I've got to run, because it took me a long time to figure out what is going on here. The more I think about it the more I like it.
Here is a new protocol for bSLIM, laid out in excruciating detail.
I've got to run, because it took me a long time to figure out what is going on here. The more I think about it the more I like it.
Here is a new protocol for bSLIM, laid out in excruciating detail.
This list isn't meant to be comprehensive, but I think maybe it could be helpful -- I'll add it as a permanent link over there --> somewhere.
Let's start with where do I go when I need help with something? Well...over the last 19 years of doing mass spec type stuff I've developed a network of contacts that I go to when I'm stuck. If you are interested in this post chances are you don't have an ill-advised tattoo that someone prominent in this field told you was a great idea after you shared a completely responsible amount of scotch that they feel just a little guilty about and that you can leverage for help extracting MS1 XICs for the rest of your career.
Have you tried Twitter? It is probably still alive right now since that smelly emerald mine heir that has tricked everyone into thinking he started Tesla and SpaceX hasn't yet taken over and tried to run that into the ground. There are a lot of helpful proteomics people on it!
Did you know there is an active proteomics reddit page? That's a great place to put questions, although r/biochemistry and r/bioinformatics have larger subscriber bases and frequently feature proteomics sample prep and data processing questions respectively.
Questions on the sample prep side? Okay....that's tougher, unless you are using a commercial solution. I use S-Traps, so when I need help I bug the support team at Protifi. If you are doing SP3 via the PreOmics stuff, bug them! If you are doing prep on the cheap, maybe try Reddit?
Software? This is much much better and I cannot stress this enough, use the Github support pages and Google groups for your software of choice!
MSFragger has a level of support that is bordering on absolutely impossible to fathom.
MetaMorpheus is just as insane...almost 600 closed issues...?....
On this topic, anyone who has a Github up will get an email if you bug them. If you're using the code they published -- bug them!
Commercial software? This is the best part of commercial software, they have support people! Don't let them sit around bored, contact them!
Need help with a method? Have you tried www.LCMSMethods.org? Not everything is in there, but over the last 5 years or so I've tried to convince other people to put their methods there. Maybe it will happen one day. I'm wonder if the reason the project hasn't really taken off is because most people with mass specs aren't confident enough in their ability to use an instrument to feel okay making them available. So... it is about 98% methods that I have personally made and since I have experience with, and access to, just about everything, the resource still plods along, gets downloaded a lot and I occasionally get positive feedback about it.
Is your question about mass spectrometry hardware? This is great. Call or email the instrument vendor! Don't have a contact? Do this!
1) Figure out who your local sales person is
2) Tell them you have questions!
This might actually be a useful application for LinkedIn. I bet your sales rep is on that thing!
Because not everyone knows this, this is largely how the system works. Your local sales rep is paid just about enough money to not lose their home. Not much more. The manufacturing company wants them as desperate as possible to sell instruments. If you are sitting there with your instrument (even an old one) and you are struggling, you don't have time to write a great grant to buy a new one, do you? Even if you do, you probably will buy something else thinking you'll struggle less with it. You struggling with your hardware is bad news for your vendor. Contact your sales rep.
They will, almost without fail (unless they've had a bad couple of years and are working nights at a hospital or spending all their time applying for jobs) get you help. You probably have local applications people and you definitely have applications support people somewhere. Now, depending on how greedy the vendor company is, when you buy that Fusion 4 instrument, your sales rep will either have a party or a really big party, because even 1% of that instrument is a pretty good day for most people in science, so it works out for everyone somehow. For real, bug them, they'll be excited to hear from you.
I feel like I forgot something else I wanted to add....maybe later!
If you haven't seen it, maybe you don't want to, but I think it is worth rambling about for a second.
Of course, my first thought was "wait. we know how a stupid proteosome works, right? RIGHT? Oh no...no...no...we don't...do we know how ANYTHING works?Probably the last one, because we're getting some really impressive returns right now from technologies like real time search and more intelligent acquisition methods, etc.,
These things have been around a looooooooooooooooooooooooooooooooooong time, though. For a really good perspective of these tools and their capabilities (to help guide all you instrument hackers out there?) and some thoughts on the future, check out this smart review on the topic!
