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Diving Deep into Cell
Signaling
New proteomics tools enable researchers to dive deeply into
signaling networks, allowing them to tease out interactions
among key molecules. But this comes with a new challenge of
increased complexity. Can cell signaling scientists balance the
bewildering complexity that comes with the discovery power of
proteomics technology? By Caitlin Smith
P
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roteomics seems a natural discipline toward which
cell signaling researchers might gravitate. Most signaling molecules studied today are proteins, after all.
But proteomics presents much greater complexity
than, say genomics, which many cell signaling scientists grew
up learning.
“Proteins are very complex, often functioning under only
a specific set of conditions, which makes multiplexing and
parallel analysis difficult,” says Chris Hebel, vice president of
business development at LC Sciences. Add to this the multifold interactions between these complex molecules, and you
have an explosion of possible questions to ask and data to be
gathered. Alterations to proteins in the form of posttranslational
modifications (PTMs) such as phosphorylation, acetylation,
myristoylation, glycosylation, and more, can further change the
functions of these proteins, their binding partners, and their
signaling properties, perhaps even initiating a cascade of other
biochemical changes across the cell.
Uncovering signaling pathways
with mass spectrometry
Cell signaling research has recently benefitted from a combination of serial enrichment methods and advances in mass
spectrometry technology. A prime example is “affinity-based
methods for selective enrichment of PTMs,” says Steven Carr,
the director of the Proteomics Platform at the Broad Institute
of Harvard University and the Massachusetts Institute of Technology. For example, recent refinements of metal-affinity enrichment methods have helped optimize samples for serine/threonine and tyrosine phosphorylation detection. When researchers
use such pre-enriched samples as their starting sample for
liquid chromatography/mass spectrometry (LC/MS), they “can
get much, much deeper into these samples,” says Carr. “It’s
very common now to get 20,000–30,000 phosphorylation sites
identified, which is really quite deep.”
The MS technologies mainly used by Carr’s lab are the new Q
Exactive systems from Thermo Fisher Scientific. “Previously it
was Orbitraps, but now we’re heavily focused on using Q Exactive Plus and Q Exactive HF instruments, because the price
per performance point is very good. We run a high throughput
laboratory with many concurrent projects so having many highperformance instruments is essential,” says Carr. Other highperformance MS systems are commercially available from other
vendors, including AB SCIEX and Waters.
“These new generations of hybrid instruments are extremely
sensitive while maintaining very high performance in terms of
mass accuracy and high resolution—we take that for granted
nowadays,” says Carr. Eight to 10 years ago, researchers had
to pay a price. “Either you gave up sensitivity for the performance factors, or you maintained sensitivity but gave up mass
accuracy and high resolution. Today, you don’t have to make
those compromises.”
And MS systems continue to be refined. “Not all of them
have what’s called ion funnels on the front end,” says Carr.
An ion funnel is a device that makes the MS system more
sensitive. Waters, Agilent Technologies, and Thermo Fisher
Scientific offer ion funnels on certain MS systems. “Another
thing that will change is more widespread availability of ion
mobility in MS systems,” says Carr. Ion mobility helps to separate peptides and proteins from one another in the gas phase
to make the sample less complex, which Carr likens to gas
phase chromatography. “This also improves sensitivity and
can increase speed,” he says.
Other new developments in MS systems include the ETD
option on Thermo’s Orbitrap Fusion. ETD stands for electron
transfer dissociation, valuable as a “type of fragmentation that
lets you map PTMs to your protein backbone,” says Andreas
Huhmer, director of 'omics marketing for life science mass
spectrometry at Thermo Fisher Scientific. Thermo also offers
kinase probes for pulling out kinases. The Thermo Scientific
Pierce Kinase Enrichment Kit uses ActivX ATP or ADP Probes
to label kinases’ active ATPase sites. The probes contain
a desthiobiotin tag that allows subsequent enrichment of
labeled kinases.
Mark Knepper, senior investigator in the Epithelial Systems
Biology Laboratory at the National Heart Lung and Blood Institute (NHLBI), part of the National Institutes continued>
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of Health, also uses recent MS advances to study phosphoproteomics. “Progress in the development of mass spectrometers allows more spectra to be collected with greater mass
resolution,” he says. “This results in remarkable increases in
sensitivity.”
