Is protein precipitation good enough when dealing with biological samples? | Trust your Science 8

Is protein precipitation good enough when dealing with biological samples? | Trust your Science 8


– So, Jonathan do we have
anything in the email today? – I dunno. You usually check. – Ugh. – I’ll check, I’ll check, no problem. – ‘Kay. – Whoa, wow, holy smokes,
we got another one on SPE. – Oh! – Yeah, one about, is
protein precipitation good enough when dealing
with biological samples? – Oh, that is a fantastic question. I’m excited to tackle this one. – [Jonathan] Yeah, yeah, we know that biologics have things in there like salts, fats, proteins, even these
things, phospholipids. – Yep, but we have to think about how those things are going
to affect our sample. Because they can cause
things like matrix effects, cause ion suppression, they can cause chromatography to not be reproducible. They can have contaminants
build up on our system, and cause us all kinds of problems. So,
– Okay. – this definitely one
I’m excited to look at. – Alright, well let’s take a look at it. – [Kim] Okay. – Aright, Kim, I’m
excited to test this myth. How we gonna do it? – Sure. So this one can be straightforward. I mean, what we’ll wanna test is the ability to just use protein precipitation and see if we can get reproducible quantification and chromatography. Right?
– Okay, right. – [Kim] So, what we can do is do a traditional protein precipitation. We’ll do a one to three protein crash We’ll use plasma. – [Jonathan] That’s good. – [Kim] And then we’ll
just keep making injections of the sample over and over and over again with a typical gradient that
you would use in bioanalysis. – Alright.
– So we’ll go to a high percentage of acetonitrile to potentially wash off the column. And then we’ll just
monitor the phospholipids, since they’re the major cause
of, you know, matrix effects. – Okay.
– Can cause issues with quantification. – Excellent.
– And then we can just see what that looks like after, when we take a look at the actual trace. – So we can look at something
like the area count, for example, and we’ll just monitor that and see if that fluctuates. – Absolutely. – It’s a good idea. – Yep, let’s set it up. – Alright, let’s do it. (cheery music) So, Kim, we talked about
one of the major problems when doing these biological
samples is phospholipids. So that’s something that I think we definitely are gonna have to monitor. – [Kim] Absolutely. – So for those that don’t know what a phospholipid is, it’s this long kind of molecule that
has a polar head group up front, and this long hydrophobic chains that tail off at the end. It’s those hydrophobic
chains that can kinda stick or bond to a most
reversed-phase column. In this experiment, we’re gonna monitor the transition, that 184, and we’ll take a look at the data over
some replicate injections. – [Kim] Awesome. – So, here we’ve done
some multiple injections. Just one after the other,
just kinda monitoring how these phospholipids
kinda build up on the column. And you can see really early on, say up to maybe injection five or
six, the phospholipid count coming off the column isn’t that much. But over time, boy you really can see how they’re really starting
to coat the column, come off the column, and not
come off at the same rate. So you get a lot of
irregularity, irreproducibility, which causes a lot of problems
for the chromatography. – [Kim] Yep, you’re exactly right, JT. And if we look at the chromatography to see how these
phospholipids would actually impact the analytical results, we can see a really interesting story. If we look at the top chromatogram here, we can see that the first
protein precipitation injection, and here we’re just
monitoring the phospholipids, but you can see that they’ve just started to build up on the
column, but they’re really not causing us any kind of a problem. – [Jonathan] Right. – [Kim] But if we look at
the bottom chromatogram, we can see that by the last injection, now those phospholipids
have really built up, and they’re starting to coelute with our risperidone and our clopidogrel, and they could be causing things like matrix effects, causing
higher column backpressure, but most importantly, causing us problems with being able to get
reproducible quantification. – [Jonathan] Yeah, so
protein precipitation just isn’t good enough to clean
up these biological samples. – [Kim] Unfortunately, it’s not. It’s easy, and, you know, sometimes you know, we scientists like to do the easiest thing.
– Yep. – [Kim] But, you know, in this case, it’s really not getting
us where we need to go. – Okay. So Kim, how do you wanna call this one? We kinda did our experiments. What do you think? – I know. Well, I think we have to call it busted because protein precipitation
really isn’t enough to make sure that you have good
reproducible quantification. – Yeah, I agree, but I don’t
feel real good about it. We are kinda leaving the customer hanging. – I know, I feel like we’re not
giving them the best advice. So, maybe we can just go a little bit further and extend this experiment. – Yeah, this calls for bonus time. – I think bonus time. – [Both] Let’s do it. – Alright I got the perfect sample, Kim, for this experiment. Synthetic cannabinoids, there’s 22 species in this sample mix, and
the sample’s whole blood. So a real challenging, complex mixture and a really tough matrix. – [Kim] Oh, definitely. So since we’re gonna do whole blood, I think let’s really go after cleaning up this sample and show our
scientists what we can do. – [Jonathan] Sure. – [Kim] Let’s do solid phase extraction, and let’s use maybe
four different sorbents. – Okay.
– To see how they all compare in performance. But let’s see specifically how they do with cleaning up what we just
looked at, phospholipids. – [Jonathan] Alright, let’s do it. – [Kim] Okay. (cheery music) – [Jonathan] So here’s the chromatogram from those 22 synthetic cannabinoids. The method is really complex. We don’t wanna be tinkering
around with it too much. You see we got everything
mostly separated. Those two isobaric compounds,
peaks nine and ten, we’re able to resolve
those chromatographically. So we don’t wanna mess
around with that at all. – [Kim] Yeah, sure, a lot of times people will try to resolve
matrix interferences away from their compounds of interest. But when you’ve got a complex
chromatogram like this, it’s better just to take
a step to remove them. – [Jonathan] Okay. – [Kim] So since we talked
a lot about phospholipids and matrix effects
specifically, let’s take a look at those four different
SPE sorbents that we used, and also the impact of our cleanup in terms of matrix effects, which is the most important thing
for a quantification. So we can see here that
for most of the compounds, everything looks pretty
good, but we definitely have a problem with compound JWH-203. We can see that there’s a lot
of ion suppression going on. – [Jonathan] Yeah, what’s
going on with that? – [Kim] Yeah, what exactly is happening? – [Jonathan] What did you do? – [Kim] (laughs loudly) So
if we take a closer look, we can see that
phospholipid 524 is actually coeluting exactly with our
compound of interest, JWH-203. So, this is definitely going to cause a problem with our matrix effects. So if we take a closer look at the impact of the phospholipids,
we can see that there’s a direct correlation between the presence of the phospholipid in our final sample, and the matrix effects. So if we take a look at
the left-hand column, we see that sample cleanup. And you can see the
presence of the phospholipid that we see on the left-hand side, directly correlates to the amount of matrix effects that we
see on the right-hand side. So you can see with the
three bottom examples, we have a lot of matrix effects. With the top example,
where the phospholipid has effectively been removed, we see very little matrix effects. – Oh, Kim, way to put in a little extra overtime there today. – I know.
– You know, I was just trying to help out the customers. – But it was worth it, it was. – So let’s get back to the original myth. You know, is protein
precipitation good enough? – (sighs) I think we have to say that one was busted, – Yep.
– like we did earlier. But, there are things you can do to take care of the problem. – Exactly right. Using some sort of SPE. – Yep, that works. – Yep, to get rid of those
pesky little phospholipids. – I know they’re sticky. – Yeah, so I’ll write
back to the customer. I’ll let them know, you know.
– Okay. – Yep. – Yep, let them know there are options. If you’d like your question to be answered on a future episode, please feel free to email us at
[email protected]