There are such great people up there doing such brilliant stuff -- plus that's a cool town) has reached out to see why this isn't working and I'm sending files now. Thanks for looking at this, Zach!

Mystery #2) Percolator in PD 2.1 HATES these files. HATES them. I only found out on accident by using the default Thermo Fusion basic ID workflow (I think it only corrects by target decoy at the peptide group level.  This is what gives me the almost 6,000 protein groups. Gotta check on that.

Throw in Percolator --- less than half the PSMs make it through the filter. knocking the BoxFahrt 400ng 90 minute HeLa runs down to less than 2,500 protein groups in 85 minutes.

Mystery #3) Are these spectra crap? Well -- they are ion trap -- so they are crap (kidding!!) -- but they aren't any worse than any other ion trap PSMs by eye -- let me know if you want to see and I'll send you the processed data. The image at the very top is my very worst MS/MS spectra (the default workflow appears to require a minimum XCorr of 2.0 -- which -- back in the day when I'd totally spend multiple days at a death metal festival and wondering when I'd run into those fun guys from the Hunt lab -- who are probably also too grown up for this stuff, I'd have considered pretty darned good.   However, I can't objectively say whether 2e5 MS/MS spectra are worse or better, but wouldn't it be cool to think that there is something important here that Percolator doesn't like about these spectra?

Maybe they're too large? Wait -- where is that picture I made it last night...? I'll find it and add it in later. I tried to overlay histograms of the charge and MH+ for peptides ID'ed with each approach. It looks like the stuff that Percolator is throwing out that Target Decoy is keeping are considerably larger and higher charged peptides, but this is inconclusive with the amount of time I have right now.

Mystery #4) Minora doesn't work AT ALL. No traces, no quan and this is a major drawback for me.

I've got some samples in I've been dying to run all year and BoxFahrt gives me loads of peptide IDs, but I need quan -- I had to resort to spectral counts (yes, I died inside a little -- but I didn't throw up or anything...I'm an adult (warning! sound)-- a spectral counting hating adult....) and they lined up with what we know from the phenotype/RNASeq for these cells-- awesome -- but I need real quan -- so the samples went back to EasyStar (IonStar for people with EasyNano and EasySprays -- see -- I'll steal method names from anyone, including my friends and collaborators. IonStar is a much cooler name. Putting results here has been on my to do list for a while. The 50cm is pretty darned close and limited runs with the 75cm EasySpray PepMap suggest that it might have more theoretical plates than the 100cm 3um column. But now I'm off topic.

Here is my best Fusion 1 BoxFahrt method iteration so far. 
Edit 5/28/18 -- here is the link. That would be useful, I guess.

It uses 60,000 resolution MS1 for 3 T-SIMs with each T-SIM getting 1.5 seconds to do as many ddMS2 ion trap MS/MS scans that it can. I use the "use all parallelizable time" AGC target over-ride feature.

If you raise the T-SIM MS1 target any higher (actually, I only tried 5e6) you lose IDs (n=1) ~10% loss.

I tried 120,000 resolution MS1 and it cost me 15% IDs.

I tried turning off the fill time over-ride and that cost me 6-8%

If you have the Fusion 2, it may be possible to alter your MS/MS isolation windows for the msxT-SIMs. I can't do it on my Fusion 1 with this tune build....bummer....

Wow. That's a lot of words -- conclusion?!?  If I can deal with the temporary loss of some of my favorite tools -- if I use staggered msxTSIM-ddMS2 on my Fusion 1 with parallelization in the ion trap, I might possibly be getting the best results I've ever seen from any instrument.

Chemical mediated proteolysis opens a whole new realm in middle-down proteomics!

Okay -- so -- as much as I love the concept of top-down proteomics, I don't actually do it in our lab. When a new and really difficult protein or proteomics project comes in I try to see if top-down is the solution. I really do. Like every time. With our best-suited equipment to the task (QE HF with protein mode and Fusion gen 1) I've got some size and complexity limitations that always make top-down seem ----just----this----far--- out of reach.

Conversely, I'm seeing more and more projects where trypsin or GluC or any of the other awesome enzymes in our freezer I always forget the keycode to -- just aren't right either.

