Expert Review of Proteomics

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ieru20

Advancing mechanistic understanding and

biomarker development in amyotrophic lateral
sclerosis

Alexander G Thompson, Patrick Oeckl, Emily Feneberg, Robert Bowser,

Markus Otto, Roman Fischer, Benedikt Kessler & Martin R Turner

To cite this article: Alexander G Thompson, Patrick Oeckl, Emily Feneberg, Robert Bowser,
Markus Otto, Roman Fischer, Benedikt Kessler & Martin R Turner (2021) Advancing mechanistic
understanding and biomarker development in amyotrophic lateral sclerosis, Expert Review of
Proteomics, 18:11, 977-994, DOI: 10.1080/14789450.2021.2004890

To link to this article: https://doi.org/10.1080/14789450.2021.2004890

© 2021 The Author(s). Published by Informa

UK Limited, trading as Taylor & Francis
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Published online: 02 Dec 2021.

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EXPERT REVIEW OF PROTEOMICS
2021, VOL. 18, NO. 11, 977–994
https://doi.org/10.1080/14789450.2021.2004890

REVIEW

Advancing mechanistic understanding and biomarker development in amyotrophic

lateral sclerosis
Alexander G Thompson a, Patrick Oeckl b,c, Emily Fenebergd, Robert Bowser e
, Markus Otto b,f
,
Roman Fischer g, Benedikt Kesslerg and Martin R Turner a
a
Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK; bDepartment of Neurology, University of Ulm, Ulm, Germany;
c
German Center for Neurodegenerative Diseases (Dzne e.V.), Ulm, Germany; dDepartment of Neurology, Klinikum Rechts der Isar, Technical
University Munich, Munich, Germany; eDepartments of Neurology and Translational Neuroscience, Barrow Neurological Institute, Phoenix, Arizona,
USA; fDepartment of Neurology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany; gTarget Discovery Institute, Nuffield
Department of Medicine, University of Oxford, Oxford, UK

ABSTRACT ARTICLE HISTORY

Introduction: Proteomic analysis has contributed significantly to the study of the neurodegenerative Received 8 July 2021
disease amyotrophic lateral sclerosis (ALS). It has helped to define the pathological change common to Accepted 8 November 2021
nearly all cases, namely intracellular aggregates of phosphorylated TDP-43, shifting the focus of
KEYWORDS
pathogenesis in ALS toward RNA biology. Proteomics has also uniquely underpinned the delineation
Amyotrophic lateral
of disease mechanisms in model systems and has been central to recent advances in human ALS sclerosis; biomarkers;
biomarker development. neurodegeneration;
Areas covered: The contribution of proteomics to understanding the cellular pathological changes, proteomics; cerebrospinal
disease mechanisms, and biomarker development in ALS are covered. fluid; TDP-43; SOD1; C9orf72
Expert opinion: Proteomics has delivered unique insights into the pathogenesis of ALS and advanced
the goal of objective measurements of disease activity to improve therapeutic trials. Further develop­
ments in sensitivity and quantification are expected, with application to the presymptomatic phase of
human disease offering the hope of prevention strategies.

1. Introduction
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenera­ perturbations [6,7]; epidemiological evidence suggests that
tive disease that causes progressive weakness due to death of there may be a summation of insults that lead to catastrophic
ventral horn motor neurons within the spinal cord and pyra­ neurodegeneration [8].
midal cells of the motor cortical areas [1]. ALS is aggressive, In common with other neurodegenerative diseases, neuro­
leading to death within 3 years of symptom onset in most nal loss accompanied by insoluble protein inclusions are core
cases, though its progression is highly variable [1]. Although pathological features of ALS, occurring primarily in the motor
most cases of ALS are sporadic, around 10% of cases report cortical regions, brainstem motor nuclei, and ventral horn of
a family history; most of these, and a small proportion of the spinal cord [9]. Our knowledge of major aggregate com­
sporadic cases, are attributable to variants in one of ponents owes much to proteomics: the identification of TDP-
a handful of genes, though variants in over 40 genes have 43 as the major component of inclusions in over 95% of ALS
been implicated in ALS [2]. Beyond monogenic causes, ALS cases (excepting those with genetic ALS due to SOD1 or FUS
shows significant heritability in twin studies, and recent mutation) and 50% of FTD cases was achieved using liquid
research indicates an oligogenic contribution to ALS suscept­ chromatography-tandem mass spectrometry (LC-MS/MS) of
ibility [3,4]. ALS has clinical and pathological overlap with urea-soluble brain fractions [9].
frontotemporal dementia (FTD) and shares genetic risk, pri­ The consequences of this landmark finding shifted etiolo­
marily through pleiotropic effects of hexanucleotide repeat gical hypotheses of ALS toward mechanisms in which TDP-43
expansion in an intronic region of the C9orf72 gene, which plays a central role, particularly relating to its functions in
constitutes the most common monogenic cause of ALS [1,5]. transcription, translation, and splicing, the stress response,
The plethora of perturbations in intracellular pathways that mitochondrial function, and the inherent aggregation proper­
has been implicated through different monogenic causes of ties of TDP-43 that might contribute to non-cell autonomous
ALS suggests that motor neuron degeneration occurs as the mechanisms of neurodegeneration [6]. It also led to the iden­
result of the final common pathway of many upstream mole­ tification of mutations in TARDBP, encoding TDP-43, as a cause
cular alterations such as defects in RNA processing, protein of a small proportion of ALS cases, spawned novel ALS disease
homeostatic processes, oxidative stress, and cytoskeletal models, and led to refocusing of biomarker study toward

