29 The Proteomics and Metabolomics of Pain: Opportunities For Systems Medicine

The Oxford Handbook of the Neurobiology of Pain
John N. Wood (ed.)
https://doi.org/10.1093/oxfordhb/9780190860509.001.0001
Published: 2018 Online ISBN: 9780190860530 Print ISBN: 9780190860509
CHAPTER
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29 The Proteomics and Metabolomics of Pain:
Opportunities for Systems Medicine 
David Gomez-Varela, Manuela Schmidt
https://doi.org/10.1093/oxfordhb/9780190860509.013.15 Pages 851–875

Published: 10 September 2018
Abstract
This article critically discusses the opportunities and challenges of proteomics and metabolomics in
the context of pain research and clinical practice. Painful pathologies are extremely complex and often
inadequately managed. In order to signi cantly improve therapeutic options of pain syndromes, a
system-wide analysis of the underlying mechanisms is fundamental. To this end, the article highlights
the potential of rmly integrating proteomics and metabolomics into the pain researcher’s toolbox.
The article introduces technological aspects of mass spectrometry and also associated stumbling
blocks for its standardization in clinical research. Lastly, it outlines the path towards personalized
systems pain medicine via the de nition of pain phenomes—from multilayered molecular data to
phenotypical pro les and integrated analysis of disease networks.
Keywords: Proteomics, metabolomics, pain phenomes, mass spectrometry, standardization, personalized

medicine, systems medicine, disease networks
Subject: Sensory and Motor Systems, Neuroscience
Series: Oxford Handbooks
Collection: Oxford Handbooks Online
Introduction
Pain, particularly when chronic, is a complex maladaptive disease with high incidence and is a major cause
of long-term disability (Breivik, Collett, Ventafridda, Cohen, & Gallacher, 2006; Price & Gold, 2017). Despite
immense research e orts, our knowledge of the mechanistic underpinnings of chronic pain syndromes
remains unsatisfactory (Grosser, Woolf, & FitzGerald, 2017; Price & Gold, 2017; Sommer, 2016; Vardeh,
Mannion, & Woolf, 2016). Consequently, today’s pain medications are best described as symptom-based,
exhibit only limited e cacy, and are often accompanied by strong side e ects. Given these limitations, the
American Pain Society identi ed the following goals in their “Pain Research Agenda” (Gereau et al., 2014):

the development of novel pain therapies with acceptable side e ects; progress in prevention, diagnosis, and
management of chronic pain; as well as optimization of the use of available treatments. The success in
reaching these goals will crucially depend on our understanding of the underlying mechanisms governing
distinct pain syndromes. To this end, a reductionist approach has been largely followed so far; that is, the
individual assessment of genes or molecules in the context of pain. Ideally, however, one would like to
identify the system-wide compendium of molecules and pathways that are speci cally modulated during
distinct chronic pain syndromes. Among those, so-called disease signatures of chronic pain (Borsook,
Becerra, & Hargreaves, 2011; Costigan, 2012; Price & Gold, 2017) could be de ned and ultimately serve as
biomarkers. However, disease signatures of chronic pain remain largely elusive to date. New avenues could
be opened by the rm integration of proteomics (i.e., the compendium of proteins in a cell, tissue, or
organism) and metabolomics (i.e., the collection of small molecules produced by a cell, tissue, or
p. 852 organism) into pain research and management. Recent technical advances towards a more reproducible and
comprehensive proteome and metabolome pro ling by mass spectrometry have made this idea feasible.
The Quest for Disease Signatures of Pain: From Genomes to

Multilayered Phenomes
Large public initiatives have searched for speci c genetic signatures underlying painful pathologies by
characterizing whole genomes of thousands of individuals. Examples include the Orofacial Pain Prospective
Evaluation and Risk Assessment (OPPERA) study on temporomandibular disorders (Slade et al., 2016) and
the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network on chronic
pelvic pain (Clemens et al., 2014). These genome-wide studies have identi ed several candidate genes
involved in chronic pain disorders (Diatchenko, Fillingim, Smith, & Maixner, 2013; Meloto et al., 2017;
Zorina-Lichtenwalter, Meloto, Khoury, & Diatchenko, 2016). However, genome-wide studies are restricted
in their power for diagnosis and causal predictions of environmental in uences (changing lifestyle,
nutritional status, comorbidities, and medical treatments) on phenotypes (Mogil, 2012; Oren & Ablin, 2013).
A growing number of studies has also explored epigenetic ((Denk & McMahon, 2012; Doehring, Geisslinger,
& Lotsch, 2011; Niederberger, 2014), gene regulatory (Meloto et al., 2017; Parisien et al., 2017; Peng et al.,
2017), and transcriptomic changes (Grace et al., 2012; Jeong et al., 2016; Simonetti et al., 2013) associated
with painful pathologies in rodents and human patients. Even so, the notion is evolving that only a limited
percentage of detectable transcriptomic alterations are translated at the proteome or metabolome level to
ultimately a ect disease phenotypes (Liu, Beyer, & Aebersold, 2016; Schwanhausser et al., 2011; Sharma et
al., 2015). This is especially true for dynamic conditions, in which protein turnover and half-lives, post-
transcriptional and post-translational cellular bu ering mechanisms may make signi cant contributions
to phenotypes (Liu et al., 2016; Williams et al., 2016).
In this context, system-wide and diverse layers of data such as the genome, epigenome, transcriptome,
proteome, and the metabolome should ideally be combined. Given the complexity of painful diseases and
the subjective nature of pain, these multilayered measurements need to be synergistically integrated with a
comprehensive characterization of phenotypes of the same individual to assemble the individual’s phenome
(Bush, Oetjens, & Crawford, 2016; Houle, Govindaraju, & Omholt, 2010; Oti, Huynen, & Brunner, 2008). The
phenome is de ned as the full set of interactions between genes, transcripts, proteins, and metabolites
shaping the phenotype of an organism. As such, it serves as a dynamic ngerprint, changing in response to
aforementioned environmental in uences. Thus, it is extremely valuable for assessing the ever-changing
p. 853
state of an organism with ample opportunities to monitor disease status, disease progression, as well as
treatment response (Bush et al., 2016; Houle et al., 2010; Oti et al., 2008; Williams et al., 2016; Zierer, Menni,
Kastenmuller, & Spector, 2015). Several recent studies have demonstrated the superior ability of
multilayered phenomics to unravel mechanisms associated with complex cellular processes. For example,
traits of mitochondrial liver metabolism were studied across a mouse genetic reference population of

