Navigating Challenges Associated with Bioanalysis of Therapeutic and Endogenous Peptides | Part 2

Navigating Challenges Associated with Bioanalysis of Therapeutic and Endogenous Peptides | Part 2


[MUSIC PLAYING] Hi. I’m Kim Haynes, a
product marketing manager in the Chemistry
Technology Center at Water’s Corporation. In part two of this
series, we will look at the five big
challenges to overcome when designing a successful
sample cleanup and enrichment protocol for peptides. These are protein binding,
non-specific binding, peptides solubility, peptide
specificity, and recovery. I’d like to discuss
each of these briefly. And you can refer to the Bio
Analysis Sample Preparation and Method Development
cards for more details. The first consideration
is protein binding. You have to take steps
to prepare the sample and eliminate binding of
the peptide to proteins or other matrix components. If you do not, the peptides
will stay bound to the proteins and pass right through the solid
phase extraction sorbent due to size exclusion effects. There are some tricks
you can use to do this, and it really depends on
how strongly the peptides are bound to the proteins. You can start with something
as simple as diluting the plasma one to one
with a 4% phosphoric acid solution or a 5% ammonium
hydroxide solution, but more aggressive
steps may be necessary. For example, denaturation with
Guanidine Hydrochloride, urea, or SDS. You may even need to do
a protein precipitation and a one-to-one ratio
with acetonitrile, but don’t go any higher or you
may precipitate your peptides right along with the proteins. The next consideration
is a tricky one, non-specific binding. Peptides are notoriously
sticky and will stick to glass and other surfaces. This leads to low
recoveries, high variability due to absorption losses
at different rates, and high detection limits. To overcome this
challenge, there are a few things you can do. Avoid using glass for sample
preparation or storage in the auto sampler. Use polypropylene or materials
designed for low binding of peptides and proteins. During the sample
cleanup and enrichment in solid phase extraction,
use the microelution format. This allows you to skip the dry
down and reconstitution step, since the elution volume is
only 25 to 50 microliters. Many peptides can be lost in the
dry down step due to absorption onto the collection container
or failure to re-solubilize. The bonus is that the
microelution format allows up to a 15 time increase
in concentration, enabling the lower detection
limits that are often necessary in these assays. When preparing stock
solutions, make them in a high concentration
to mitigate losses and saturate the surfaces. Then do subsequent
serial delusions. You can use things like
organic solvents, detergents, and carrier proteins
to your advantage. Use organic solvents
or detergents like SDS or guanidine hydrochloride to
promote peptide solubility. Finally, you can
add carrier proteins to solutions that have low
protein concentrations, such as neat solutions
or cerebral spinal fluid. This saturates the binding
sites of the surfaces that the sample may
come into contact with, and therefore, prevents
absorption losses. 5% rat plasma or a 40 microgram
per mil solution of VSA works well for this purpose. The third challenge is one
that afflicts chromatography as much as sample preparation,
and that is peptide solubility. Those of you that have
worked with small molecules in the past may have learned
the hard way that peptides behave very differently. From the initial solubilization
from powder through the sample preparation and
chromatography steps, we suggest limiting the
organic concentration to no more than 75%. Otherwise, you risk
precipitating your peptides, and that will show up in terms
of low recovery in sample preparation and
possibly even carry over in your chromatography. You can use modifiers
to promote solubility, but it may require
higher concentrations than what you have used
with small molecules. Examples that have
worked include a range from 1% up to 10% acid or
base, such as TFA, formic acid, acetic acid, or
ammonium hydroxide. The fourth challenge
is specificity. Let me explain. In peptide bioanalysis,
we need to create highly sensitive and
reproducible assays. In many cases,
protein precipitation, while very popular with
small molecule bioanalysis, will just not be good
enough for peptides. Protein precipitation
uses high concentrations of organic solvent, which can
result in peptide precipitation losses, right along
with the proteins. In addition, protein
precipitation does not remove any other
matrix interferences, which can ultimately
lead to matrix effects. Protein precipitation
typically requires a dry down and reconstitution
step which, again, can lead to peptide losses due
to absorption to the container or failure to
re-solubilize the peptide. The specificity I
refer to here is gained by creating a sample
preparation method that use orthogonal clean up
approaches, both reverse phase and ion exchange to
selectively capture your target peptide while allowing you to
wash away the unwanted matrix interferences. Then you can
concentrate your peptide in a small elution
volume that can be diluted with a little
water and directly injected into the LC-MS system. The last challenge
is low recovery. In reality, low recoveries
or variable recoveries are usually just a symptom
of one of the four problems I just discussed previously. When you have low
recoveries, think about the steps you can take
to reduce protein binding, non-specific binding, peptide
solubility, or increase peptide specificity in your
sample cleanup protocol. There are also
some steps you can take to make sure that you’re
using your sample preparation device properly, and
you can learn more about those in the
troubleshooting section of this guide. We really hope
this module helped shed some light on the
five big challenges to overcome when designing
a successful sample cleanup and enrichment
protocol for peptides. For more information about
this and other related topics, you can always visit
the Bioanalysis Bootcamp to learn more and increase
your peptide and protein quantification fitness. Thank you. [MUSIC PLAYING]

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