Everyone, I think it is time to admit that the biologists have different opinions about what is important in proteomics. And maybe we're the ones that are wrong. This field originated largely in analytical chemistry where they drilled accuracy and precision into us. Sure, there are reasons for accurate protein measurements, like when you're in clinical chemistry, and maybe my time in those dark basement labs ruined my brain to think that when we measure a protein we actually really want to know how much of that protein is there.
The biologists want to detect a protein and they want to be able to say that in condition 1 vs condition 2 one of those conditions might possibly maybe have more protein. They don't care at all how much more protein. And - again - I'm the one here who is probably wrong. Hannah did this phenomenal thesis project in my lab and she worked out the nanomolar concentrations of 7k or 8k proteins at the blood brain barrier. We were operating under the assumption that absolute concentrations have value. Like - if your are doing medical imaging you know that proteins below xxnM just can't be visualized with any of today's technology. Don't try. And maybe that's just one outlier where we absolutely have to know the protein concentration.
Maybe the other clinical assays, like CRP and troponin and ALT/AST ratios are also outliers. Sure - whether you're going to get a wire jabbed into a blood vessel might be determined by the absolute amount of troponin in your blood right now as compared to 30 minutes ago. But it really appears that for the vast majority of new people in proteomics they want to know - is there probably less protein here and more protein there?
So if you really just want to detect proteins and you truly do not care how much of the protein is around - and you've got a lot of money - do I have a technology to show you!
SOMASCAN COUPLED TO NEXT NEXT GEN SEQUENCING! It's called Illumina Protein Prep!
For real, it's a real thing.
First of all - let's look at what aptamers are and what they do. I'll back way up because some people looked at me like I was out of my mind when I talked about the proteomics assay with the lowest quantitative dynamic range.
Let's start with this review from ancient history (2010). Don't worry, this is a physical limitation of protein oligonucleotide interactions. Not much has changed but there are more modern references below.
Aptamers are oligonucleotides and it's really really cool that one of the cheapest and easiest molecular reagents to make in a customized way can bind proteins at all! Not joking. That's cool stuff. And despite what companies will charge you, they are pennies to manufacture.
The binding, however, is calculated through either the dissociation constant or association constant and this functions in a linear way over an extremely narrow dynamic range.
This was taken from the review above. Please note the fluorescence intensity of the blank. In this solo interaction of one aptamer vs. one protein we see a relative increase in aptamer binding from 10nM to 150nM. At 150nM of protein, however, you no longer get a linear response. Lots of reasons for this and I haven't taught stoichiometry since....let's go with a long time.....and I don't want to get into it.
Imagine you have patient A and patient B. And one has 5nm of IgE in their blood? Well....that's probably about where the blank is, so you get a zero. What happens if you have 1,000nM of IgE? Well...you probably register at about 150nM, maybe a little bit more? Again, maybe you do not actually care in any way whether you have 150nM or 10,000nM? Maybe you're just weird for wanting to know.
What's important here, though is that each aptamer is like this. It is designed for a very specific protein and each one has it's own binding and dissociation constants. It's also important to know that in a complex solution, you're dumping in (in the case of Illumina protein prep) about 10,000 of these different aptamers! It is very very likely that the 1 order linear quantitative dynamic range represented in this figure in an isolated 1 vs 1 system is perturbed and not quite as successful as the above.
Edit -5/3/2025 because I'm self conscious about the crazy number of views this 20 minutes of typing has gotten in a single day.
This is how a pile of aptamer measurements work.
True concentration of protein X - 0 nM - Aptamer readout - Not zero
True concentration of protein X - 5nM - Aptamer readout - Same as blank
True concentration of protein X - 10nM - Aptamer readout - 2x blank
True concentration of protein X - 20nM - 2x of 10nM - This is good! You're in your dynamic range!
True concentration of protein X - 50nM - 3x of 10nM - It's still higher, but you've already left that little window where you're aptamer binding corresponds linearly to the amount of proteins (your linear quantitative dynamic range).
True concentration of protein X - 100nM - 4x of 10nM.....It's still higher but you are now need fancy math to have some way of estimating how much of the protein is there based on the aptamer binding response.
True concentration of protein X -1000nM - about 5x of 10nM.... You've maxed out your concentration and all you know is that you've got more than 100nM
True concentration of protein X - 10000nM - about 5x of 10nM - same as above.
This is important because as you'll see at the very last panel, it s pretty common in mass spectometery to get a linear concentration /signal increase across this ENTIRE range.
So - in aptamer measurements -
A) You almost always see a signal whether or not there is any protein there at all. So....when someone tells you they can detect 1,000 or 10,000 or 100,000 proteins in a sample you need to keep in mind that that is simply how many aptamers they put into the mixture. That doesn't mean they actually detect that number of proteins. They love to mix those terms up. And maybe you see a measurement for each protein aptamer. That does not necessarily mean protein detection.
B) You can trust that signal corresponds to how much protein is present in only a very narrow concentration range.
C) Above that 10x concentration range the value you see has no relationship AT ALL to the amount of protein present. You've simply maxed out.
End 5/3/2025 edits.
Again - the figures and review above are old - what can we do in 1 vs 1 relationships in 2025? Here is what I'd consider the high water mark today.
I should go get some lunch and if you want to read this you should, because it is a solid advance in aptamer binding
measurements - that last word is key because aptamer binding is not going to change. These are limited by physics and chemistry and they simply won't change. Yes, you can select for more efficient aptamers for your protein, but you aren't going to change the fundamentals of dissocation constants and maintain the proteins in a state in which they can be measured.
How did they do? Pretty darned good! About 1 order!
To be fair this study is focused on measuring aptamer binding over a course of time in a single molecule context. This isn't about extending the linear dynamic range of protein measurements. There are things out there about that. In some techniques what they do is have one aptamer that is good at one concentration and another that is better at others. Then you combine the measurements of both to get a better range. There is a preprint somewhere, but I've spent too much time on this.
So....imagine my disappointment when knowing that I couldn't talk about what I knew regarding an illumina - somalogic partnership (I just assume I'm under NDA with every proteomics company in the world now and I just don't share anything until I can google search it) - and I discover that does not appear to be what they did?
They appear to simply throw in the requirement to own a NovaSeq 6000 or NovaSeq X system to generate - get this - data on up to 384 samples per WEEK, which is 1/3 the speed of O-link? And even slower than mass spectrometry?
And if you're new here and aren't familiar with the quantitative dynamic range of mass spectrometry - here is the first thing I found searching my desktop. It's a Sciex app note, but this isn't extraordinary data. I can show you real data like this all day. It's actually surprising because normally you think vendor app notes are going to be crazy unachievable data and this is just very normal.
You spike 0.l ng/mL of this peptide in rat plasma - you can see it. If you put in 5ng/mL you get a peak that is 6e4 tall. If you put in 500ng/mL (100x more) you basically get a peak that is 100x taller. So...if you want to know how much protein is in your sample, you always have mass spectrometry to fall back on!