recombinant inbred strains under two dietary conditions on the genome, transcriptome, proteome, and
metabolome levels (Williams et al., 2016; Wu et al., 2014). Also, a phenome-wide association study in mice
and humans revealed novel molecular associations with disease risks such as bone mineral density (Wang et
al., 2016). Altogether, these ndings indicated that the integrated analysis of multiple layers provided more
mechanistic insights than could be derived from any layer by itself. This nourishes the hope that
implementation of proteomics and metabolomics approaches can signi cantly expand our knowledge of
mechanisms underlying painful pathologies in animal models and humans.
Assembling Multilayered Phenomes of Chronic Pain: Integration of

Proteomics and Metabolomics
Whereas the genotypical basis for phenotypical variation in painful experiences has been explored
(Diatchenko et al., 2013; Meloto et al., 2017; Zorina-Lichtenwalter et al., 2016), there has still been relatively
little work done on the integration of proteomics and metabolomics to mechanistically explain phenotypes
related to pain.
Technical Considerations in Reproducible Mass Spectrometry
Mass spectrometry (MS) is the method of choice for proteomics and metabolomics experiments. In a classic
bottom-up proteomics experiment (Aebersold & Mann, 2003, 2016), protein extracts obtained from tissues
or liquids of interest are cleaved into short peptide sequences using enzymatic digestions (e.g., trypsin).
These are separated by chromatography and analyzed in a mass spectrometer. Similarly, in the case of
metabolomics, metabolites are extracted from complex samples and prepared for analysis by mass
spectrometry (Patti, Yanes, & Siuzdak, 2012). Depending on the intended goals, distinct types of mass
spectrometry technologies can be used (please see Figure 29.1 for an overview) (Aebersold & Mann, 2016;
Sajic, Liu, & Aebersold, 2015). To date, most of the proteomics/metabolomics experiments in the context of

pain (please see sections “Proteomic Studies in Pain Research” and “Metabolomics Studies in Pain
p. 854 Research”) have been performed using data-dependent
p. 855
p. 856
acquisition (DDA) mass spectrometry. As shown in Figure 29.1, in a typical DDA experiment, the mass
spectrometer selects a subset of peptides/metabolites (in general referred to as precursors) for
fragmentation and identi cation. Due to its high sampling breadth, DDA enables the large-scale discovery
of changes in the relative abundance of peptides/metabolites across conditions (e.g., health versus disease)
(Aebersold & Mann, 2016; Geyer, Kulak, et al., 2016; Zamboni, Saghatelian, & Patti, 2015). Despite its
common use, DDA is variable in selecting peptides/metabolites for measurements, leading to imprecise
quanti cation and limited reproducibility across replicates (40–70%) (Michalski, Cox, & Mann, 2011; Sajic
et al., 2015). With the emergence of novel MS approaches and optimizations in bioinformatics data analysis,
these limitations can be overcome to achieve extensive proteome coverage and reliable quantitation. Among
these techniques, targeted MS (Picotti & Aebersold, 2012; Sajic et al., 2015) and data-independent
acquisition (DIA)-MS (Panchaud et al., 2009; Sajic et al., 2015; Venable, Dong, Wohlschlegel, Dillin, & Yates,
2004) hold great potential for hypothesis-driven experiments and biomarker discovery/validation
(Cerciello et al., 2013; Huttenhain et al., 2012; Takadate et al., 2013). Targeted MS modes like selected
reaction monitoring (SRM; Figure 29.1) feature the a priori (i.e., “targeted”) selection and subsequent
fragmentation of multiple (on average 50–100 per run) peptides/metabolites of choice. When combined
with reference peptides/metabolites (of known concentrations and spiked in as internal standards), highly
accurate and absolute quanti cation of chosen peptides/metabolites across many samples is achieved. Yet
this comes at the expense of preceding labor-intensive and time-consuming experiments required to
establish the analyte-speci c targeted selection. On the other hand, in DIA strategies, no
precursor/metabolite selection takes place, and most of the peptides present in a complex sample are
fragmented (in a given mass/charge [m/z] range and above the detection limit of the MS instrument; Figure
29.1). This permits the continuous and unbiased acquisition of peptides/metabolites, thereby increasing the
data dimensionality. However, the resulting DIA datasets (stored in digital maps) are too complex to be
analyzed by a classical database search (Figure 29.1). For this reason, diverse analytical methods have been
developed for DIA digital maps: computational work ows independent from or dependent on reference
spectral libraries (Figure 29.1) (Bruderer, Bernhardt, et al., 2017; Bruderer, Sondermann, et al., 2017; Chen et
al., 2017; Li et al., 2015; Schubert et al., 2015; Tsou et al., 2015; Wang et al., 2015). Along the same lines,
metabolite reference spectral libraries have been introduced for DIA metabolomics (Bruderer et al., 2018).
Reference spectral libraries represent a compendium of peptides/metabolites with the necessary
physicochemical information to uniquely identify single peptides/metabolites in the aforementioned
complex spectra of DIA digital maps.
Figure 29.1.