PROSIT now does deep learning prediction of TMT labeled spectra.
WHAT??? I know!!
How to do it? Okay, well this worked for me. I basically just followed this old tutorial I made:
Then I opened the EncyclopeDIA output file and made a new column (or row, I still don't know the difference) and I think I just put in HCD in every entry, saved it and loaded it to Prosit.
I made the data an MSPepSearch compatible file and loaded it into a copy of PD where the free software developed by NIST wasn't disabled due to there being no money being funneled to a big company for that particular PC!
This isn't the first archaeological forensics via proteomics paper, but it's the first one I can think of that used old bones(!!) and had this much evidence to support identifications -- old identifications!
Lots to digest here, this isn't a small paper, but worth the time.
Moral of the story might be: we can digest and identify just about anything, and it doesn't really matter how old it is, there is probably trace proteins around and that's all you need today?
I have been buried in work linked to maintaining my employment for several weeks. So buried that I scheduled a lot of blogposts for 2032. I'll leave them there so they'll post again later.
The annual congress of EuPA is something I've always wanted to attend and it has never quite worked out, so I conned some guest bloggers into providing some insight about what happened at the conference. (Sorry for the month long delay).
At Proteomics
Forum/EuPA2022 in Leipzig, we got word of the shout out from across the ocean
to provide a little update on the most exciting stuff that was presented. In
search of a brief format, Ghent attendees (#GhentRepresent) spread out and each
of us came up with a personal perspective on the most exciting stuff, a
predominantly ECR perspective:
Arthur
Declercq: “EuPA2022 was the conference of tackling
problems in specific proteomics niches such as single cell or
immunopeptidomics. It was very nice the see many people showed up for the
immunoproteomics symposium where Michal
Bassani-Sternberg gave her talk about identifying neo-epitopes for
personalized cancer immunotherapy with their proteogenomics pipeline NeoDisc,
showing how they bring state-of-the-art research from bench to bedside (10.1038/s41587-021-01072-6).”
Ben commentary: Hey! This is one of the two immunopeptidomics splicing groups! Despite the controversy about whether HLA peptides splice or not, there are some very successful clinical trials going on right now centered on weird spliced peptides.
Tine Claeys: “The
diversity of proteomics and its wide variety of instrument, tools,
applications, … shows the creativity of scientist in tackling very different
problems. I was intrigued by the talk from Jennifer
Van Eyk on how her group approaches clinical applications and biomarker
discovery integrating multi-omics analyses. Combining already existing clinical
data with the new advancements in the mass spec field to determine your
molecular twin demonstrated again the importance of big-data analysis,
especially in a clinical context. The
talk from Ileana Cristea on
spatiotemporal organelle remodeling ( 10.1016/j.celrep.2020.107943) blew my mind on how the location of a
protein can have a huge impact and can be very altered upon viral infection but
we're still mainly focused on protein abundance. This will definitely impact
the way I approach my own research. I've had some amazing interactions with
amazing people and expanded my scientific horizon in ways that would've been
impossible without Proteomics Forum 2022!”
Toon Callens: “As I
just started my PhD in proteomics (bioinformatics) it was great to see what
everyone is working on and to have a broad overview of the field and the
state-of-the-art. It was also interesting to see overlap with my own projects,
which gave me some new ideas.”
Annelies Bogaert: “Single cells, single cells, single
cells! It is great to see how much progress has been made in this field. Also
great to see how proteomics is contributing to help conquer the covid-19
pandemic. Chris Overall his talk about how they used TAILS to identify
substrates of the SARS-CoV-2 3CLpro protease which gives insights in
how the virus uses this to its benefit, is just one great example (10.1016/j.celrep.2021.109892). While Albert
Heck gave a nice presentation showing that we are evolving to personalized
diagnostic as top down comparisons of IgG antibodies from one person over time
could in the future help diagnose medical conditions (10.1016/j.cels.2021.08.008). Since
I spend some of my time trying to understand better the functionality of
proteoforms (thanks to Neil Kelleher
for introducing this term [10.1038/nmeth.2369]) it was
nice to hear that so many talks also considered them and stressed their
importance as they are often still neglected! And as a last note, this conference
really showed that DIA is becoming the standard over DDA.”
