A proteomic platform that can also detect SNPs

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SOMAscan
Assay: A proteomic platform that can also detect SNPs
Sheri K. Wilcox, Alex Poole, Deborah Ayers, Eduardo Tabacman Dominic A. Zichi
SomaLogic, Inc., Boulder, CO/United States of America ABSTRACT WHAT IS A SOMAMER? Slow Off-rate Modified Aptamers (SOMAmer® reagents) are a novel class
of affinity binding reagents made from single-stranded DNA engineered
with hydrophobic side chains. These modifications on the 5-position of
uracil greatly expand the physicochemical diversity of the large
combinatorial SELEX libraries from which they are selected, resulting in
binding molecules to more proteins, and with higher affinity, than
observed with traditional aptamers. The hydrophobic nature of these
interactions results in exquisite shape complementarity between the
reagents and their protein targets. SOMAmer reagents have proven
effective as reagents for biomarker discovery, diagnostic products, and
research tools. SomaLogic has developed a proteomic assay called
SOMAscan™ assay for biomarker discovery that transforms protein
concentrations in a biological sample into a corresponding DNA signature
that can be measured using any DNA quantification technology.
SOMAmer reagents are a new and unique class of protein binding
reagents that consist of single stranded DNA with nucleotides modified at
the 5-pyrimidine position to mimic amino acid side-chains and other
small molecule structures that bind proteins. SOMAmer reagents are
selected in vitro in a process called Systematic Evolution of Ligands by
EXponential enrichment (SELEX), which is a combinatorial method that is
unique in its ability to screen libraries of enormous size that typically
contain up to 1015 molecules.
The introduction of these modified
nucleotides imparts SOMAmers with novel properties by expanding their
chemical diversity. These modifications dramatically improve affinity and
specificity. The co-crystal structures are shown below, highlighting the
exquisite interactions between the reagents and their targets, the unique
structures adopted by these reagents, and the extensive surface areas of
each interaction.
Recent SOMAscan data have suggested that certain SOMAmer reagents
can distinguish between proteins resulting from single-nucleotide
polymorphisms (SNPs) and wild-type proteins in human plasma. One
such protein is the Low affinity immunoglobulin gamma Fc region
receptor II-a. We have demonstrated that the SOMAmer reagent
selected against the H167R mutant of the protein, resulting from a SNP,
does not bind the H167 form. As a result, proteomic measurements in
the SOMAscan assay can be utilized to not only detect protein levels,
but, in some cases, identify individuals expressing alternate forms of
proteins.
The SOMAscan assay measures over 1000 human proteins in biological
samples. For some analytes, the SOMamer reagents are sensitive to
protein changes resulting from SNPs. The ability to measure these types
of changes in a proteomic assay provides a valuable tool across many
research areas.
Platelet-­‐derived growth factor (PDGF) Nerve growth factor (NGF) Interleukin-­‐6 PLOTS Certain coding sequence single-nucleotide polymorphisms (SNPs) result
in a change in the amino acid sequence for the encoded protein. We
have observed with SOMAscan many instances where such a single
amino acid change in a protein sequence dramatically affects analyte
measurements (Lourdusamy, Newhouse et al. 2012). Loss of SOMAmer
binding due to a SNP is a good example of the specificity that can be
obtained with SOMAmer affinity reagents. We present here one example
of SNP and differential SOMAmer binding for the protein termed Low
affinity immunoglobulin gamma Fc region receptor II-a, or FCGR2A.
In addition, the SOMAmer reagent selected to bind Low affinity
immunoglobulin gamma Fc region receptor II-b, or FCGR2B, which is
93% identical to FCGR2A, including Arg at the equivalent position, does
not display a bimodal distribution of RFU values in the same population.
Purified proteins with either His 167 or Arg 167 were used to examine
binding to the reagent selected against FCGR2A. The affinity for the
H167R mutant is two orders of magnitude higher than that for the wildtype protein, illustrating a striking specificity among two proteins with a
single amino acid substitution. Binding of the FCGR2A and FCGR2B
proteins was also measured against the SOMAmer reagents selected to
both proteins. Interestingly, the reagent selected against FCGR2B is able
to bind both proteins (93% identity), but the reagent selected against
FCGR2A only binds the FCGR2A with high affinity. The H167 form of
FCGR2A does not bind well to either SOMAmer reagent, suggesting that
this mutation results in a significant structural change in the protein.
Since SOMAmer reagents recognize structural epitopes on proteins,
these data suggest the FCGR2A reagent recognizes a unique epitope not
present on the SNP mutant of FCGR2A or on FCGR2B. The FGCR2B
reagent appears to recognize a shared epitope on H167R FCGR2A and
FGR2B.
F C G R 2A S O M A m er R eagent
1 .0
1 .0
F C G R 2A H 167
F C G R 2A H 167
F C G R 2A H 167R
0 .8
B ou nd
RFU values for plasma (x-axes) and serum (y-axes) from healthy
volunteers measured in the SOMAscan assay. A bimodal distribution is
clearly present in both plasma and serum using the SOMAmer reagent
selected against H167R FCGR2A (left), with many signals well below 500
RFU.
A continuous distribution is seen with the SOMAmer reagent
selected against FCG2B, with the lowest signal around 900 RFU.
F C G R 2B S O M A m er R eag ent
F C G R 2B
0 .6
F r a c tio n
FCGR2A contains a SNP that changes His at position 167 in the protein
sequence to Arg. The minor allele frequency for this SNP is quite high,
~44%. In fact, the protein for FCGR2A used in in vitro selection
contained an Arg at position 167. It was observed that in a population of
healthy individuals FCGR2A signaled ~ 4,000 – 10,000 Relative
Fluorescent Units (RFU) for 2/3 of the individuals while ~1/3 of the
population had a signal ~200 RFU, presumably corresponding to baseline
in plasma. If the SOMAmer reagent loses affinity to the protein with His
at position 167, such a protein would occur ~1/3 of the time in a
population (0.562 = 0.3), and the remaining 2/3 of the population would
express one or both copies with Arg resulting from the SNP.
BINDING DATA CONFIRM SNP DISTINCTION B ou nd
SOMASCAN DATA SUGGESTS BIMODAL POPULATION The SomaLogic SOMAscan assay converts target protein concentration to
DNA concentration. SOMAmer reagents are captured on a bead and
equilibrated with a biological sample.
After equilibration, unbound
proteins are removed. Complexes are released from the bead, and fast
off-rate complexes dissociate. Remaining proteins are captured on a
second bead, removing unbound SOMAmer reagents.
Since the
complexes are non-covalent, SOMAmer reagents are then released under
denaturing conditions. The eluted DNA can be measured using any DNA
quantitation technology. We use hybridization arrays with fluorescently
labeled SOMAmers for biomarker discovery, although QPCR, Luminex,
and MS are also viable readouts depending on the desired application.
The resulting signal is proportional to the amount of target protein in the
initial sample.
F r a c tio n
The SOMAscan assay measures over 1000 human proteins in biological
samples. For some analytes, the SOMamer reagents are sensitive to
protein changes resulting from SNPs. The ability to measure these types
of changes in a proteomic assay provides a valuable tool across many
research areas.
THE SOMASCAN ASSAY 0 .4
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F C G R 2B
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For more information, please visit www.somalogic.com, call 303-625-2094, or email [email protected]