Overview of mass spectrometry in proteomics/metabolomics. Prototypical examples of three major types of mass spectrometry
approaches. Alongside, data analysis pipelines (P denotes “used for proteomics,” M denotes “used for metabolomics”) and an
overview of major properties are listed.
The data-dependent acquisition (DDA) method is shown using a quadrupole-orbitrap instrument. The first quadrupole (Q1)
selects precursor ions dependent on their abundance. These are further fragmented in the collision cell (Q2), and the fragmentsʼ
mass/charge (m/z) is analyzed by the orbitrap mass analyzer, yielding MS/MS spectra. The peptide identity of these spectra is
then determined by searching protein databases such as Uniprot (http://www.uniprot.org/). Similarly, the identity of features
represented by MS/MS spectra in a metabolomics experiment is determined by searching metabolite databases such as the
Human Metabolome Database (http://www.hmdb.ca (Wishart et al., 2013). Examples of data analysis pipelines: MaxQuant (Cox &
Mann, 2008) and Perseus (http://www.coxdocs.org/doku.php), (meta)XCMS (Tautenhahn et al., 2011; Tautenhahn et al., 2012),
and Mzmine (Katajamaa, Miettinen, & Oresic, 2006).In selected-reaction-monitoring (SRM), a distinct precursor is selected in Q1
based on predetermined m/z values. This precursor is fragmented in Q2, and specific fragments (transitions) are further selected
in Q3 for quantification. The intensity of each transition is then recorded over the whole chromatographic time; i.e., the
instrument (shown here is a triple quadrupole mass spectrometer) continuously scans the sample for the predetermined
peptide. Examples of SRM data analysis pipelines: Skyline (MacLean et al., 2010), mProphet (Reiter et al., 2011), and (meta)XCMS
(Tautenhahn et al., 2011; Tautenhahn et al., 2012).Several variations of DIA approaches have been implemented. As an example,
sequential window acquisition of all theoretical fragment-ion spectra (SWATH)–DIA-MS is depicted. The mass spectrometer
(shown here is a quadrupole-orbitrap instrument) sequentially cycles through ranges (windows) of m/z values (typical window
width is 25m/z units). All peptides eluting in these windows from the chromatography are fragmented, and MS/MS spectra of all
fragments are acquired in the mass analyzer. The instrument rapidly and continuously scans these windows over the whole
chromatographic time and m/z precursor range. In this way, all detectable precursors entering the mass spectrometer can be
measured generating multiplexed MS/MS spectra. Examples of data analysis pipelines: OpenSWATH (Rost et al., 2014),
Spectronaut™ (Biognosys, Switzerland), DIA-Umpire (Tsou et al., 2015), (meta)XCMS (Tautenhahn et al., 2011; Tautenhahn et al.,
2012), and MetaboDia (Chen et al., 2017).Based on their distinct properties regarding analytical breadth, DDA and DIA
approaches are especially suitable for discovery proteomics/metabolomics aiming at identifying large sets of proteins.
Conversely, SRM approaches represent the gold standard for highly accurate quantification and are therefore used for validation
of distinct candidate molecules in biomarker research and in clinical samples. Irrespective of the MS workflow, statistical data
analysis includes the determination of false-discovery rates and diverse clustering algorithms for intra- and inter-group
comparisons.
A valuable feature of DIA digital maps is that they can be re-currently queried in silico, reducing the need for
new biological material—an immense advantage when biological material is limited, as is the case with
some patient samples (e.g., invasive biopsies; Ebhardt, Root, Sander, & Aebersold, 2015). Combined with
storage of reference spectral libraries and MS datasets in growing public repositories (e.g., PeptideAtlas,
peptideatlas.org; PRIDE archive of the ProteomeXChange consortium, www.ebi.ac.uk/pride; the Human
p. 857 Protein Reference Database [HPRD] of Humanproteinpedia, www.humanproteinpedia.org; iProX of the
Human Protein Project [HUPO], www.iprox.hupo.org), the integration of MS technologies into clinical pain
research and management would open immense opportunities: longitudinal monitoring, hypothesis
generation, and patient strati cation. Importantly, standardization and quality control of MS results for
routine clinical studies are currently discussed (Collins et al., 2017; Kolker et al., 2014; Rocca-Serra et al.,
2016; Rosenberger et al., 2017). Overall, technical advances in MS have already excelled in proof-of-concept
studies interrogating pathology-related proteomes. For example, in patients, robust tumor protein
signatures could be identi ed (Addona et al., 2011; Cerciello et al., 2013; Huttenhain et al., 2012; Takadate et
al., 2013). Hopefully, similar success stories will emerge in relation to painful pathologies in the upcoming
years.
Methodological Challenges: Standardized Sampling, Analyte Abundance, and

