REvIEwS

Monitoring protein communities and

their responses to therapeutics
Hanna G. Budayeva and Donald S. Kirkpatrick ✉

Abstract | Most therapeutics are designed to alter the activities of proteins. From metabolic
enzymes to cell surface receptors, connecting the function of a protein to a cellular phenotype,
to the activity of a drug and to a clinical outcome represents key mechanistic milestones during
drug development. Yet, even for therapeutics with exquisite specificity, the sequence of events
following target engagement can be complex. Interconnected communities of structural,
metabolic and signalling proteins modulate diverse downstream effects that manifest as
interindividual differences in efficacy, adverse effects and resistance to therapy. Recent advances
in mass spectrometry proteomics have made it possible to decipher these complex relationships
and to understand how factors such as genotype, cell type, local environment and external
perturbations influence them. In this Review, we explore how proteomic technologies are
expanding our understanding of protein communities and their responses to large- and
small-molecule therapeutics.

Department of Microchemistry,
Proteomics and Lipidomics,
Genentech, South San
Francisco, CA, USA.
✉e-mail: donaldk@gene.com
https://doi.org/10.1038/
s41573-020-0063-y
A single human cell contains an estimated 10 billion
individual proteins1. The molecules that comprise the
cellular proteome derive from the mRNA expression of
approximately 12 thousand distinct genes2–6 and range in
abundance from one to upward of 10 million copies per
cell7. Protein molecules may exist within a subcellular
compartment or may transit between compartments as
part of their dedicated functions (Fig. 1). Like a citizen
residing in a crowded metropolis, a protein molecule will
encounter innumerable others during its lifetime.
When considering the state of a cell and its response
to therapeutic agents, the concept of protein com-
munities provides a valuable framework for forming
hypotheses and interrogating data. The largest protein
community within an organism is its proteome — the
superset of all proteins that may be expressed in a given
cell. If we zoom in to the cellular level, a proteome can be
envisaged as a multidimensional array of protein com-
munities related through physical interactions, subcel-
lular colocalization (spatial communities), coordinated
regulation (co-regulatory communities) or biochemical
activity (functional communities) (Fig. 2). Just as with
human communities, the members of a protein com-
munity may physically interact, or they could equally
well reside passively in the same subcellular location.
Certain functional protein communities are defined as
the recipients of input from a common source, as in the
case of phosphorylation substrates downstream of an
activated kinase. In other cases, proteins from a func-
tional community may be connected not by proximity
or signalling, but rather by a common origin. This would
be the case for a community of proteins expressed down-
stream of a transcription factor or the enzymes involved
in a broad-acting cellular process (for example, protein
degradation). Integrating these concepts, we define
a protein community as a set of proteins within a cell
that are interconnected through one or more physical,
spatial, co-regulatory or functional relationships.
The composition and dynamics of protein communi-
ties are controlled by integrated regulation at the DNA,
RNA and protein levels (Fig. 2). The genome of a cell,
its epigenetic state and the corresponding gene expres-
sion profile define the repertoire of proteins that may
be present. Downstream of gene transcription, mRNA
splicing and translation further shape the emergence
of proteins, whereas lysosomal proteolysis, proteaso-
mal degradation and secretion maintain proteostasis.
Amongst the available mechanisms, protein–protein
interactions (PPIs) frequently control the activity, sta-
bility and localization of proteins within a community.
Similarly, post-translational modifications (PTMs) con-
trol protein activity, stability and localization, in addi-
tion to modulating PPIs. From common marks, such
as phosphorylation, acetylation and ubiquitylation, to
rare or cell-type-specific events like hypusinylation8 or
crotonylation9, the complexity imparted to the proteome
by PTMs is immense. Conditionally co-regulated PPIs
and PTMs represent key dimensions with which to
define protein communities.
Questions involving where proteins reside within
cells and which other proteins they physically inter-
act with, have long been of interest in proteomics and

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Stromal cell

EGF
a Analysing activation
versus inhibition of
EGFR signal transduction

Cancer cell P P Ras

P P P P
Endosome
RAF
d Studying b Studying
changes in biochemical
protein location c Analysing changes inhibition
in protein levels MEK P
Ub Ub Lysosome
Ub
Ub
P
ERK

Golgi

ER

ETV Gene expression

Fig. 1 | Proteins function within multiple protein communities. Extracellular activation of a plasma membrane receptor
(such as epidermal growth factor receptor (EGFR)) initiates signalling events that propagate across multiple protein
communities within a cell. A phosphorylation-mediated signalling pathway delivers the message to the nucleus, where
the transcriptional machinery controls downstream gene expression. The receptor can then redistribute across multiple
cellular compartments: endoplasmic reticulum (ER) and Golgi trafficking regulate folding and delivery of the receptor
to the plasma membrane, whereas ubiquitylation can initiate endosomal recycling or lysosomal degradation. In a tissue,
cell-to-cell communication and changes in the extracellular environment result in the initiation of cell-specific signalling
cascades. Comparing signal transduction downstream of a receptor that results from either activation or inhibition —
by, for example, antibody-based therapeutics (a) — can elucidate the members of physical, spatial, co-regulatory and
functional protein communities. Studying perturbations in signalling following biochemical inhibition (b), as well as
profiling drug–protein interactions, can help elucidate the drug mechanism of action. Changes in global protein levels
can indicate on- and off-target effects of molecules that trigger inducible protein degradation (c). Proximity-labelling
approaches and proteomic analysis of organelle fractions can determine changes in protein localization (d), such as
receptor endocytosis and recycling to the plasma membrane.

cell biology. Increasingly, now, this is also the case in
drug development. Cell surface proteins can only sense
extracellular signals directly if they are on the plasma
membrane. Transcription factors only drive RNA
expression if they are present in the nucleus. Likewise,
multicomponent enzyme complexes must come together
and engage their substrates if they are to control cellu-
lar processes. An emerging body of evidence points to
‘non-canonical’ functions of certain organelle-specific
proteins when they become localized to different com-
partments10,11. As such, tumour-promoting functions of
metabolic enzymes that are independent of their cata-
lytic activities may be targeted through modulation of
their specific subcellular localizations (reviewed in11).
Defining the community of proteins that reside at the
synapse of a neuron12, the identities of receptors on a
brain endothelial cell13 or the subcellular distribution of
oncogenic signalling modules in a cancer cell14 can be
early steps in establishing novel therapeutic targets.
As drug development progresses against the next
generation of challenging targets, understanding the
interconnected protein communities that they partici-
pate in is critical. This Review covers the growing role
of mass spectrometry proteomics in defining the phys-
ical, spatial, co-regulatory and functional relationships
of proteins, the ways in which therapeutics affect protein
communities, and the value of these experiments in drug
development.
Advancements in proteomic technologies
Steady improvements over the past two decades in
analytical mass spectrometry and protein biochemis-
try have propelled the proteomics field by facilitating
the characterization of cellular and tissue proteomes
in great detail15 (Fig. 3; Table 1). Advances in the speed
and sensitivity of mass spectrometry instrumentation,
along with requisite improvements in downstream data
analy­sis pipelines, have been at the heart of this progress.