Tim Van Den Bossche:
“I was really honored to present the flagship manuscript of the Metaproteomics
Initiative and give a short introduction about the Initiative itself. Where the
field 10 years ago was often overlooked, it's amazing to see that there's now a
dedicated metaproteomics session at the major European proteomics conference”
Ralf Gabriels: “Proteomics
is done with simple setups; more challenging workflows is where the interest is now: Single cell, subcellular/spatial, immunopeptidomics, metaproteomics,
proteogenomics, open modification... And quantification labels are coming to
DIA!? Both non-isobaric with plexDIA (10.1101/2021.11.03.467007) and isobaric with DIA-TMT (10.1016/j.mcpro.2021.100177).”
Ben commentary:
Neil Kelleher is doing stuff with single ions I will never understand because I am a QTOF person, but he is so right that “we should measure what we must, not what we can!”.
Markus Ralsner suggested ordering your differential proteins according to chromosome localization, because you might in fact be looking at an aneuploidy event in your cell lines. That reminds me of a shrimp project where we once were thinking along the same lines, but we never really showed it as beautifully as Markus did!
And OMG, Kathryn Lilley is leaving us to look at nucleotide biomolecules?! Nooooooooo! But then again: what she is noticing on subcellular RNA localization is beyond cool. Please Kathryn, come back when you finished throwing over their world view and throw over ours a few more times?
Vadim Demichev got across the message that Match Between Runs (MBR) is making a significant difference in the current DIA-NN implementation. Therefore, he does upfront peak picking and alignment. And hey, that reminded me of something we were doing a while ago...
In fact, just talking about it became the biggest breakthrough for me personally: our ion-networks could finally be revived (10.1101/726273), all because of the return of in-person interactions and how these are so different from digital meetings! I am convinced that Bruker data will turn out to be the perfect match for ion-networks. So, if ion-networks are a “sleeping beauty”, then Proteomics Forum/EuPA2022 sure woke her up!”
Ben commentary: whoa....check out the highlighted stuff....this happens all the time in cancer, particularly when cells are treated with platinum based therapies. Worth considering, for real...
Lennart Martens: “Lots of great interactions and creative new ideas at the first in-person meeting in ages. Also… CAKE!”
CONCLUSION: The conference hall was part of the Zoo of Leipzig. After the conference, we visited the zoo and found a fish that perfectly illustrated how our heads felt after a full week of in-person conference!
Guest reporters:
Arthur Declercq (@DeclercqArthur) 1,3
Tine Claeys (@TineClaeys1) 1,3
Toon Callens (@ToonCallens) 1,3
Annelies Bogaert (@AnneliesBogaert) 2,3
Tim Van Den Bossche (@tvdbossche) 1,3
Ralf Gabriels (@RalfGabriels) 1,3
Maarten Dhaenens (@MaartenDhaenens) 4
Lennart Martens (@CompOmics) 1,3
1. CompOmics,
VIB-UGent Center for Biotechnology, VIB, Ghent, Belgium
2. Gevaert Lab, VIB-UGent Center for Biotechnology, VIB, Ghent, Belgium
3. Department of Biomolecular Medicine, Ghent University, Belgium
4. ProGenTomics, Department of Pharmaceutics, Ghent University, Belgium
I just stumbled on this while working on a tough deadline or three, and now I can cite it! It is a preprint from a couple of years ago.