Complexity of Samples
The variable quality of biological material used in research and clinical routine represents a major

bottleneck for reproducibility. For example, venipuncture used to obtain human blood samples is highly
variable in regard to the use of sampling vials, temperature, and time of storage. These variables have a
huge impact on the stability and abundance of quanti able proteins (Chambers, Percy, Hardie, & Borchers,
2013; Percy, Parker, & Borchers, 2013; Rosskopf et al., 2015) and metabolites (Breier et al., 2014). Ultimately,
this in uences the statistical power and clinical utility of biomarker discovery projects. The lack of sampling
standardization contributes to the well-known gap between the amount of preclinically identi ed
proteomic/metabolomic biomarkers and the actually validated ones available for clinical practice (Drucker
& Krapfenbauer, 2013). In light of these di culties, minimally invasive and reproducible procedures that
enable preservation of easy-to-degrade molecules (e.g., proteins and metabolites) are mandatory. Only in
this way can reliable monitoring of analytes useful for preventive, diagnostic, and prognostic management
of chronic pain be achieved. The development of devices for reproducible capillary blood collection points in
® TM
this direction (e.g., Tap by SeventhSense Biosystems, and HemoLink by Tasso INC). Standardized
sampling procedures are also key requirements for the utility of samples stored in biobanks and for
longitudinal studies. Longitudinal studies are designed to measure alterations of particular analytes from
individual baselines, rather than using one-time measurements and population-based cuto values. In this
line, it is noteworthy that the proteome of blood plasma has been shown to be more constant within an
individual over time than between individuals (Geyer, Holdt, Teupser, & Mann, 2017; Geyer, Wewer
Albrechtsen, et al., 2016; Liu et al., 2015). Consequently, alterations from individuals’ baselines have been
successfully exploited to reveal personalized disease- or lifestyle-associated changes in the plasma
proteome (Geyer et al., 2017; Geyer, Wewer Albrechtsen, et al., 2016; Liu et al., 2015). Certainly, manifold
p. 858 pain-related aspects could be examined by longitudinal and “before–after” studies, such as treatment
response and probability for pain chroni cation after surgery (Backryd, 2015; Sommer, 2016).
In addition to biosampling standardization, several biological issues must be considered when analyzing
proteomes and metabolomes. Protein and metabolite abundance varies with cell type, developmental stage,
and disease stage (Geyer et al., 2017). Moreover, likely disease-associated proteins are only found at minute
concentrations in blood, composing the so-called “hidden blood proteome.” In contrast, serum proteins are
very abundant. This very wide dynamic range of ten to eleven orders of magnitude (from <pg to mg/µl
concentrations) poses substantial practical challenges to the comprehensive analysis of potential biomarker
signatures (Geyer et al., 2017; Geyer, Kulak, et al., 2016). Furthermore, a tissue/liquid biopsy is rarely
composed of only one cell type. This leads to the fact that generally mixed samples are analyzed, in which
protein/metabolite (and certainly also transcript-level) changes in one cell type may be masked by opposing
changes in another one (Berta, Qadri, Tan, & Ji, 2017; Krames, 2014). This challenge also applies to samples
harboring a ected and una ected cells side-by-side, such as preclinical mouse models of chronic pain
involving only partial injury of the sciatic nerve (e.g., the chronic constriction injury [CCI]-model and the
spared nerve injury [SNI]-model; Minett, Quick, & Wood, 2011), and painful conditions in patients such as
peripheral neuropathies and disc herniation (Costigan, Scholz, & Woolf, 2009; Price & Gold, 2017).
Interestingly, pronounced di erences in the transcriptome pro le of injured and uninjured sensory neurons
have been found (Berta, Perrin, et al., 2017; Reinhold et al., 2015). While assays speci c for cell types and
subcellular compartments are being developed (Kim & Roux, 2016; Sharma et al., 2015; Usoskin et al., 2015),
they should ideally be complemented by cell labeling and separation re ecting their pathophysiological
state; for example, injured versus uninjured cells.
A complementary and clinically applicable solution to dealing with sample heterogeneity may be o ered by
MS-based imaging of formalin- xed para n embedded (FFPE) specimens. Sample preservation through
para n embedding is commonplace in clinical routine, such as minimally invasive skin biopsies to
diagnose patients with small ber neuropathy, bromyalgia, and painful diabetic neuropathies (Levine &
Saperstein, 2015; Themistocleous et al., 2016). When coupled with laser microdissection and MS high
resolution, spatial localization of proteins/metabolites can be achieved and correlated with tissue
pathology, as demonstrated by several oncological studies (Ebhardt et al., 2015; Steiner et al., 2014).