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Spectral counting
Quantitative analysis based on
comparing the total number of
spectra identified per protein
in a mass spectrometry
analysis.
Multiplexed isobaric
tagging
Mass spectrometry (MS)-based
quantification approach, in
which labelled peptides from
multiple samples are mixed
together and analysed by mass
spectrometer. Upon peptide
fragmentation, reporter ions of
different masses are released
from the isobaric tags, and
their relative intensities are
measured within individual
MS2/MS3 scan events.
Chemoproteomics
Proteomics approaches that
use chemical probes to
elucidate small-molecule–
protein interactions in the
proteome.
Proximity labelling
Proteomics approaches that
use genetically engineered
enzymes for the labelling and
enrichment of proteins within
subcellular regions and protein
communities.
Nature Reviews | Drug Discovery
Together, these developments make it feasible to survey
most of the proteome in a matter of hours3,6,16. Even
the more neglected regions of the proteome can now
be explored using alternative proteolytic enzymes17 or
enrichment protocols. For PTMs such as phosphoryl-
ation18,19, acetylation20,21, ubiquitylation22,23 and other
processes, peptide enrichment strategies have yielded
extensive catalogues of marks that can occur on each
protein24. These approaches extend to features such as the
reactive cysteines, which convey information about
the activity of proteins or their availability for engaging
with a ligand25,26. Whereas many PTMs occur at low fre-
quency and require specialized enrichment in order to
be assessed, recent advances in instrument performance
have opened the door to directly profiling certain modi­
fications6,27. As such, a recently reported shotgun pro-
teomic workflow that did not include PTM enrichment
resulted in the identification of 7,000 N-acetylation
and 10,000 phosphorylation sites in HeLa cells6. In
parallel, high-throughput studies evaluating thou-
sands of proteins as baits in immunoaffinity pull-down
experiments have driven systematic cataloguing of
the physi­cal relationships between constituents of the
human proteome28,29. One such study revealed more
than 1,300 protein communities that display physical
(and presumably spatial and functional) relationships
upon overexpression in cultured human cells28.
Alongside improvements in sample handling and
analytical mass spectrometry have come improved
methods to quantify proteins across many samples
(Box 1). Notable amongst these are protocols for per-
forming quantification based on spectral counting, inte-
grated peak areas or multiplexed isobaric tagging30. Global
proteome profiling, PTM profiling, activity-based
protein profiling (ABPP) and chemoproteomics studies
have benefited from multiplexed quantification and have
been used for characterizing the mechanisms of drug
action21,31–33. Quantitative methods have also helped elu-
cidate the organizational structure of cellular compart-
ments and the signalling modules in them34–37. Spatial
proteomic approaches that involve density centrifu-
gation and proximity labelling provide data for a broad
range of proteins in a single analysis, and thus com-
plement imaging-based methods that examine protein
Input Proteome
Genotype
Cell type
Environment
Pertubation
Fig. 2 | Protein communities from the prism of mass spectrometry-based proteomics. Genotype, cell type,
environment and extracellular perturbations define the state of the proteome and its communities at a given point in time.
Mass spectrometry-based proteomics can decipher protein relations within physical, functional, spatial and co-regulatory
protein communities.
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localization for small numbers of proteins in situ38.
Protein quantification across conditions was at the core
of the initial mechanistic studies for communities of
proteins, including receptor tyrosine kinases (RTKs) and
G protein-coupled receptors (GPCRs), laying the foun-
dation for proteome-profiling studies with applications
to drug discovery.
Although much has been learned from sequencing
technologies, the characterization of DNA and RNA, by
definition, cannot provide information about the phys-
ical, spatial or functional relationships of most protein
communities. Systematic studies that compare mRNA
and protein expression profiles have shown that gene
expression holds only partial regulatory control over
protein levels4,39,40. Even for co-regulated gene sets, the
inducible degradation of a single component can desta-
bilize the remaining complex subunits, rendering gene
expression futile. As drug development moves headlong
into the inducible degradation of disease-causing pro-
teins41, understanding the relationships between protein
communities becomes particularly important.
Targeting protein communities
Mechanistic studies that involve therapeutic agents and
their targets have revealed many important tenets con-
cerning the dynamic organization of cellular protein
communities as controlled by PTMs and PPIs. On the
drug development side, model systems used to study
the biochemistry and cell biology of these communities
have been valuable in differentiating between early ther-
apeutic candidates. Building upon these foun­dations,
mass spectrometry proteomics is helping define the
composition and dynamics of protein communities
relevant to drug development (Fig. 1; Table 1).
RTKs and the proteins that they engage with have
been central to building our understanding of how pro-
tein communities respond to therapeutic agents. RTKs
localize to the plasma membrane and generally func-
tion as sensors of the extracellular environment (Fig. 1).
Classically, engagement of an RTK with its ligand ini-
tiates autophosphorylation of the intracellular tyrosine
kinase domain, signal propagation through a commu-
nity of effector molecules and, ultimately, modulation
of the expression of a target gene42,43. Signal duration
Proteomics Protein communities
Physical
Functional
Spatial
Co-regulatory
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Liquid
Peptide chromatography Target protein
Cancer cell Protein mixtures Immunoaffinity resin mixtures mass spectrometer community

Affinity Lysis • Physical

purification interaction

Label

Affinity resin B
Proximity • Physical
Lysis B interaction
labelling
• Functional
B interaction
B

Fractionation Fractions
Organelle • Spatial
Digestion
proteome interaction
profiling • Co-regulatory
interaction