Due to the time of flight (effect, not instrument) coming out of the quadrupole on the TIMSTOF you can't really scan low and high mass fragment ions simultaneously. They take longer to get there and you didn't get fined by the union that "maintains" your building for altering your ceiling tiles because you wanted to scan slooooow. (BTW: I'm 100% pro-union, labor unions are the only thing between lots of members of my family dying in coal mines in West Virginia so people like the...governor....of the state can save $10. He is really really really rich, btw, largely by operating coal mines unsafely and owes millions of dollars in safety violations that he just doesn't pay because billionaires in the US can literally do anything they want with absolutely no repercussions. I have personally known people who have died underground in mines owned by that walking pile of dogshit, though, thankfully no family, so please pardon the emotion here.)
What was I....oh yeah! This preprint!
Okay, so don't quote me on this preprint at all. The data isn't publicly available anywhere and it's been sitting on a preprint server a couple of years, but I think the concept is cool.
I was looking to see if anyone who has shared with me how they are doing glycoproteomics on these ceiling disrupting monsters has published their approach so I can cite it. Why I'm interested is that I really fine tune out my pre-pulse storage conditions for my two TMT scan events and crank the collision energy to 11 on the scan that liberates my reporter ions.
The end result is that in my MS2 spectra I often see crazy high intensity reporter ions and then a great big gap up to around 230 or so before we see the peptide signal. (They look like the spectra at the very top -- see where the signal starts? Ain't gonna see that 204 or 183. It looks like there is nothing in the 204 range. Which causes a lot of the glycoproteomics programs to output nothing at all because a lot of them only consider spectra for glycan ID if they see oxonium ions. This group uses PEAKS and they just don't think about oxonium ions. Boom. Problem solved, maybe?
I've often wondered who is using which search engines. I've also made guesses and they don't seem to be anywhere near accurate based on this Google Scholar analysis by Dr. Felipe da Veiga Leprevost who I finally met in person at US HUPO this year.
He shared this on social media and was clear that it wasn't comprehensive (who can keep up with the 1,000 plus proteomics tools out there? I mean, @PastelBio is doing an incredible job, but I don't think it's at 1,000 yet.)
This is by citations, so there are lags, and it doesn't tell us what companies that don't publish are doing (and there are a lot of instruments in those places). Click to expand.
I haven't seen much success with double barrel type chromatography in a while -- but here is a demonstration of a successful application in ultra low levels.
Setups like this are jus as fun to set up as they look, but the idea is that you are trapping your peptides on trap number 2 while your peptides are eluting off of analytical column #1. When everything goes onto column 2 trap and analytical columns number 1 are re-equilibrating. The goal is to wipe out that downtime between injections. The results are often having two distinct sets of files with very different looking chromatography, but this group seems to have the recipe for sorting it out!
There is an entire glossary of definitions available here (thanks for the link, Fengchao!)
I hadn't updated DIA-NN in a while AND I'm running it on a newer PC, so I can't say for sure what is making it seem a whole lot faster than before for dia-PASEF.
Woohooo! You can check out this protocol here!
One of the best things that has happened for my work recently has been the addition of increased multiplexing in the TMTPro sets. Sure, on the Orbis we can 18-plex, but on the fast instruments we can now 10-plex! I saw some misunderstandings of how this works on the Twitter thing recently, so this is how it works.
What we most recently did was buy a 16-plex kit + the two extra tags. Now there are full 18-plex kits available in just the huge 5mg aliquots (enough to label approximately 1.8 million single human cells) that you can aliquot out. We preprinted a super short and fast protocol to aliquot out TMT kits using an inexpensive robot last summer that saved us enough money in one single kit to more than pay for the robot. I bet you can find it. That paper also received a response from a peer reviewer that was so negative that I'm having it framed for my office. Though it might spend some time above my heavy bag for a while. Motivation!
We use the N-tags for the TOFs and we keep the C-tags for when we make TMT spectral libraries or when a collaborator has just a few samples. We may need to shake this up a little because there are a lot of c-tag aliquots around.
Since this is unit resolution, you can use these tags for 10-plex multiplexing on ion traps or triple quads as well. AND the collision energy is so much closer to the peptide bond energy that I can't imagine ever using the TMT6/10/11-plex reagents ever again.