Proteomic Studies in Pain Research
While a full list of proteomic studies in pain would be beyond the scope of this chapter, representative
studies focused on di erent aspects will be highlighted. On one hand, pathology-related changes of
proteomes were interrogated in diverse tissues and preclinical rodent models of pain (Huang et al., 2008;
Komori et al., 2007; Lee et al., 2003; Rouwette, Sondermann, Avenali, Gomez-Varela, & Schmidt, 2016; Sui
p. 859 et al., 2014; Vacca et al., 2014; Zhang et al., 2008). Despite many discrepancies across these studies
(probably due to aforementioned technical and biological factors), regulations in distinct cellular pathways
have meanwhile reproducibly emerged. For example, proteins implicated in oxidative stress or its
prevention were found to be regulated in hyper-excitable neuromas (Huang et al. (2008), in a dorsal root
ganglia (DRG) proteome after spinal nerve ligation (Komori et al., 2007), and also in DRG membrane
proteomes after in ammatory and neuropathic pain (Rouwette et al., 2016). The latter study represents the
rst to apply DIA-MS in pain research and reproducibly quanti ed approximately 2,500 membrane-
associated proteins across two pain models. Besides, several studies promote pain research by de ning
proteomes in regions along the pain-axis of naïve rodent models, such as the sciatic nerve (Lu, Wisniewski,
& Mann, 2009), DRG (Rouwette et al., 2016), the spinal cord (Lu et al., 2009), as well as various areas of the
mouse brain (Sharma et al., 2015). Ultimately, this knowledge will serve as a stepping stone to assess
proteome alterations under painful conditions.
Di erential proteome analysis also suggested prominent changes in synaptic transmission, as inferred by
alterations in Synapsin 1 (SYN1), N-ethylmaleimide-sensitive factor attachment protein (NAPB), and
synaptophysin (SYP) in the dorsal horn of spinal cords upon spinal nerve ligation in rats (Sui et al., 2014).
These results are in line with the crucial role that several types of voltage-gated calcium channels play for
presynaptic neurotransmitter release in the spinal cord, such as Cav2.2 (Brittain et al., 2011; Brittain et al.,
2009). Using antibody-mediated protein immunoprecipitation followed by MS, several Cav2.2 binding
proteins (i.e., members of the Cav 2.2 interactome) were identi ed. Among those, the collapsin response
mediator protein 2 (CRMP-2) increases Cav2.2 surface expression and activity, contributing to nociceptor
hyperexcitability and chronic pain (Brittain et al., 2011; Brittain et al., 2009). Remarkably, this nding
prompted the design of analgesic CRMP-2 peptides. Their in vivo injection uncoupled the CRMP-2–Cav2.2
interaction, and ultimately relieved acute, in ammatory, and neuropathic pain in various mouse models
(Brittain et al., 2011; Brittain et al., 2009). Hence, MS-based identi cation of protein–protein interactions
can o er elegant strategies to interfere with dysregulated proteins for pain relief—at least in rodent models
of pain. Moreover, antibody-mediated protein immunoprecipitation followed by MS has elucidated novel
pain players:
(1) Interacting proteins of transient receptor potential (TRP) ion channels; e.g., gamma-aminobutyric
acid type B receptors subunit 1 (GABAB1) suppressing transient receptor potential vanilloid 1 (TRPV1)
ion channels during in ammatory pain (Hanack et al., 2015), annexin A2 (ANXA2) controlling
transient receptor potential ankyrin 1 (TRPA1) -dependent nociceptive signaling (Avenali et al.,
2014), and transmembrane protein 100 (TMEM100) potentiating TRPA1 by weakening its association
with TRPV1 (Weng et al., 2015);
(2) Interactions between the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)

receptor subunit 2 (GLUR2) and glutamate receptor-interacting protein (GRIP), protein interacting
p. 860 with C-kinase (PICK1), and N-ethylmaleimide sensitive fusion protein (NSF), all of which are
linked to hypersensitivity upon nerve injury (Garry et al., 2003; Katano et al., 2008; Wang et al.,
2010);
(3) Interaction partners of the voltage-gated sodium channel NaV1.7, such as the G protein-regulated