PTM affinity resin • Physical

PTM P P interaction
Lysis P Digestion P • Spatial
profiling
interaction
P P P • Co-regulatory
P
P interaction
P
Drug
Inhibitor resin

Chemoaffinity
enrichment Lysis • Functional
interaction

Soluble
fractions
Thermal
proteome Lysis Digestion
profiling • Functional
interaction
Temperature
curve

Fig. 3 | Proteomic approaches for basic biology and drug discovery. Immunoaffinity purification identifies protein
interactions upon antibody-mediated isolation of a target protein with its complexes. Proximity labelling in cells captures
transient and functional interactions, as well as subcellular colocalization, by labelling with a reactive substrate followed
by affinity purification (such as streptavidin-mediated isolation of biotin-labelled products). Organelle proteome profiling
identifies the spatial protein distribution and co-regulation of proteins enriched in organellar fractions. Post-translational
modification (PTM) profiling can indicate protein association, localization and co-regulation through the enrichment and
quantification of modified peptides (for example, phosphorylated peptides). Chemoaffinity enrichment uses chemical
inhibitors (such as kinase inhibitors) as an affinity matrix to profile biochemical activities in the presence or absence of
a drug (such as another kinase inhibitor of interest). The expression levels, activity and affinity of an isolated enzyme for
the drug can be inferred from such study. Thermal proteome profiling leverages the concept of protein thermal stability
to interrogate small-molecule binding to direct targets and the affected protein communities, united by a common
biochemical response to a drug.

is regulated by internalization of the activated recep-
tors from the plasma membrane and, in some cases, by
their degradation44. Activators and inhibitors of RTKs
reshape numerous proteins through controlling syn-
thesis, activity, localization and stability. The dynamics
of both the initial and downstream processes depend
greatly on the abundance of the RTKs, the composition
of the protein communities they engage with and the
PTM states of community members at various stages.
By extension, the dynamics of signalling within the
interconnected RTK community can directly affect
the efficacy of therapeutic agents (Fig. 1).
Two heavily studied representatives of the RTK com-
munity are the epidermal growth factor receptors EGFR
(encoded by EGFR) and HER2 (encoded by ERBB2)43.
Both proteins are mutated and overexpressed in a variety
of tumours and have been targeted by small-molecule
inhibitors and therapeutic antibodies, such as lapatinib
and trastuzumab, respectively43,45. Proteomic technology
was applied early on to elucidation of the relationships
between members of the ERBB community44,46,47, as
well as their responses to tyrosine kinase inhibition32.
Mass spectrometry proteomics combined with path-
way activation or inhibition has been used to elucidate

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Phosphoproteomics
Proteomics approaches used
for the characterization of
proteins post-translationally
modified with a phosphate
group by kinase enzymes.
the protein communities downstream of many RTKs,
including SRC family kinases (SFKs)48, FLT3 (ref.49),
MET50, BCR-ABL51 and vascular endothelial growth
factor (VEGF) 52. Because compensatory signalling
between these communities is frequently implicated in
drug resistance, these studies provide a framework for
evaluating and developing novel combinations53.
Another area in which proteomics-based methods
have proved valuable is in determining the mechanisms
of acquired resistance that occur through rewiring of
otherwise silent signalling nodes (Table 1). Rexer et al.
used phosphotyrosine profiling of lapatinib-resistant
breast cancer cell lines48 to reveal activation of SFKs, and
Boyer et al. demonstrated context-dependent changes
in members of the SRC and MAPK pathways, as well as in
FAM83A in trastuzumab-resistant, HER2-amplified
breast cancer cell lines54. In HER2-positive, oestrogen
receptor (ER)-positive breast cancer, overexpression of
the kinase AXL has been tied to acquired resistance to
HER2-targeting therapeutics55, and in gastric cancer,
profiling of trastuzumab-resistant cancer cells revealed
activation of the mTOR pathway and sensitivity to
mTOR kinase inhibition56. In the case of lung cancer
cells treated with the tyrosine kinase inhibitor dasat-
inib, both chemoproteomics and phosphoproteomics
together revealed broad-spanning inhibitory responses,
manifested in decreased phosphorylation events
amongst members of the kinase community and their
substrates57. Several putative targets of dasatinib were
identified, such as a community of kinases including
non-RTKs (such as ABL and SFK family members),
RTKs (such as ephrin receptors and EGFR) and serine–
threonine kinases (such as G-associated kinase and the
QIK subfamily)57.

Table 1 | Applications and limitations of proteomic approaches for the characterization of protein communities
experimental approach
Affinity purification
Proximity labelling
Organelle proteome profiling
Global proteome profiling
Chemoaffinity enrichment
Thermal proteome profiling
PTM profiling
PPI, protein–protein interaction; PTM, post-translational modification.
community Applications in drug discovery Limitations
Physical and Identification of drug-dependent PPIs Tag-mediated purification requires bait tagging
spatial and functional validation
Identification of drug–protein interactions Endogenous protein purification requires
validated antibody reagents
Characterization of protein complex Artificial interactions can be introduced during
stoichiometry cell lysis
Physical, spatial Characterization of unbounded cellular Technique requires bait tagging and functional
and functional compartments validation
Capture of transient PPIs Method frequently involves exogenous bait
expression. Alternatively, a proximity-labelling
enzyme can be inserted within the endogenous
gene locus via cellular engineering
Spatial and Characterization of protein communities Sample preparation is laborious
co-regulatory of all major organelles in one dataset
Identification of drug-induced translocation Technique requires advanced bioinformatics
events analysis in order to translate mass spectrometry
data into protein distribution between organelle
Identification of secreted signalling factors fractions
Co-regulatory Protein co-regulation in disease models or Method is limited by the wide dynamic range
upon drug treatment of protein abundances, which complicates
quantification of the lower abundance proteins
Functional Identification of potential drug targets and Technique may require optimization for drug
off-targets conjugation to the beads
Drug binding does not necessarily reflect activity
in cells
Functional Identification of potential drug target and Changes in protein thermal stability in cells can
off-targets result from upstream events (such as changes in
PPIs or PTMs) and may not necessarily indicate
direct drug–protein interaction
It is difficult to ascertain the affinity of the
molecule to its target
Physical, spatial, Elucidation of time- and dose-dependent Enrichment of some PTMs (such as acetylation or
functional and signalling events mediated by PTMs tyrosine phosphorylation) relies on high-affinity
co-regulatory and high-specificity antibodies
High recovery of the modified peptides requires
careful protocol optimization
Large input material requirements can limit the
analysis of tissue and clinical samples
Bottom-up approaches can suffer from ambiguity
in PTM localization assignment

