Choosing the Right Protein Biomarker Discovery Tool

Comparison of Mass Spectrometry and SOMAmer Reagent-Based Assays

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INTRODUCTION

The word proteomics was coined in 1997 to describe the entire

complement of an organisms proteins, and the study of their structure and
function. In the two decades since, several complementary technologies
have emerged to dissect the enormous complexity of the proteome to help
understand fundamental and disease biology, and to accelerate new clinical
diagnostic and therapeutic approaches.

This document briefly summarizes two of the most promising technologies

mass spectrometry and the SOMAmer reagent-based SOMAscan
assay for hypothesis-free protein biomarker discovery. Each technology
has particular strengths and limitations and, for some purposes, synergies
between the approaches may allow discoveries that neither technology can
yet achieve independently.

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Mass Spectrometry and
the SOMAscan Assay in
Biomarker Discovery
Mass spectrometry (MS) is currently the most widely used
discovery tool in the field of proteomics, having an impact in a
number of research areas, including basic biology, biomedical
research, and systems biology. The applications of MS-based
proteomics range from descriptive to quantitative, and it can be
used for quantitative analysis of up to several hundred proteins
in a single liquid chromatographymass spectrometry (LC-MS)
experiment. MS instruments and supplies, though generally
expensive and requiring significant expertise to operate
reliably, are widely available.
The scientific literature for MS-based protein biomarker
discovery is dominated by a bottom up approach. In this
approach, proteins in a biological matrix are enzymatically
digested, and the resulting mixture of peptides is separated
chromatographically prior to analysis by MS The peptide
masses are then compared to databases, and the
observation of 2-5 key peptides confirms the presence of a
particular protein. The concentrations of the peptides can be
approximated by comparison to isotopically labeled standards,
and the peptide concentrations are used as surrogates for each
parent protein concentration.
The SOMAscan assay, which shares the goal of quantitation
of protein concentrations in biological fluids, takes a
fundamentally different approach than MS. Similar to
most antibody-based assays, SOMAscan relies on protein
conformational epitope recognition. However, unlike antibodies,
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SOMAmer reagents can be highly multiplexed in a single
experiment (currently 1,310 in the commercial version, and
over 5,000 in proprietary versions), with no upper limit yet
encountered. Throughput is currently several hundred samples
per day, limited only by the resources available to run the assay.
Finally, the SOMAscan assay is not susceptible to interference
from high abundance proteins, making sample preparation a
much simpler proposition than the fractionation required in MS.
The different starting points of these two technologies (i.e.,
peptide fragment separation and detection vs. protein epitope
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binding) confer the particular strengths outlined briefly above,
and also underlie the distinct technical limitations of each
approach.
The current number of proteins that can be measured from a
single sample using a bottom up approach with stable isotope
standards is in the range of 50-200 proteins. This is far fewer
than the number assayed in a single SOMAscan run, though
recent development of data acquisition methods with relative
quantitation (e.g., SWATH) has increased this to ~500 in serum
or plasma, and thousands in cell extracts.
Furthermore, the inability to completely chromatographically
separate thousands of peptides in a digested serum or plasma
sample precludes detection of many proteins, particularly those
in low abundance, some of which are likely to be critically
important biomarkers. In addition, many of the approaches that
enhance mass spectrometry sensitivity, such as high-abundance
protein depletion, can inadvertently alter the measured
concentration of the proteins of interest. Finally, only proteins
that are present within the databases can be identified using
this methodology. In 2012, the Human Proteome Organization
(HUPO) had a core dataset containing 3,020 unique proteins,
identified based on two or more peptides. Recent work has
increased this number to >10,000, but the possibility that
important biomarkers can be missed is still likely (ongoing work
is trying to address this issue).
The inherent challenges of conformational epitope-binding in
SOMAscan is that any change to the strength of the interaction
leads to a change in signal strength, with no direct ability to
distinguish between causes for the change. For example, a loss
in SOMAscan intensity could be due to a true change in protein
concentration (e.g., down regulation). It could also be due to
modification of the protein (e.g., glycosylation, phosphorylation,
etc.) in a way that partially or totally alters and/or blocks the
epitope, or results in a conformational change of the protein
that blocks or changes the epitope. In addition, many protein
signaling pathways involve a change in complexation with other
blood plasma proteins or cofactors, which could also change or
alter the availability of the target epitope. These challenges for
SOMAscan are, conversely, a particular strength of MS (e.g.,
detection of post-translational modifications, or PTMs). The
SOMAscan assay is also limited in the number of proteins it can
detect by the number of individual SOMAmer reagents available
for inclusion in the assay. Finally, unlike mass spectrometry,
SOMAscan is currently available at only a limited number of
institutions, though that number is growing.
Both technologies are dependent on careful sample collection
and preparation, an issue that has challenged proteomics
studies from the beginning. Recent work with SOMAscan
has begun to uncover particular protein changes that indicate
sample quality issues (e.g., hemolysis or protein degradation),
fundamental findings that could be used to enhance both
technologies.
Table 1 summarizes important comparisons of SOMAscan and
MS-based proteomics.
In summary, the main advantages of SOMAscan over MS
for protein biomarker discovery are sensitivity, dynamic
range, multiplexing (especially in serum or plasma due to the
dynamic range advantage), and throughput/turn-around-time.
The advantages indicate that SOMAscan is much faster in
discovery mode. MS is a better choice when the goal is absolute
quantitation and/or understanding of PTMs.
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Table 1
Highlighted comparisons of SOMAscan with mass spectometry