Is it middle-down time??  It can't be. That only works for monoclonal antibodies, right?

No longer, thanks to this awesome paper from Kristina Srzentic et al.,!

This team details the use of a number of chemicals that can be used for middle down applications all the way down to plotting the sizes of the fragments produced and -- so important, and often overlooked --- provide a table of the mass shifts incurred by such reagents.

Okay -- at the risk of annoying my dear friends at ACS and JPR, I'm putting this table here as my civic duty to people to maybe make it just a little easier to find (ACS, please contact me directly if this is a real problem (orsburn@vt.edu), p.s., you're the best!)

This is just another great new tool for our utility belts for when the next weird protein (or, more likely, PTM) comes through the door!

New bioRxiV paper shows how the EvoSep works!

I've mentioned the EvoSEP on here at least once before, but it has been a mystery how this ultrafast and low to zero- carryover system works. I know that it uses disposable columns for each sample but this new open source pre-print finally shows how it works.

It's is really smart and a nice step forward for clinical proteomics or anything where any carryover is going to sink you.

Jailbreak 2.0!

Are you feeling a little limited with your awesome and super easy to use EasySpray source? Want to power it up?

Check out EasySpray JailBreak 2.0! (unless it's illegal -- then don't. and don't tell anyone I sent you to this site)

The original JailBreak kit let you put any nano columns into your EasySpray that you wanted to -- 2.0 lets you go up to MicroFlow -- WITH SHEATH GAS CONTROL!

We're hearing lots about MicroFlow right now -- which -- depending on how you define it, your high flow LC or your nanoflow LC can probably do it (even EasyNanoLCs -- just make sure your gradient is short enough that you don't have to restroke the buffer pumps).

This source addition is the missing link.

Worth noting, the manufacturer does have MicroFlow columns and emitters now, but if they don't have the solution that works for you this looks like a viable option.

Analysis of PNGase F-resistant glycopeptides with SugarQb!

Is it glycoproteomics week? Sacred Bos taurus, there have been so many awesome papers this week.

If this is your field -- or if you know it is coming your direction, you'll be happy to know that every aspect of it appears to be developing rapidly!

I recommend checking out --

The GlycoPeptide Decoy Generator (and a new Glycopeptide CID library!)

This new metal based enrichment strategy for glycopeptides!

This new proteoglycan deep sequencing paper (they use a some neat enrichment with a QE HF and process the data with PEAKs in conjunction with an awesome software package GlycReSoft that they developed. While you're there, check out the gold mine of other neat little algorithms they have posted!)

Told you!! One heck of a week for glycoproteomics!!

The title of the post is about this one, though! 

I'm out of blogging time today, but if that title doesn't interest you (cryptic specificities? what?) you're probably here for the jokes -- but if nothing else it's great to see SugarQb being put to use. In case you don't have this free node installed in your copy of Proteome Discoverer 2.1 -- you should -- you can get it here.

Addressing more BoxCar/BoxFahrt comments!

It can be hard to both post and to find comments that are made on this dumb blog. Especially when they go on separate posts. It helps to address them directly sometimes! In no particular order

Q1) Is the Xcalibur add-in available yet?

--Not yet, I don't think. I'll probably run around screaming when I find out it has

Q2) How can you process the data currently?

-- For the TMT stuff we've been doing (LFQ runs are on this weekend when I did the math and realized there were a few (very rare in our lab) open hours on something!) Proteome Discoverer 2.1 has no problems with the data. Actually -- I know the peptide ID is great, but I need to do the quan comparisons later. I haven't tested PD 2.2 (I'm using IMP-PD nodes for this project and they aren't all available in PD 2.2 yet)

-- The MaxQuant version in the BoxCar paper is specifically equipped for real BoxCar data. I don't know yet (maybe next week) if it can handle BoxFahrt.

-- Testing is in progress right now for RAWQuant --- which, honestly, deserves it's own post. It's REALLY cool and I think it is something that we need to integrate into our data processing immmediately.