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Box 1 | common methods of quantification in mass spectrometry-based proteomics

Bottom-up proteomics is a commonly used workflow for the mass spectrometry-based characterization of proteins
following proteolytic digestion. Peptides are typically separated by reverse-phase liquid chromatography and introduced
to the mass spectrometer through electrospray ionization of the eluting peptides, where mass-to-charge ratios (m/z)
are measured during the first stage of mass spectrometry (Ms1). in data-dependent acquisition, the most intense intact
precursor ions are selected for fragmentation and detection in Ms2 (see the figure). the Ms2 spectra are typically
matched to peptide sequences through database searching. Label-free quantification, which does not utilize isotope
labelling of the peptides, can be performed on the basis of the Ms1 or Ms2 scans, with the relative quantification coming
from counting peptide spectral matches or assessing signal intensities.
an inherent drawback of bottom-up proteomics is incomplete sequence coverage for many proteins, even with
high-sensitivity protocols and instrumentation. Data-dependent sampling can lead to missing quantitative data between
samples. awareness of this limitation allows for the incorporation of steps to address ambiguity, such as assuring that
distinguishing peptides are quantified when differentiating between protein isoforms128.
stable isotope-based multiplexing approaches can partially overcome missing-data issues by comparing conditions
within a single mass spectrometry run. stable isotope-based approaches include metabolic (such as stable
isotope labelling of amino acids in cell culture (SILAC)67) or chemical (such as, tandem mass tags (tMt)129) labelling.
Metabolic labelling typically allows for analysis of 2 or 3 experimental conditions (such as drug-treated versus untreated
cells), whereas isobaric chemical labelling now allows for comparison of up to 16 different conditions. reporter ions from
isobaric chemical labels can be generated during Ms2 or Ms3. Quantification during Ms3 allows for selective isolation
and fragmentation of the product ions in complex Ms2 mixtures, where fragments from multiple precursor ions with
the same intact m/z may be present.
in targeted quantification, peptides are monitored by mass spectrometry based on a predefined list precursor or
fragment-ion m/z ratios. isotopically labelled internal standards (such aQua peptides130) can improve quantification
when they are introduced during sample processing, especially for low-abundance features (such as phosphopeptides
or isoform-defining peptides). Data-independent acquisition is an increased-throughput-targeted approach, in which
swaths of ions are co-isolated and co-fragmented for concurrent Ms2 analysis131, allowing systematic quantification of
many peptides across samples.
Peptide Precursor ion Product ion
separation dissociation dissociation
Liquid MS1 MS2 MS3
chromatography Intact peptide Peptide fragment Reporter ion
detection detection detection

100 100 100

75 75 75
Intensity

Intensity

50 50 50
25 25 25
0 0 0
400 405 410 415 250 750 1,250 1,750 126 127 128 129 130 131
m/z m/z m/z

Peak area Reporter ion intensity

abundance

abundance
Relative

Relative

Retention 126 127

time (min) m/z

An emerging theme across many studies is that con-
served communities of kinases function interchangeably
to mediate resistance to kinase inhibition. This phenom­
enon of adaptive kinome response is common for
single-agent kinase-targeted therapies and was observed
during proteomics-based kinome profiling in treatments
with HER2, MEK and PI3K inhibitors58–60. For instance,
lapatinib-resistant HER2-positive cell lines elicit a
hetero­geneous adaptive response and show sensitivity
to inhibition of transcriptional reprogramming medi-
ated by bromodomain and extra-terminal domain (BET)
chromatin readers59. Interestingly, dual agents capable
of targeting both kinases and bromodomain-containing
(BRD) proteins may already exist, with Ciceri et al.
reporting on several clinical kinase inhibitors that dis-
play polypharmacological properties, including BRD4
inhibition (such as the protein kinase PLK1 inhibitor
BI-2536 and the JAK2–FLT3 inhibitor fedratinib)61.
GPCRs are another family of signalling proteins that
multitask within numerous protein communities. GPCR
signalling is initiated upon ligand binding and is propa-
gated downstream to effector molecules through hetero-
trimeric G proteins62. Owing to their essential regulatory
functions and accessibility on the cell surface, GPCRs
are targeted by a wide array of approved and emerging
therapeutic agents62. Certain GPCR-targeting agents can
differentially impact downstream signal propagation
through a mechanism called ‘biased agonism’, which
is defined as the property of a molecule to activate one
signalling pathway while antagonizing another63–65.
Selective modulation of GPCR signalling has been
studied with quantitative proteomics46,66. Christensen et al.