Knepper’s lab builds models of signaling networks by
identifying specific protein kinases and phosphatases. Using
the CRISPR-Cas9 genome editing system, the group can
delete a particular kinase or phosphatase gene, and then study
the subsequent engineered clones. “Phosphoproteomics in
CRISPR clones can potentially allow conclusions about the role
of specific gene products in specific signaling pathways,” says
Knepper.
The power of good antibodies
At their best, antibodies are indispensable for molecular tagging, identification, and affinity purification. But antibodies
come with a downside. “There’s just not enough of them,” says
Carr. “Most of them don’t work in all contexts, and some of
them don’t work at all. So reliance on antibodies remains very
challenging.” As with MS, recent improvements in antibody
technology have helped advance cell signaling and proteomics
research. Yet according to Carr, “mass spectrometry has played
a major role in unraveling the off-target effects of antibodies.”
Not all antibodies are created equal—some work well in some
experiments, such as Western blotting or immunohistochemistry, but not for others, like immunoprecipitation.
Antibody technology has improved greatly for targets with
PTMs, led mainly by “motif antibodies” from Cell Signaling
Technologies (CST). “CST developed a class of antibodies
that were designed around a particular PTM sequence motif
as opposed to a site-specific PTM epitope,” says Jeffrey Silva,
KinomeView and PTMScan proteomics service manager at
CST. Examples include phosphorylated serine/threonine targets
and phosphorylated tyrosine targets. Although it does not make
a motif antibody for glycosylation, CST does have antibodies
against acetyl-lysine, methyl-arginine, methyl-lysine, succinyllysine, and phospho-histadine in the works.
Silva explains that CST developed the PTMScan Direct
reagents—combinations of CST’s motif antibodies—in
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Deciphering signaling networks
with microchips
One of the advantages of using microchips for cell signaling
research is that only small amounts of reagents and samples
are needed. LC Sciences’ new phosphopeptide microarray is
designed on a microchip to assess changes in expression levels of key signaling proteins in different pathways—all at once.
It can “map tyrosine-phosphoproteome interaction networks by
detecting the expression level of proteins containing [the] corresponding phosphoprotein-binding domains,” says Hebel. This
is unique in that researchers can see at a glance where their
experimental manipulations are affecting signaling pathways.
Hebel says that there are multiple reasons to use phosphobinding domains on a custom microarray instead of the more
traditional antibody binding. “The probe density is much higher,
which translates into higher specificity,” he explains, and the
technique does not depend “on the availability of high-affinity
antibodies [or] require many different antibodies with different
affinities and optimal binding conditions—which would require
complicated redevelopment and validation if any design change
is made.”
Phosphoproteins are also the topic of study for James
Heath, professor of chemistry and director of the NanoSystems
Biology Cancer Center at the California Institute of
Technology. Heath uses single cell proteomics to study
phosphoprotein signaling cascades, with glioblastoma cells
from brain tumors as a model system. Like many types of
cancer, these tumors are not made of one kind of cancer cell,
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At their best,
antibodies are
indispensable for
molecular tagging,
identification, and
affinity purification.
response to customers’ requests for tools to pinpoint six
different signaling areas: tyrosine kinases, serine/threonine
kinases, apoptosis, cell cycle and DNA damage, AKT and PI3
kinase signaling, and a Multipathway Reagent that enables the
identification of approximately 19 major signaling pathways.
This lets researchers sample key nodes from a number of
different critical signaling pathways in a multiplex LCMS
assay. “It’s an efficient way to ask just which key pathways
are affected by the stimulus—then they can quickly focus their
efforts on the affected modulated proteins,” says Silva. All in
all, the Multipathway Reagent allows researchers to identify
and quantify 409 proteins and 1,006 unique phosphopeptides.
CST has also developed new PTM antibodies for ubiquitination,
acetylation, and cleaved caspase substrates.
Antibodies to G-protein-coupled receptors (GPCRs), which
are important in multiple signaling pathways, are in high
demand, but historically they have been difficult to generate.
The biggest challenge is “finding antibodies of sufficiently
high affinity to distinguish between closely related receptors
and receptor subtypes,” according to Lora Tebbetts, product
manager at Enzo Life Sciences, which offers over 120
antibodies for GPCRs and associated proteins. Tebbetts says
that more specific antibodies are made using “peptides from
the more sequence-diverse areas of N- and C-terminal regions
[of GPCRs], or the third intracellular loop combined with carrier
proteins as immunogens.”
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but rather a heterogeneous
Featured Participants
population. This
AB SCIEX
heterogeneity is thought to
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explain why single cancer
drugs that target one
Agilent Technologies
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signaling protein often fail.