inducer of neurite outgrowth (Gprin1) and also aforementioned CRMP-2 (Kanellopoulos et al., 2018).
Proteomics on tissue biopsies of human patients is in its infancy. Nevertheless, several studies have
revealed the rst mechanistic insights into disorders like chronic widespread pain (CWP) (Olausson et al.,
2015; Olausson, Ghafouri, Ghafouri, & Gerdle, 2016), complex regional pain syndrome (CRPS) (Oki et al.,
2012), and chronic musculoskeletal pain; i.e., myalgia (Hadrevi, Ghafouri, Larsson, Gerdle, & Hellstrom,
2013; Olausson, Gerdle, Ghafouri, Larsson, & Ghafouri, 2012). For example, Olausson and colleagues
identi ed 17 di erentially regulated proteins in the trapezius muscle of female patients diagnosed with CWP
compared to healthy controls (Olausson et al., 2015). Functionally, these proteins have been implicated in
glycolysis and gluconeogenesis, muscle damage and recovery, stress and in ammation (Olausson et al.,
2015). These results were later corroborated by examining the cerebrospinal uid (CSF) proteome of female
CWP patients (Olausson, Ghafouri, Backryd, & Gerdle, 2017), which suggested a prominent role of
in ammation in CWP.
Liquid biopsies such as saliva, urine, CSF, and blood are of high clinical relevance. Especially the last holds
great promise for easy implementation of longitudinal studies designed to assess diverse aspects such as
surgery-induced changes, treatment success, and disease progression (Backryd, 2015). MS-based
proteomics on plasma samples from farmers with work-related musculoskeletal disorders suggested
alterations in several proteins such as hemopexin, apolipoprotein A1, and antithrombin, to name a few
(Ghafouri, Carlsson, Holmberg, Thelin, & Tagesson, 2016). Interestingly, hemopexin correlated with pain
intensity and physical disability upon low back pain (LBP) due to intervertebral disc degeneration (DD) in an
unrelated MS-based examination of CSF (Lim et al., 2017). Furthermore, the analysis of CSF from patients
with peripheral neuropathic pain uncovered several di erentially regulated proteins compared to healthy
controls (Backryd, Ghafouri, Carlsson, Olausson, & Gerdle, 2015). Among those, seven proteins (e.g.,
angiotensinogen isoforms, haptoglobin, and alpha-1-antitrypsin) exhibited high discriminative power
between patients and healthy controls. Furthermore, numerous regulated proteins were also increased in
rat serum ve weeks after the induction of neuropathic pain in the CCI-model (Bellei et al., 2017); for
example, prostaglandin-H2-D isomerase (PTGDS), transthyretin (TTR), apolipoprotein E (APOE), and
apolipoprotein A-1 (APOA1). These similarities across species reinforce translational e orts in pain
research.
Insights into proteome changes in response to diverse treatment options could also be gathered. In
neuropathic pain patients, electrical neuromodulation by spinal cord stimulation was described to alter
numerous CSF proteins in a before–after trial (i.e., each patient served as his/her own control) (Lind et al.,
2016). Modulated canonical pathways included neuroprotection (e.g., gelsolin, angiotensinogen), synaptic
p. 861 plasticity (e.g., apoliprotein C-1 and APOE, similar to aforementioned across-species ndings on
neuropathic pain; Backryd et al., 2015; Bellei et al., 2017), and nociceptive signaling (e.g., neurosecretory
protein VGF). A comparison of CSF proteomes of patients with postherpetic neuralgia (PHN) before and
after treatment with intrathecal methylprednisolone and lidocaine four times every week yielded 14
prominently regulated proteins (Lu et al., 2012). As shown for peripheral neuropathic pain (Backryd et al.,
2015), prostaglandin metabolism seemed to exhibit prominent regulation, as evidenced by a reduction of
lipocalin-type prostaglandin D synthase (L-PGDS) upon treatment (Lu et al., 2012). Yet, this did not
correlate with treatment-induced pain relief (Lu et al., 2012).
Taken together, these studies o er promising and at times intriguingly overlapping insights into chronic
pain conditions. However, the validity and clinical utility of identi ed protein changes need to be critically
assessed in large-cohort multi-center studies. This is especially necessary as these studies were performed
on a small scale in few patients, using varying sample preparation methods, MS, and data processing
technologies.
Metabolomics in Pain Research

The importance of metabolites as triggers for adaptive and dysregulated responses in biological systems has
increasingly been revealed. Even though many small molecules are still uncharacterized (Patti, Yanes, &
Siuzdak, 2012), metabolic dysregulations have been shown to be involved in many chronic diseases,
including painful ones (Patti, Yanes, Shriver, et al., 2012; Patti, Yanes, & Siuzdak, 2012; Williams et al., 2016;
Zierer et al., 2015; Zub et al., 2015). These studies are supported by storage of detected metabolites and their
biochemical characteristics in reference databases such as the Human Metabolome Database
(http://www.hmdb.ca; Wishart et al., 2013) and METLIN (Guijas et al., 2018; Smith et al., 2005). These
databases are of signi cant value, as MS/MS metabolomics data analysis software (Tautenhahn et al., 2011;
Tautenhahn, Patti, Rinehart, & Siuzdak, 2012) only identify features, not metabolites (Figure 29.1). The
identi cation of metabolites relies on accurate matching of MS/MS spectra with databases of known
metabolites (Patti, Yanes, Shriver, et al., 2012; Patti, Yanes, & Siuzdak, 2012).
The use of metabolomic pro ling in pain research is in its initial stages. Yet MS-independent methods have
provided evidence that painful conditions correlate with changes in distinct metabolites. For example, Finco
and colleagues used nuclear magnetic resonance (NMR) spectroscopy to compare the urine metabolome
across healthy controls and patients su ering from pain. Distinct metabolome pro les allowed them to
classify patients and discriminate between nociceptive and neuropathic pain (Finco et al., 2016). Hence, this
study provided a proof-of-concept for metabolome-based patient strati cation. In another study, elevation
of salivary cortisol (detected by enzyme immunoassay) was found to be associated with pain
catastrophizing in the context of experimental pain (Quartana et al., 2010).
p. 862 Few pioneer studies have systematically analyzed the metabolome by MS to gain comprehensive
mechanistic insights into chronic pain. Patti and colleagues discovered pronounced changes of metabolic
features in several tissues and blood plasma of a rat model of neuropathic pain (Patti, Yanes, Shriver, et al.,
2012). In particular, their data indicated that neuropathic pain alters sphingomyelin–ceramide metabolism,
resulting in increased levels of distinct ceramide catabolites (N,N-dimethylsphingosines; DMS) with
functional consequences for rat pain behaviors. These ndings are in line with previous reports on the
importance of ceramide and its metabolite sphingosine-1-phosphate for nociceptor hyperexcitability and
pain (Chi & Nicol, 2010; Zhang, Vasko, & Nicol, 2002). In another study, the same work group presented an
improved meta-analysis software for metabolomics experiments (Tautenhahn et al., 2011). Aided by this
software, they identi ed several hundred dysregulated features in the plantar skin of three etiologically
di erent mouse models of pain (compared to their respective controls): the acute pain model of noxious
heat application to the hind paw, the in ammatory complete Freund´s adjuvant (CFA) model, and a
spontaneous arthritis model of pain (animals intraperitoneally injected with serum from K/BxN mice
representing a model for in ammatory arthritis-). In addition, histamine and two so-far-uncharacterized
metabolites were found to be commonly dysregulated in all three pain models (Tautenhahn et al., 2011). It
will be interesting to decipher the identity of these dysregulated features, as metabolite databases are
constantly increasing their breadth.
Su and colleagues assessed the response of patients diagnosed with primary dysmenorrhea (PD)—painful
menstrual cramps—to herbal treatment (speci cally Shaofu Zhuyu formula concentrated-granule, SFZYFG)
(Su et al., 2013). Longitudinal (before and after a three-month treatment) MS-based metabolomics
identi ed alterations in 35 metabolites in blood plasma and urine of patients compared to healthy controls.
Remarkably, the levels of several metabolites were normalized after herbal treatment, providing promising
mechanistic insights into PD pathophysiology and potential treatment options (Su et al., 2013). Another
study employed MS-based imaging of the spinal cord to investigate the regulation of spinal lipids upon
neuropathic pain induced by peripheral nerve injury in mice (Banno et al., 2017). In this way, arachidonic
acid containing phosphatidylcholine was found to be increased in the ipsilateral dorsal horn and correlated
with behavioral hypersensitivity and activation of microglia.