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Thermal-shift assay
A method used to quantify
changes in protein stability
under thermal denaturation in
response to different
treatments or conditions, such
as binding of a small molecule.
Nature Reviews | Drug Discovery
used stable isotope labelling by amino acids in cell cul-
ture (SILAC67) and quantitative phosphoproteomics to
analyse the responses elicited by [Sar1,Ile4,Ile8] angio­
tensin II (SII Ang II), a biased agonist that activates
only Gαq-independent signalling, versus Ang II, a full
agonist of GPCR Ang II type 1 receptor (AT1R)68. The
study revealed a divergence between Gαq-dependent
and Gαq-independent signalling, in which, for example,
Gαq-mediated phosphorylation of some of the MAPK1/3
substrates was reflective of nuclear translocation down-
stream of Ang II, but not of SII Ang II, stimulation68.
Mass spectrometry proteomics revealed qualitatively
and quantitatively different GPCR signalling down-
stream of β2-adrenergic receptor stimulation by two dif-
ferential agonists (isoprenaline and salmeterol). Despite
targeting the same signalling nodes, there were differ-
ences in cAMP production, in initial rates of endocytosis
and in the phosphorylation of downstream targets, but
not in the late (transcriptional) phase of signal propa-
gation65. These differences may have important impli-
cations in the contexts of interindividual differences in
efficacy, unintended activities and resistance to therapy.
Because dynamic protein communities shape the
behaviour of diseased cells and tissues, novel therapeutic
targets can emerge from proteomic assessment of sig-
nalling nodes, epigenetic regulators and protein degra-
dation pathways. With these goals in mind, Bantscheff
et al. investigated the effects of histone deacetylase
(HDAC) inhibitors on PPIs using chemoproteomics69.
Isolation and quantification of proteins bound to an
HDAC inhibitor-coupled resin enabled comparisons of
drug potencies, binding kinetics and off-target binding
across the community of HDAC complexes, including
SIN3, nucleosome remodelling deacetylase (NuRD)
and co-repressor to the RE1-silencing transcription
factor (CoREST)69. Separately, the community of pro-
teins engaged by the HDAC inhibitor panobinostat
were studied in living cells via thermal proteome profil-
ing through thermal-shift assay70,71. In that work, protein
stability was monitored over a range of temperatures at
a fixed compound concentration — or over a range of
compound concentrations at a fixed temperature — in
order to reveal direct and indirect effects of drug binding
on members of the community71,72.
The aetiology of off-target effects has been extensively
explored with ABPP and chemoproteomics (Table 1;
Fig. 3). The fatty amide hydrolase inhibitor BIA 10-2474
displayed off-target interactions with several lipases that
have important metabolic functions in neurons33 and
may help explain the neurological phenotypes observed
in clinical trials involving this inhibitor73. Similarly, the
proteasome inhibitor bortezomib exhibits certain neuro­
toxic side effects in patients undergoing treatment for
multiple myeloma74. An assessment of serine hydrolase
activities in this context demonstrated inhibition of
several additional serine hydrolases by bortezomib74.
For the CDK4/6 inhibitors, chemoproteomic profil-
ing revealed inhibition of additional protein and lipid
kinases by palbociclib in liver cancer cells and primary
tumour tissues, indicating that its polypharmacology
may exert clinical effects not exhibited by the more spe-
cific ribociclib75. Together, these studies demonstrate the
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ability of discovery proteomic assays to assess cryptic
protein communities in order to predict and explain the
unintended side effects of therapeutic agents.
Drugs affect protein localization
Many cellular processes are controlled through regulated
subcellular localization of key proteins. In one promi-
nent example, the 14-3-3 proteins — which are dedicated
to recognizing and sequestering proteins in the cytosolic
compartment — control the expression of a broad com-
munity of signalling molecules76. Similar mechanisms
regulate localization of the signalling receptor NOTCH77,
the transcriptional regulators YAP and TAZ78, and the
histone-modifying enzyme HDAC4 (ref.79). The recep-
tor stability and assembly of membrane-associated
signalling complexes is also regulated by PPIs80–82. For
the transcriptional regulator and tumour suppressor
p53, localization to the mitochondrial outer mem-
brane may facilitate a novel transcription-independent
apoptotic programme (reviewed in10). Beyond abun-
dance, proteomic analysis can provide information on
changes in protein localization upon drug treatment
(Fig. 1; Table 1).
Within the proteome, plasma membrane proteins
serve as first-line responders to environmental changes.
Owing to their accessibility, many members of this
community also serve as therapeutic targets for
small-molecule and antibody-based therapeutics43,45,83.
Amongst these, extracellular interactions between
immune checkpoint receptor programmed cell death
protein 1 (PD-1) and its ligand PD-L1 have emerged as
central targets in cancer immunotherapy (reviewed in83).
Antibody-based therapies that block PD-1 interaction
with PD-L1 have been tested across many cancer types
and have proved successful for a subset of patients83.
A complementary approach to the existing antibody-
based strategy has emerged from proteomic studies
of cancer cell lines that were deficient chemokine-
like factor-like MARVEL transmembrane domain-
containing family member 6 (CMTM6), a protein with
previously unknown function80,81. Depletion of CMTM6
in tumour cells specifically destabilized PD-L1 at the
plasma membrane and restored the cytotoxic activity of
PD-1-expressing T cells80, suggesting a new entry point
for modulating the immune checkpoint. In the context
of T cell receptor (TCR) activation, a combination of
ABPP and proximity labelling showed translocation
of the deubiquitinase USP12 from the nucleus to the
cytosol and stabilization of the TCR complex82, confirm-
ing the utility of mass spectrometry-based approaches
for shedding light on novel inputs to therapeutically
meaningful protein communities.
Fractionating subcellular compartments. Even when pro-
tein levels within affected cells are seemingly unchanged,
the spatial distribution of a protein community can be
dynamic14,37,84. Drug-induced and disease-initiated redis-
tribution of proteins can be studied using different types
of microscopy, although reagent availability, multiplexing
and quantification between samples still present chal-
lenges38. These approaches are complemented by meth-
ods that combine biochemical separation and mass
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Synaptic cleft
Space or gap at the synapse
between a neuron and an
effector cell (such as a neuron
or a muscle cell).
