Researcher translation from xkcd

Original comic here.

Problems with antibodies?

Edit:  Severely edited this post after reading some more and discussing with someone who did take immunology AND got an A in it.

SO, I'm gonna direct you to this article in Nature called "Reproducibility crisis: blame it on the antibodies" until I have a chance to gather more information to support my ramblings on this topic.

Oh. And I think I actually have permission from Nature to use that image up there.  They have kind of an intense permissions form and I think I did it right!

Control your EasyNLC from your desktop

Man, I swore I wrote this a long time ago, but I can't find it.

Okay, on the same tilt as the post this week so you can observe your flow remotely: What if you want to control your EasyNLC remotely?

Well, you should download the file Easy NLC VLC from my FTP site.  Now, depending on your security configurations this might or might not be easy.  You may need to get one of those IT nerds up to put in their passwords and things.  I've had a ton of trouble getting it installed on some high security networks, even with the help of IT nerds.

Shout out to Shan for this and Fred for finding this convenient Zip file!  I work in a pretty awesome team these days!

Challenges still present in getting phosphoproteomics to the clinic

This is a great perspective paper!  It is from Anton Iliuk et. al., and came to my desktop thanks to the hard work of @PastelBio in making sure I always have cool stuff to read while caffeinating.

I do love introducing Proteomics to people who are new to the field.  One of the big reasons is that I'm a little indoctrinated in the inside perspective.  When you talk to someone who has the audacity to want to use our awesome toys as simply a tool to solve a biological problem that they have...well...it makes some of our big papers with their big lists seem kind of silly.  Yes, maybe we can see 14,000 phosphosites.  But what the heck do you do with all of that??

In this paper, this team wants to take our ability to find tons of phosphorylations and translate them to clinical relevance.  I.e. can we use these patterns to discern a disease state and/or the progression of that state before more traditional assays can?  My first thought would be :

But, you know what? We aren't quite there yet.  And this review really stomps down on the reasons why.

Massively upgrade your FASP with MStern blotting!

I admit it. I HATE western blots. There's nothing like coming in with data from one of the most sensitive/accurate analytical devices every conjured up by physics and being told to "validate" these observations with funny colored rabbit blood.

Fortunately for us, MStern blotting has nothing at all to do with Western blots at all, except that it makes use of PVDF membranes to massively speed up our ability to do FASP-style digestions.

I stole the figure above from this paper by Sebastien Berger et. al., that is currently open access at MCP here. (oh, some Steens were involved here as well!)

The idea is this: you can do FASP in 96 well plates (a very nice JCVI paper can be found on this blog). The problem is that it takes a long time with most filters.  By substituting PVDF membranes for MW cutoff filters they were able to get great digestions an cleanups in much faster times using speeds that are compatible with 96-well plates.

Awesome?  Absolutely.  And not just because I don't have to do a western blot!  Definitely check this one out!

Told you!!! When it comes to targeted quan, a Q Exactive can hang with the QQQs!

I catch some flak once in a while for this assertion that I will now restate:  Depending on what you want to quantify, the Q Exactive (family of instruments, so...also the Fusion and Lumos) can be as sensitive, if not more, than any other mass spec on earth.  Don't quote me on this, but I think I could go with more sensitive, but there are caveats.

To support this statement, I bring to the court the evidence proposed in this brand new paper from Graziella Ronsein et. al., which is titled: "Parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) exhibit comparable linearity, dynamic range and precision for targeted quantitative HDL proteomics". Should I go on?

Here is the thing.  Sure, the QQQ is killer fast and sensitive.  I've gotten to mess around here and there with the next gen QQQs like the Quantiva.  If I need to accurately quantify 2k things that I have already characterized, its my pick. But when do I have 2,000 fully characterized things to quan?  If I'm validating something, its the 3 cool things from my discovery experiment that I'm iffy about.

If I've got a limited number of targets, I can set the quad on the Q-exactive to filter for my ion of interest and fill the C-trap for just about as long as I want (upper limit is 6 seconds, I think).  If that isn't enough to collect your ion...well...it probably doesn't exist.  The sensitivity you would get from  that would be orders of magnitude higher than what you could get from a straight beam quadrupole transmission.  Add in PRM so you can select your high resolution fragment ions, and you've got the selectivity of your SRM (MRMs, or whatever) beat completely.

Very cool paper that proves something I've been saying for years!  Yes, I feel really smart right now.

Get your nanosource camera view on your PC screen

For some of us out there, we have the ability to log into our instruments from home.  Is there anything more comforting than going to bed knowing that you queued everything up properly, the QCs looked fine and the spray stability is awesome?  Or...seeing that something is messed up so you can shut it down and fix it when you get back in?!?

The missing link for most of us these days is the source camera.  If you've got an EasySpray or a nanoFlex ion source your video likely shows up on a little TV screen on top your mass spec.  That isn't too useful if you're using Remote Login.

Well, Sheng Zhang at the Cornell Proteomics Core has a brilliant solution for us!  You can buy Dino-Lite USB controlled microscopes on Amazon and they fit right in where your normal cameras fit into your source!  He has a different one on his Elite and his Fusion and we couldn't find the exact part numbers. The one on the Elite is a little better quality, but both are just awesome.  The little camera even comes with a small program that projects what it sees onto the PC screen. So no extra software necessary.  It doesn't appear to consume many resources at all.

There are tons of options (check this Amazon search page), but one that definitely works with USB 2.0 is this guy for $150!