An additional level of complexity arises from functional interactions between proteins and metabolites
(PMIs). These interactions may modulate diverse enzymes, membrane proteins, and transcription factors
(Changeux & Christopoulos, 2016; Gerosa & Sauer, 2011; Piazza et al., 2018), and thereby control a variety of
cellular processes. Thus, PMIs can be exploited to determine the (patho)-physiological status of an
organism by using large-scale MS-based approaches (Piazza et al., 2018). This knowledge may ultimately
lead to the identi cation of novel allosteric sites (Nussinov & Tsai, 2013) with potential relevance for future
drug discovery related to chronic pain.
p. 863
Multilayered Phenomics in Personalized Systems Pain Medicine
Abovementioned studies highlight the immense opportunities that could be provided by a rm integration
of proteomics and metabolomics into the pain researcher’s toolbox: in essence, (1) mechanistic insights into
pain-associated phenome changes, (2) the identi cation of chronic pain signatures, and, ultimately, (3)
novel therapeutic targets. Overall, the assembly of multilayered phenomes can pave the way towards
systems medicine (Barabasi, Gulbahce, & Loscalzo, 2011; Menche et al., 2015; Zhou, Menche, Barabasi, &
Sharma, 2014). Systems medicine aims at revealing the multilayered relationships among di erent disease-
causing factors to obtain a comprehensive understanding of pathologies. Its application to pain would
transform currently practiced empirical pain therapies into personalized, preventive, and predictive care;
that is, personalized systems pain medicine (Borsook, Hargreaves, Bountra, & Porreca, 2014; Borsook &
Kalso, 2013; Bruehl et al., 2013; Price & Gold, 2017; Vardeh et al., 2016).
From the operational point of view, the following four pillars are required to implement this paradigm shift:
(1) Proper, statistically supported study design and objective phenotyping: Well-controlled and
unbiased study design is key to deriving clinical decisions based on multilayered phenomes (Pepe,
Feng, Janes, Bossuyt, & Potter, 2008; Ransoho , 2005). Traditionally, biomarker discovery involves
only few sample numbers at the initial proteome/metabolome discovery stage. During veri cation
and validation steps the sample number is steadily increased until large clinical cohorts are analyzed
by orthogonal methods, such as immunoassays. Due to the small initial sample number, these
approaches lack statistical power (Diamandis, 2010; Hanash, 2011; Pepe et al., 2008; Ransoho ,
2005) and are limited by well-known technicalities of speci city and sensitivity associated with
immunoassays (Geyer et al., 2017; Hanash, 2011). In contrast, in a so-called rectangular work ow,
the discovery phase would already be performed on an adequately composed and large cohort (Geyer
et al., 2017). In this way, statistically supported validation can be achieved in a straightforward
manner and on a faster time-scale (Dunn, Wilson, Nicholls, & Broadhurst, 2012; Geyer et al., 2017).
In this context, objective phenotyping tools enabling patient strati cation are urgently required.
Several e orts along these lines have been made for painful conditions; notably, standardized
assessment of sensory characteristics by quantitative sensory testing (QST) (Arendt-Nielsen &
Yarnitsky, 2009; Baron et al., 2017; Landis et al., 2014; Themistocleous et al., 2016) paired with
p. 864 elaborate pain questionnaires (Ruscheweyh, Marziniak, Stumpenhorst, Reinholz, & Knecht, 2009;
Ruscheweyh et al., 2012), neuroimaging approaches (Landis et al., 2014; Woo & Wager, 2015), and
aforementioned blood protein signatures, once identi ed.
(2) Standardized biosampling procedures as discussed (please see “Methodological Challenges”

section).
(3) Reproducible and accurate analysis technology as discussed (please see “Technical Considerations”
for MS-based analysis in this article).