Postsynaptic density
Protein-dense compartment at
the postsynaptic membrane of
a synapse between two
neurons.
Xenobiotics
Substances that are foreign to
the biological system.
BioID
A mass spectrometry-based
method for studying the
associations of a protein of
interest fused to a biotin ligase
that labels other proteins in its
close proximity.
spectrometry-based quantification, which can provide
protein localization on a global scale14,36,37,85,86 (Fig. 3).
The localization of organelle proteins by isotope tagging
(LOPIT) approach, developed by Lilley and colleagues,
couples density centrifugation or differential centrifu-
gation with mass spectrometry to determine the pro-
tein composition of intracellular organelles36,86. Building
on this technique, Itzhak et al. introduced isotopically
labelled organelle fractions as internal references, to
improve reproducibility and enable the identification
of protein translocation events between conditions14.
Protein translocation events associated with EGFR sig-
nalling were reported, although these were limited to
cell lines. These events included localization of EGFR
to endosomal compartments upon receptor stimulation
with EGF14, as well as the sequestration of EGFR adaptor
proteins (such as growth factor receptor-bound protein 2
(GRB2) and SHC-transforming protein 1 (SHC1)) in the
cytosol away from the secretory organelles upon treat-
ment with the EGFR inhibitor gefitinib37. Subcellular
fractionation also revealed changes in protein distribu-
tion that did not manifest as changes in global protein
levels, including upregulation of mismatch repair pro-
teins in the microsomal and cytosolic fractions and their
downregulation in the mitochondrial compartment of
glucosamine-treated human Hodgkin lymphoma cells84.
Organelle fractionation and mass spectrometry
analysis were recently paired with immunofluorescence
microscopy and transcriptomic analysis in order to
gene­rate a subcellular map of a human cell38. It is yet
to be demonstrated whether subcellular fractionation
coupled to mass spectrometry can sensitively and repro-
ducibly detect protein translocation events between
closely related compartments, such as endosomes and
lysosomes — an ability that could be important for both
large- and small-molecule drug development.
Proximity-based labelling. Proximity labelling is
another way to investigate protein communities, par-
ticularly those that are hard to isolate using biochem-
ical approaches because they are not enclosed within
a membrane (Fig. 3) . Such communities include a
synaptic cleft , postsynaptic density (PSD) and other
non-membrane-bound cellular compartments (for
example, nucleoli in the nucleus and stress granules in
the cytoplasm). In the case of neurological synapses,
proximity labelling has been informative in several
recent studies12,87. Synaptic proteins were tagged with
enzymes that catalyse biotinylation of proteins in their
close proximity, such as horseradish peroxidase (HRP)
or biotin ligase (BirA), which enabled protein isolation
and identification by mass spectrometry12,87. Uezu et al.
fused proteins that reside specifically in excitatory and
inhibitory regions of the PSD of mouse brain with BirA,
in order to label the synaptic complexes in the vicinity
of the PSD87. Although inhibitory PSD is especially hard
to characterize, owing to its thin structure88, proximity
labelling revealed remarkable complexity87. In a similar
way, the protein composition of synaptic clefts in rat
cortical neuron cultures was investigated by Loh et al.,
using HRP-conjugated proteins in combination with a
membrane-impermeable substrate12. The purification
of biotinylated protein complexes using denaturing
and non-denaturing conditions enabled the recovery
of labelled cleft proteins and cytoplasmic constituents
from the PSD12. The identification of community mem-
bers in these unbounded structures can elucidate basic
biology and suggest potentially novel therapeutic targets
in neurological disorders. Looking ahead, a combina-
tion of proximity labelling with fast kinetics and mass
spectrometry-based multiplexing has the potential to
resolve the temporal events occurring at neurological
synapses.
One challenge at the intersection of proteomics and
drug development is the transient nature of the PPIs that
regulate immediate responses to xenobiotics. Although
they are dynamic and complex, the interaction net-
works of oncoproteins such as MYC and KRAS have the
potential to reveal new therapeutic modalities89,90. Both
oncoproteins have essential functions in normal physiol­
ogy and development, and both lack an enzymatic
activity that can be easily manipulated. These dynamic
mechanisms must be thoroughly understood in order
to minimize the side effects associated with therapeutic
targeting. Historically, however, affinity purification
coupled to mass spectrometry (AP–MS) approaches have
provided only a static picture of human protein commu-
nities28 (Fig. 3). These approaches have often required the
optimization of conditions for each target and have been
susceptible to artificial interactions introduced by cell
lysis. A notable example comes from AP–MS studies of
SCF ubiquitin ligases that have described an exchange
of regulatory factors and substrate receptors in a cell lysis
buffer within minutes87. To estimate the composition of
SCF complexes in cells accurately, Reitsma et al. intro-
duced a molecular sponge that effectively suppressed
post-lysis exchange by absorbing unbound exchange
factors91. In another example, SILAC-based quantifica-
tion of interaction stability by isotopic differentiation of
interactions as random or targeted (I-DIRT)92 revealed
fast-exchanging interactions of class I and class II
HDACs93. Proximity-labelling approaches can also
capture snapshots of the dynamics within protein com-
munities in living cells, such as disruption by ibrutinib
of the complex formed by MYD88, Toll-like receptor 9
(TLR9) and B cell receptor (BCR) in subtypes of B cell
lymphoma94. The BioID and AP–MS methods have been
used in concert to dissect regulatory interactions within
the Hippo pathway95. Low-solubility membrane- and
chromatin-bound proteins such as the transcription fac-
tors TEAD1 and TEAD3 were identified in association
with YAP1 by BioID, but not by AP–MS95. In the same
study, Couzens et al. used phosphatase inhibitor (oka-
daic acid) treatment to test the hypothesis that Hippo
pathway interactions are regulated by phosphorylation95.
However, slow biotin ligase activity (with signal typically
being accumulated over more than 12 h) made BioID
incompatible with acute phosphatase inhibitor treatment
(2.5 h). By contrast, phosphorylation-dependent regula-
tors of the Hippo pathway were successfully identified by
AP–MS95. Together, these studies convincingly demon-
strate the applicability of proximity-labelling proteom-
ics to studying the mechanisms of drug sensitivity and
resistance at the level of protein communities.
www.nature.com/nrd