Having the ability to leverage these reagents for the super cool nPOP method is going to be a major win for us. The way this is configured it looks like we can prep almost 1,500 multiplexed single cells at a time.
What we most recently did was buy a 16-plex kit + the two extra tags. Now there are full 18-plex kits available in just the huge 5mg aliquots (enough to label approximately 1.8 million single human cells) that you can aliquot out. We preprinted a super short and fast protocol to aliquot out TMT kits using an inexpensive robot last summer that saved us enough money in one single kit to more than pay for the robot. I bet you can find it. That paper also received a response from a peer reviewer that was so negative that I'm having it framed for my office. Though it might spend some time above my heavy bag for a while. Motivation!
More and more cool learning and prediction tools to boost our confidence in our IDs! There are two others that I'm dying to talk about. I was looking to see if either was officially launched and found this new one.
Okay...I'm going to post this, but I'll lead with the fact that I'm on the fence about leaving this in the drafts folder.
It looks really cool and the idea seems smart but if you aren't running on Linux and ready to do a decent amount of command line typing this set up for your GPU it isn't for you. If you're like "omg, I love typing while being strictly penalized for every tiny mistake I make and I didn't know there were operating systems outside of Linux" this one IS for you:
You can get all this at GitLAB here.
Trying to figure out processing that ZenoTOF IDA data? Me too! MaxQuant 2.1.0.0.0 has native support and you don't have to use my buggy fix for TMTPro data analysis. It's default. I bet there are other features to support the jump all the way to 2.something!
For what is going on at PRIDE check out this one!
And for what is happening at IProX here!
I haven't had a chance to read this one all the way through yet, but we'll be talking about it in lab a lot when everyone gets back from Experimental Biology but I've read far enough to know that a lot has changed since the original preprint (almost 2 years ago? I think it was right around ASMS 2020?)
The blog hasn't had a head-to-head deathmatch in a while, but wooooo-- what a great topic for one!
I gotta move fast -- and I've mentioned this preprint in passing a while back, but it keeps coming up at meetings and in calls, and it is absolutely worth revisiting.
SomaScan is an aptamer based proteomics technology (nucleotide thingies are specifically designed to bind to whatever you want) that has been around for several years now. It has been getting a lot of attention recently thanks to a couple of big studies and the amazing amount of capital that the company pours into advertising revenue. I think a deep dive into ad costs vs investment capital for this tech would be a lot of fun. Strangely, you can't find a picture of the device that generates the "data." Many of the searches direct you back to this blog, and I'm probably not being all that helpful, but I'll try harder.
O-link is a newer proteomics technology that is antibody powered, but actually pretty clever for a technology based on a reagent that millions of years of evolution has worked very very hard to make unpredictable. To get around the fact that antibodies will randomly bind to whatever they want to because binding to new things is legitimately their biological task, O-link requires that two matched antibodies bind to a target protein. When that happens the oligonucleotides on antibody A and antibody B will hybridize (bind together into double stranded nucleotides -- amplification of that sequence can only happen if hybridization happens. Only antibody A? No signal. Same for B. This should rule out some false positives, but due to the unpredictable nature of antibodies, I expect an increase in false negatives also must occur.
Deathmatch time! Ready?
How'd they do this deathmatch? They looked at 871 proteins that both technologies could quantify.
In 10,000 individuals(!?!?!?) These things do seriously have some throughput capabilities!Good news? Neither technology appears to have a systematic bias toward specific pathways. That suggests there isn't a copy number bias or anything.
Bad news?
One does much worse with membrane proteins than the other, there is a thorough evaluation of that in the study.
Correlation of results between the two? 0.38, you know, a little bit closer to a few things match here and there to NOTHING MATCHES.
Which is right? Are either? Well, these authors go to the trusty pQTLs and genetic elements to see how they agree? Which is...ummm.....
For years and years and years we've waited for this to happen.