(4) Integrated data analysis pipelines: Thorough proteome/metabolome pro ling of large cohorts will
result in highly complex and big datasets with speci c demands on MS informatics infrastructures.
Issues related to data processing, reporting, sharing, and quality control need to be addressed. Along
these lines, diverse databases and public repositories have been created (please see “Proteomic
Studies in Pain Research” and “Metabolomics Studies in Pain Research” sections). Ideally, these
databases should additionally integrate multilayered phenomes of large cohorts; that is, molecular
data linked to phenotypical data and electronic medical records (observing legal and ethical
precautions) (Borsook & Kalso, 2013; Bowton et al., 2015). Advanced bioinformatics tools for “big
data” analysis will then be needed to aid with the interpretation of integrated data. Several
laboratories have contributed to developing algorithms of network theory aimed at nding disease
modules in large datasets (Barabasi et al., 2011; Kitsak et al., 2016; Shi et al., 2015; Zhang et al., 2009).
In this way, disease signatures associated with distinct pathologies (cardiovascular diseases, cancer,
and diabetes) could be identi ed (Barabasi et al., 2011; Kitsak et al., 2016; Oti, Snel, Huynen, &
Brunner, 2006; Zhou et al., 2014). In addition, the correlation of two diseases can be mapped based
on the similarity of symptoms and the connectivity of underlying protein interaction networks (Zhou
et al., 2014). Remarkably, network-based algorithms have successfully been employed to predict
human disease commonalities, even though our knowledge of the human interactome is fairly
incomplete (Menche et al., 2015). A study by Guney and colleagues systematically compared the role
of protein networks for the interplay of 238 drugs with 78 human diseases (Guney, Menche, Vidal, &
Barabasi, 2016). It revealed that therapeutic drug e ects are rather focused on a distinct subnetwork
of disease modules (therefore, e ective drugs are called “proximal drugs”). In contrast, ine ective
drugs are more likely to target proteins “distant” from disease modules (hence they are called
“distant drugs”), as is the case for ibuprofen in the context of rheumatoid arthritis (Guney et al.,
2016). Moreover, machine-learning methods are rapidly evolving to assist in the search for disease-
associated predictive features in multilayered datasets of large cohorts (Miotto, Li, Kidd, & Dudley,
2016; Soualmia & Lecroq, 2015; Tenenbaum et al., 2016). Ultimately, the goal is to derive
personalized medical decisions by comparison of individual multilayered phenomes with existing
reference pro les in these ever-growing datasets.
p. 865
Conclusion
To date, most of the studies summarized here (please see “Proteomic Studies in Pain Research” and
“Metabolomics Studies in Pain Research”) concentrated on the generation of one type of “-omics” data to
compare di erent conditions; e.g., health versus disease. However, the strong interdependencies of
di erent “-omics” data need to be kept in mind when aiming at a comprehensive understanding of pain
syndromes. This is especially true for the study of chronic pain, due to its multifaceted nature, pronounced
subjectivity, and high degree of inter-individual variability. The collection and integration of all collectable
data (molecular—from genomic, epigenomic, transcriptomic, and proteomic to metabolomic—and clinical

phenotypical data) into a multilayered phenome would allow the identi cation of biomarkers and the
development of systems pain medicine. This would open novel avenues to address currently unmet needs in
pain management. Of special interest will be the application to the elds of patient strati cation, drug
development, and treatment. Analyzing large cohorts for phenomic disease signatures underlying distinct
chronic pain disorders will promote the strati cation of patients by re ning diagnosis, and also enable
e ective companion diagnostic strategies. All of this may expedite drug development.
In this new paradigm, the individual patient will be placed at the center of the stage and can play a crucial
participatory role (Borsook & Kalso, 2013; Bruehl et al., 2013). Personalized care is already advocated by
patient associations. This re ects the increasing determination of patients to better manage their own
health by demanding more personalized and clinically meaningful information.
Future Directions
To make this vision come true, numerous hurdles in respect to sample preparation, data collection
(standardization, sensitivity, throughput, and storage), and data processing still have to be overcome. In
addition, legal, ethical, and political guidelines related to collection, clinical use, and storage of extensive
personal data will need to be addressed. Nonetheless, if the technological advances of multilayered
phenomics are embraced by the pain community (basic/clinical researchers and medical practitioners
alike), it is conceivable that the next decade will bring about a signi cant change towards mechanism-based
and personalized pain therapies.
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29 The Proteomics and Metabolomics of Pain: Opportunities For Systems Medicine

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The Oxford Handbook of the Neurobiology of Pain

John N. Wood (ed.)

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https://doi.org/10.1093/oxfordhb/9780190860509.013.15 Pages 851–875

Keywords: Proteomics, metabolomics, pain phenomes, mass spectrometry, standardization, personalized

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The Quest for Disease Signatures of Pain: From Genomes to

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Assembling Multilayered Phenomes of Chronic Pain: Integration of

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Methodological Challenges: Standardized Sampling, Analyte Abundance, and

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(2) Interactions between the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)

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Metabolomics in Pain Research

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(2) Standardized biosampling procedures as discussed (please see “Methodological Challenges”

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Costigan, M. (2012). Painʼs peptide signature. Pain, 153(3), 509–510. doi:10.1016/j.pain.2012.01.004

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Niederberger, E. (2014). [Epigenetics and pain]. Anaesthetist, 63(1), 63–69. doi:10.1007/s00101-013-2274-7

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