Box 2 | How to pick a time point and a drug concentration
appropriate time points and drug concentrations for proteomic analysis of signalling
must be defined for each individual experiment. Prior knowledge of the targeted
pathway or a related process can serve as a reference for selecting initial time points.
For example, receptor tyrosine kinase (rtK)-mediated phosphorylation can be detected
within seconds after stimulation132,133 but is typically profiled within 2–10 min after
ligand binding, and occasionally up to several hours later44,134. similarly, changes in
lysine acetylation135 and ubiquitylation136 levels have been reported within 1 min of
rtK activation. rtK internalization was observed within 8–20 min after ligand binding,
and its re-emergence on the cell surface was observed after an hour42,44. Continuous
treatment with tumour necrosis factor (tNF) induces oscillations in the nuclear–
cytoplasmic translocation of transcription factor nuclear factor κB (NF-κB) with
a period of 100 min, as well as waves of target gene expression at 1, 3 and 6 h137,138.
these time points seem to be cell-specific, which indicates that it is necessary to
optimize time courses on the basis of the model system137,138.
several levels of evidence can help determine an appropriate drug concentration for
cell-based experiments. Biochemical IC50 values for a compound and for structurally
related molecules can provide a starting point. small-scale preliminary studies with
western blotting (wB) or immunoprecipitation followed by wB (iP–wB) readouts
provide evidence on the appropriate time points and drug concentrations in cells
prior to large-scale experimentation. although preliminary studies can be cumbersome
— especially for poorly characterized processes in which the reagents require
optimization — they can save weeks to months in large-scale proteomic experiments
by maximizing the chances that the key biological question will be addressed by the
experimental design.
Insights into transient PPIs can reveal novel pathway-
targeting strategies. The SOX2 transcription factor is
frequently amplified in squamous cell carcinoma and is
itself not readily druggable96. BioID revealed that SOX2
interacts with the EP300 transcription co-activator,
whose inhibition suppressed the growth of lung cancer
cells96. Split-BioID, which uses the fusion of two inactive
halves of BirA to interacting proteins of interest, suc-
cessfully identified transient dimerization-dependent
protein interactions of PP1 phosphatase 97 and
miRNA-mediated gene-silencing complexes98, and this
seems a suitable technique for evaluating compounds
that disrupt PPIs. A complementary approach is to fuse
a protein of interest or a small-molecule-interacting
protein to the HIV Gag protein99. Gag stimulates the
production of virus-like particles that trap protein com-
plexes inside themselves, for isolation and subsequent
identification by mass spectrometry (Virotrap)99. Proof
of concept for the Virotrap approach has been demon-
strated for innate immune-signalling complexes100,
small-molecule binders100 and a RAS-associated ubiq-
uitin ligase substrate adapter, leucine-zipper-like
transcriptional regulator 1 (LZTR1)101.
Recent advances in proteomics have enabled near-
comprehensive assessment of protein communities
that can be isolated biochemically, by affinity purifi-
cation or via proximity labelling (Table 1; Fig. 3). Such
methodologies are now regularly revealing mecha-
nisms of drug action that would be invisible at the level
of the global transcriptome or proteome, and they hold
promise for otherwise intractable targets96,101–103.
Proteomics in space and time
With global proteome-profiling studies revealing
changes in the composition of protein communities,
questions of temporal regulation arise. What are the pri-
mary and secondary events in signal propagation, or the
Nature Reviews | Drug Discovery
Reviews
immediate and delayed responses to drug treatments?
Events that occur within minutes or seconds, such as
the immediate mechanistic response of an activated
receptor upon engaging its effector molecule, can pro-
foundly influence downstream signalling and pathway
druggability. Is it feasible to track these acute changes
using proteomics?
The correlation between drug dosage, substrate con-
centration and response duration represents a litmus
test for the efficiency and specificity of target engage-
ment. When these local events are placed in the con-
text of signalling, proteomic tools can help trace signal
propagation across a protein community (Fig. 1; Box 2).
By extension, time course and dose–response analy-
ses of phosphorylation dynamics can reveal direct and
secondary effectors (Figs 1,3; Table 1). Upon treatment
with lapatinib, dynamic phosphorylation of the down-
stream signalling nodes (such as adaptor proteins,
transcription factors or cytoskeletal proteins) reflects
their physical proximity to the initial EGFR or HER2
signal104. In melanoma, a time course analysis of phos-
phorylation revealed activation of the DNA damage
response (DDR) pathway upon simultaneous inhibition
of RAS–RAF–MEK and PI3K–AKT–mTOR pathways
with cobimetinib (MEK inhibitor) and pictilisib (PI3K
inhibitor) and highlighted the opportunity for enhanc-
ing cytotoxicity through dual pathway inhibition105. In
diffuse intrinsic pontine glioma, for which limited thera-
peutics currently exist, strategies have emerged in recent
years, such as concurrent inhibition of PI3K–MEK106,
BET107 and the histone–lysine methyltransferase EZH2
(ref.108). Whether individually or in combination, these
putative targets affect signalling within multiple protein
communities and would benefit from systematic pro-
teomic profiling in preclinical models and beyond.
Beyond identifying key signalling proteins, time-
resolved analysis of proteins and PTMs can help sub-
divide them into communities that act early, midway
through and late in signal propagation. This temporal
blueprint can establish connections to unappreciated
biological processes and their points of regulation. In
one example, segregation of phosphorylation-mediated
signalling events was demonstrated following glu-
cose stimulation of insulin-secreting beta cells109. The
phosphopeptide profiles clustered into four distinct
groups, and the presence of enriched phosphoryl­
ation motifs among differential responders highlighted
the roles of distinct kinases109. Temporal resolution
of global proteome changes was used to investigate
antibiotic resistance at sub-lethal doses of rifampicin
during early exposure, at the onset of bacteriostasis
and during recovery from treatment110. This analysis
revealed protein communities that act as the driving
forces at each stage of antibiotic resistance: an initial SOS
response, upregulation of transcription and translation,
and the expression of drug-inactivating enzymes110.
Differential signalling triggered by two EGFR ligands,
EGF and transforming growth factor alpha (TGFα), was
resolved by, respectively, interactome, phosphoproteome,
ubiquitinome and proteome analyses across time, so as
to charac­terize the transient and sustained responses
induced by each ligand44. These signal propagation
Reviews
Single-shot mass
spectrometry
A mass spectrometry
experimental setup wherein
single sample injections are
performed, as opposed to
multiple injections of
fractionated peptide mixtures.
maps can be superimposed on the community of
proteins regulated by drug-induced changes, in order
to highlight transient regulatory events.
One area where global proteome profiling is set
to have a profound impact is in the development of
proteolysis-targeting chimeric (PROTAC) molecules.
These and other degraders seek to induce the recruit-
ment of target substrates to an E3 ligase in order to trig-
ger their ubiquitylation and degradation, most recently
demonstrated for RTK targeting as an alternative to
enzymatic inhibition111 (Fig. 1). In this emerging field,
mass spectrometry-based proteomics is ideally suited to
characterizing on- and off-target effects112. Winter et al.
used proteomics to demonstrate the selectivity of dBET1,
a tool compound composed of JQ1 and an E3 ubiqui-
tin ligase complex-binding thalidomide moiety that is
capable of reducing the protein levels of BRD2, BRD3
and BRD4, as well as downregulating gene expression
through the MYC oncogene102. The dBET1 molecule
outperformed JQ1 in triggering apoptosis in primary
AML cells and in reducing tumour growth in a human
leukaemia xenograft model102. Similarly, Bondeson et al.
demonstrated the specificity of RIPK2- and oestrogen-
related receptor (ERR) α-targeting degraders in THP-1
and MCF-7 cell cultures, respectively103.
Temporal profiles of the cellular proteome can resolve
changes in drug-induced signalling, but they do not
provide information on the spatial changes within and
between protein communities. Although frequently
overshadowed by shifts in protein levels, these events can
themselves underlie significant outcomes. One example
is the regulation of PD-L1 levels at the plasma membrane
by CMTM6 through the inhibition of degradation80,81.
Proteomic characterization of individual organelles pro-
vides a ‘zoom-in’ feature for more thorough exploration
of dynamic protein community maps.
The localization, PTMs and abundance of proteins
can now be resolved across space and time using orga-
nelle separation or proximity labelling combined with
multiplexed quantitative methods. For example, prox-
imity labelling with fast kinetics nicely resolved the
timing of local events in the GPCR community113,114.
Single-shot mass spectrometry with isobaric labelling
has been used for whole-organelle profiling, as demon-
strated through the characterization of protein and PTM
dynamics occurring in isolated mitochondria23,115, and
this method holds promise in combination with rapid
organelle isolation116. Protein redistribution across
organelles can also be temporally profiled, as has been
demonstrated for human cytomegalovirus infection
(HCMV)117,118. For example, Beltran et al. demonstrated
the translocation of scavenger receptor class B member 1
(SCARB1) from lysosomes to the plasma membrane,
whereas the unconventional myosin MYO18A trafficked
from the plasma membrane to the HCMV assembly
complex at later stages of the infection118. In the future,
integrating chemoproteomics and ABPP into spatiotem-
poral assessments of protein communities holds pro­
mise for profiling the effects of drugs on their direct and
unintended targets.
Although their development has been challeng-
ing, in vivo spatiotemporal proteomic methods are
maturing. For instance, postsynaptic densities isolated
from mouse brains by immunoaffinity have been charac­
terized throughout development119. Following spinal
cord injury, soluble factors were quantified across several
spinal cord segments120. This analysis revealed the inhi-
bition of regenerative and the activation of inflammation
processes in the first segment posterior to the spinal cord
lesion, presenting a potential new therapeutic target for
stimulating regenerative processes120. In another study,
brain region-specific phosphosignalling maps were
obtained for five structurally different GPCR kappa
opioid receptor (KOR) agonists121. KOR ligands are major
therapeutic candidates for neurological diseases, owing
to their analgesic effects, but some molecules (for exam-
ple, U-50488H) can produce undesirable effects, such as
dysphoria and aversion121. A study revealed activation of
mTOR signalling by the KOR ligands with undesirable
side effects and demonstrated that pre-treatment with
mTOR inhibitor can abolish conditioned place aversion
in mice treated with U-50488H121. With the development
and accessibility of high-sensitivity mass spectrometry
instrumentation, such spatiotemporal proteomic studies
will hopefully further extend into in vivo drug discovery.
Many developmental and clinical targets function at
the intersection of signalling pathways. Characterization
of the drugs that target these proteins at intersections
may now be useful for the prediction of off-target
effects and tissue-specific phenotypes. For example, the
phospholipase D isoenzymes PLD1 and PLD2 generate
phosphatidic acid and act downstream of many plasma
membrane signalling receptors122. These isoenzymes
have been studied as targets in cancer, neurodegenera-
tion and infectious disease122. However, the involvement
of phosphatidic acid in multiple signalling pathways pre-
sents a challenge, owing to toxicity122. The combination
of chemoproteomics, proximity labelling and cell line
engineering (for example, introduction of an ascorbic
acid peroxidase (APEX) tag at the endogenous gene
locus) should enable systematic evaluation of com-
pounds targeting phospholipase D isoforms and their
associated protein communities.
Cell-to-cell communication in tissues with hetero­
geneous cell populations provides another unique
challenge. Proteomic studies of extracellular inter-
actions, secreted molecules, and the corresponding
intracellular responses are valuable first steps in dis-
covering pharma­codynamic biomarkers. Co-culture
of T cells with antigen-presenting B cells was used to
study extracellular signalling via proximity-based inter-
action tagging123. Tian et al. also used T cell and B cell
co-culture to characterize CD28 receptor signalling in
the presence of the rheumatoid arthritis drug CTLA4-Ig,
which can block interaction of CD28 with the B7 ligand
on B cells124. Temporal analysis of reciprocal phospho-
signalling between stromal and pancreatic tumour
cells revealed that the signals exchanged between cell
populations interact in complex ways125. In vivo stud-
ies of tumour–stroma interactions in cancer cell lines
and patient-derived xenograft models may further
differentiate the tumour proteome from the micro­
environment, on the basis of human- and mouse-specific
protein sequences126,127. This approach could be used to
www.nature.com/nrd
 Reviews