SOMAscan Mass
VS. Spectometry

3 Sensitivity >1,000
(pg/mL, median)
<5 Coefficient of variation 5-20
(% median, plasma)
65 Sample volume 30-100
requirements (L)
1310 (relative) Proteins measured (#) 50-200 (absolute)
in complex matrices < 500 (relative)

8 Dynamic range 4 (without dilutions)

(orders of magnitude)
Days to weeks Throughput time Weeks to months
(large projects)
No Identifying protein-protein No
complexes
No Identifying proteomforms Yes
(e.g, PTMs, sequence variants)
No Identify unknown proteins No

Days to weeks Turnaround time Weeks to months

(large projects)
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Synergistic uses of mass spectrometry
and SOMAscan/SOMAmers
There are circumstances where the use of both MS and
SOMAscan will provide more information than either one alone.
For example, comparison of biomarkers discovered by both
methods independently will likely provide a broader, clearer
picture of the biology involved, and leading marker candidates
discovered independently by both techniques will likely be the
most robust. On a related note, antibodies/ELISA assays are
often used as a validation of biomarker discovered via MS.
SOMAscan or an individual SOMAmer reagent could fill that
same role, particularly when antibodies are not available or not
reliable for the potential biomarkers being confirmed, or when
consistent measurements over long time frames are needed.
Another use of individual SOMAmer reagents may be in
enrichment- or depletion-based approaches for MS. Traditional
enrichment or depletion steps have used immobilized
antibodies specific for key peptides or proteins, which has been
very successful for quantitation of low-abundance proteins,
but is limited by the availability of antibodies. SOMAmer
reagents could easily be substituted for antibodies in these
assay steps, while offering the additional advantages of lot-
to-lot reproducibility and shelf-life stability, as well as lower
backgrounds in some applications.
Finally, MS can be useful in further understanding of some
SOMAscan assay results. For example, because SOMAmer
reagents are generally selected against recombinant proteins
expressed in bacteria, MS could be employed to provide
insight into to the distribution of glycoforms and other modified
versions of the protein that the individual SOMAmer reagent
can bind. MS could also help determine epitope-binding sites
for individual SOMAmers, or even clarify whether a SOMAscan
signal decreases because a particular binding site is not
available rather than a decrease in the protein concentration.
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Summary

Neither MS nor the SOMAscan assay provides a complete

solution for all proteomics needs, though SOMAscan has a
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significant edge in hypothesis-free protein biomarker discovery
in terms of throughput, multiplexing, sensitivity and dynamic 2945 Wilderness Place
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range. In addition, individual SOMAmer reagents can enhance
MS-related methods that currently rely on antibodies. As both
technologies continue to evolve, the individual researcher
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