Heath studies the signaling
Broad Institute
cascades of glioblastoma
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cells to learn how to
California Institute of
combine cancer drugs to
Technology
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make effective therapies.
Heath’s group uses
Cell Signaling Technologies
homemade microfluidic
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chips to study individual
Enzo Life Sciences
cells, which are positioned
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in their own tiny chambers
inside the microchip. Each
cell is separately lysed, so
that its contents are captured by the waiting array of antibodies that lie within each chamber. The team then uses quantitative sandwich ELISAs that are calibrated to measure the copy
number of proteins per cell. The array includes antibodies
against proteins known to participate in glioblastoma signaling
pathways, such as phosphoAKT, phosphoERK, phosphoSRC,
and phosphoEGFR. Heath uses “targeted drugs to hit signaling
pathways, like those driven by EGFR, that maintain the tumor
and help it grow,” he says.
Treating a tumor with a cancer drug may cause it to stop
growing, or even to shrink. But in all cases of glioblastoma,
says Heath, the tumor soon develops resistance to the drug
and starts growing aggressively. Yet for some tumors, this isn’t
the result of Darwinian selection; Heath found that the resistant
cells were not simply survivors—they were adapters.
“The same cells that were responding to the drug, actually
developed resistance to the drug,” says Heath, by activating particular signaling pathways—the same interactions that
Heath saw when analyzing single cells before and after drug
treatment. “If you can identify those other pathways that are
activated by the drug, that tells you in principle what combination therapies you would use to treat that resistance,” says
Heath. In other words, if you can find a second drug to stop the
adaptation response, then you can kill the tumor.
Heath’s results hold promise for cancer therapy. For example,
if he treats a tumor with an ERK inhibitor, or an EGFR inhibitor,
the results are unimpressive. But if he treats a tumor with two
inhibitors that can work together to prevent drug resistance, he
says the tumor “completely shuts down.”
From single cells to tissues
Studying single cells also has great value for better
understanding tissues since they are communities of cells,
according to Garry Nolan, director of the NHLBI Proteomics
Center for Systems Immunology, and professor in the
Department of Microbiology and Immunology at Stanford
University. “They are interacting and living in the context
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of each other,” he says.
“Their individual biology is
Fluidigm
really about what makes us
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normal or dysfunctional.”
He sees cancer as an
LC Sciences
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example of a tissue that
takes on a life of its own
National Institutes of Health
and creates its own
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context or environment.
Stanford University
“If we are to hope to
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understand the complexity
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of [the environment], and
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how drugs might act
Waters
on that, then we need
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to understand how this
community of players are
interacting with each other,”
Nolan says. “That means
you have to assay as many things as you deem relevant
without drowning yourself in information.”
Nolan pioneered a technique called mass cytometry to
measure the complement of proteins expressed in single cells
simultaneously. Using antibodies against multiple proteins of
interest labeled with isotopic mass tags—rather than, say, fluorescent tags—he can multiplex to a greater extent than with
microscopy because spectral overlap is not an issue. When an
individual cell enters a mass cytometer, it vaporizes and the
isotopic tags are read by a mass spectrometer. Using this system, Nolan’s lab was able to generate snapshots of signaling
networks using 35–40 markers. Today they can measure about
50 parameters.
Nolan’s lab uses a mass cytometry system called CyTOF 2
from Fluidigm. Recent improvements to the CyTOF 2 make
it easier to “do experiments to help better understand the
signaling properties of the proteins in different cell types,”
says Olga Ornatsky, principal scientist at Fluidigm. Fluidigm
is adopting the CyTOF 2 system for imaging of immunohistochemistry-type stained fixed-tissue sections. While conventional immunohistochemistry assays measure three or four
fluorescently labeled markers at a time, the CyTOF version
can measure more than 30 markers at once. “This will widen
the scope of questions that can be asked,” says Ornatsky.
Increasingly, proteomics researchers are using such technologies to study more than just changes in protein levels, as
they can now look into the complex interactions of signaling
molecules and networks. How PTMs regulate and alter such
pathways brings an additional layer to this research, with scientists having to ask: “How do these modifications affect one
another?” says Carr. And as progressively more powerful proteomics tools become available, such as better antibodies and
MS technologies, researchers will be able to dive even deeper
into such questions.
Caitlin Smith is a freelance science writer in Portland, Oregon.
DOI: 10.1126/science.opms.p1500093
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