identify the community of proteins that correlates with
tumour subtype, drug response or acquired resistance.
Future efforts will be necessary in order to capitalize on
the available proteomic technologies in the context of
tissues.
Conclusions
Studies from the past decade have demonstrated a
continually evolving array of applications for mass
spectrometry-based proteomics. These investigations
have expanded from exploring the fundamentals of
cellular signalling to aiding in drug discovery and
spatio­temporal profiling in response to drug treatment.
Although much of the available data in the community
remain proprietary, we hope that this Review provides
perspective for those in the proteomics and drug develop-
ment communities on the opportunities afforded by the
available technology. Looking ahead, the mass spectro­
metry proteomics community should aim to leverage the
existing methods towards building a rich understanding
of protein communities in vivo, to embrace and system-
atize automation, to measure the stoichiometric rela-
tionships between proteins and to improve proteomic
methods for low-level clinical samples. On the drug
development side, the challenge is to start thinking of
connections between phenotypes and protein communi-
ties, rather than just between individual proteins. Doing
so will require using proteomics systematically to under-
stand the full sequence of events following target engage-
ment and the connections of these events to efficacy,
unintended activities and resistance. Our understanding
of protein communities and their relations has grown
in large part from proteomic investigations of existing
therapeutic agents and has set the stage for the next
round of impactful contributions to the development
of safe and effective therapeutics.
Published online xx xx xxxx

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Acknowledgements
The authors thank members of the MPL group for their input
and support.
Author contributions
The authors contributed equally to all aspects of the article.
Competing interests
H.G.B. and D.S.K. are employees of Genentech Inc., a wholly
owned subsidiary of Roche. D.S.K. is a shareholder in Roche.
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