NOT A Q!!!)  Okay -- so -- thank you Chris -- I didn't know that the Fusion has features to allow you to select individual isolation windows. I will evaluate this immediately.  If you can optimize your isolation windows to spread out the densest regions of ion current (like BoxCar does) -- the results I'm getting on the Fusion right now might just be the beginning of the improvements I'm seeing!!

Q3) How does this differ from WiSIMDIA? It's got some similarities in that we're doing gas phase fractionation for the MS1 -- and WiSIMDIA is probably a good starting template for how BoxCar can be adapted to DIA. BoxCar staggers the isolation in the MS1 and allows for a more even distribution of MS1 ions than WiSIM -- and that even distribution allows lower intensity ions to come up out of the noise and be selected for fragmentation.

Finally learned the XlinkX workflow and nodes!

High on my "to do" list for 2017 was to learn how to use the new XlinkX workflow and nodes. I didn't get to it -- and there I was just wandering through Mount Ember minding my own business and --

I hadn't battled crosslinked proteins in half a decade, but we recently got the XlinkX workflow added into one of our PD boxes --- 2 hours later (mostly because I was reading stuff I didn't actually need to)

(Not my sample -- just being cautious) but HOW COOL IS THAT OUTPUT? Here is what we think it is (very top) -- here is the MS2 evidence (first spectra). Bottom panels -- Here is the MS3 evidence for each side of the DSSO crosslink!

If you did crosslinking in the past and you still wake up from nightmares of the experience, I really recommend you check out this new generation of crosslinking reagents, instrument methods and data processing software. For real. I think the nodes for Proteome Discoverer are $500 in the US. The DSSO reagent set us back $100?

EASI-tag -- some lab in Germany is working on new reporter ion technology!

What a busy week! Some labs would be content with identifying a major point of inefficiency in every mass spectrometer in the world and demonstrating a strategy to confront it -- and maybe take some time to sit around and feel smart about it.  This new preprint shows that this isn't how they do things. 

If you're thinking "hey! we have lots of reporter ion tagging technologies already. what could this one add?"

What if I said that you could take 3 samples and label them and mix them in a ratio of 1 to 12 to 144 -- and when you process the data that your output was 1 to 12 to 144? In MS2 -- no funny, time wasting, MS3 tricks, no isotope suppression!

It is worth keeping in mind that this was a single shot of yeast digest on a 95 minute 45cm Dr. Maisch column on a QE HF -- this setup alone (sharp peaks, sample with only around 4,000 proteins on a fast instrument) would probably cause some reduction in ratio suppression, but when TMT10 first came out, Dr. Min Du and I 2D fractionated some human protein digest we labeled in 1:2:4:8:16 -- and 16 doesn't look like 16 -- it looks like 8-10 on a QE Plus. (This is the example set in any of the TMT/iTRAQ Proteome Discoverer processing videos I've made over the years.) If you could really see a 144 fold difference in MS2 scans? This is huge.

How's EASI-Tag do it? The reporter fragments off at significantly lower energy than it take to break the peptide backbone. The authors use a 2 stage collision energy, one low, and one normal.

They also do two things to the QE HF I'm not sure I understand the logic behind.

#1 -- They offset the isolation window for MS/MS
#2 -- They use a special setting they've developed for the QE HF software to preferably isolate the monoisotopic peak. Since we're adding a tag to these peptides, this is about the opposite of what we normally do -- for example --

When looking at a peptide greater than around 1600Th -- the M+1 peak becomes the most intense species, statistically, on something as large as this peptide, the M+2 is almost as abundant as the monoisotopic.  Since I'm signal starved and the heavy isotopes are distributed evenly across the peptide (and the Orbitrap onboard computer can identify the monoisotopic -- regardless of what is fragmented), I generally want the M+1 to be fragmented....

OH -- They describe the reason why they did this in Figure 1 c. Both the preferential selection of the monoisotopic and the offset. I think it has a lot to do with the particular characteristics of this tagging technology and doesn't mean I should start reoptimizing my other reporter ion experiments.

The co-first author on this great new study is one of my fellow instructors at the Advanced Proteomics summer school in Vienna in July! I can compartmentalize it in my brain (forget about this entirely -- there isn't all that much space in here...) and ask a million questions this summer!