CONTACT Alexander G Thompson alexander.thompson@ndcn.ox.ac.uk Nuffield Department of Clinical Neurosciences, University of Oxford, Level 3 West
Wing, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom
© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
978 A. G. THOMPSON ET AL.

protein or coordinated protein network changes [10] that

Article highlights can be applied to tissue, disease models, and directly in ALS
● Proteomics has contributed across the field of ALS in study of post
patients.
mortem tissue, disease models and identification of biomarkers This review highlights the contribution of proteomics to
● Identification of TDP-43 as the major aggregate component in 95% of the study of ALS, in pathological tissue specimens, in disease
cases of ALS was brought about by proteomics as well as identifying
disease-specific truncation and posttranslational modification of TDP-
models, and in the biomarker field (highlighted in Figure 1). It
43 also discusses the potential future contributions of proteomic
● Proteomics has helped delineate the effects of ALS-causing gene techniques at the leading edge.
mutations on protein interactions and in cellular and animal models
● Major advances in ALS biomarkers have come about through the
identification of chitinase protein alterations in cerebrospinal fluid of
ALS patients 2. Proteomics of human tissue in ALS
● Future advances are likely to include spatial proteomic methods,
improvements in proteomic depth through data independent acquisi­ Proteomic analysis of post mortem tissue from ALS patients
tion and through the study of ‘at-risk’ populations to understand early has driven progress in our understanding of underlying dis­
events in ALS
ease mechanisms. Studies using proteomic techniques to ana­
lyze human ALS tissue are summarized in Table 1. Although
protein aggregation has long been recognized as a core
pathological feature of ALS, it was not until the publication
in 2006 of LC-MS/MS analysis of urea soluble, detergent inso­
measuring and characterizing full-length, truncated, and phos­ luble fractions from neuropathological tissue of patients with
phorylated TDP-43 forms. ALS and FTD, that TDP-43 was the major aggregate compo­
Whether ALS occurs through alterations in protein–protein nent in over 95% of ALS cases, including ubiquitinated, abnor­
interactions leading to aberrant stress response, excitotoxicity, mally truncated, and hyperphosphorylated TDP-43 species
decreases in protein degradation mechanisms leading to [9,11]. Immunoblotting of urea-soluble fractions and immuno­
aggregation, changes in axonal transport mechanisms or histochemistry indicates a pattern of full length and truncated
gene splicing, or due to non-cell autonomous mechanisms – peptides that mostly represent C-terminal peptides at 20–35
as the major biological effector molecules, proteins are impli­ kDa in brain tissue from ALS and FTD patients [12]; interest­
cated in every proposed pathological mechanisms in ALS [6]. ingly, this is a much less consistent finding in spinal cord [13].
Proteomics is therefore ideally placed to disentangle these Subsequent work has utilized proteomics to further define the
mechanisms by providing a platform to identify individual nature of TDP-43 in aggregates.

Figure 1. Insights into ALS pathophysiology from proteomics.

Table 1. Proteomic studies of human tissue samples in ALS. ALS – amyotrophic lateral sclerosis; CSF – cerebrospinal fluid; EV – extracellular vesicle; FT-ICR – Fourier transform ion cyclotron resistance; iTRAQ – isobaric tags for
relative and absolute quantitation; LC-MS/MS – liquid chromatography tandem mass spectrometry; MALDI – matrix-assisted laser desorption-ionization; TOF – time-of-flight.
Reference Tissue type Participants Instrument Quantitation labeling Main findings
Brain
Neumann Brain (FTD proteomics, ALS and FTD FTD 35 LC-MS/MS 2D electrophoresis and TDP-43 is major protein constituent of inclusions in FTD-U and ALS cases with pattern of truncatd and full-
2006 immunoblots, and ALS-FTD 19 identification of 25kDa length forms. Nuclear clearing of TDP-43 observed in neurons with inclusions
immunofluorescence) ALS 17 peptides
Alonso Brain ALS 3 LC-MS/MS Peptide identification Fungal peptides identified in ALS brain tissue; no control samples included and FDR for peptide identification
2015 not detailed
[14]
Kametani Brain, sarkosyl insoluble aggregates; ALS 2 LC-MS/MS Spectral counting Mutliple semi-tryptic and semi-chymotryptic peptides identified in ALS cases. Multiple phosphorylation
2016 TDP-43 bands excised from SDS- Vascular (serine 242, 292, 305, 317, 332, 333, 373, 375, 379, 387, 289, 393, 395, 403, 404, 407, 409), one
[19] PAGE dementia ubiquitination (lysine 79), and one acetylation (lysine 89) site identified.
1
Umoh Dorsolateral prefrontal cortex ALS 19 (8 LC-MS/MS MS1 LFQ WGCNA module eigenprotein expression upregulated in ALS for antigen binding and zinc ion binding in ALS,
2018 C9orf72 ALS-FTD, and FTD; downregulation of synapse, mitochondrion, and neuron differentiation module
[25] HRE) eigenproteins in ALS-FTD and FTD; and upregulation of RNA splicing, homeostatic process, and response to
ALS-FTD 10 biotic stimulus (microglial and astrocytic proteins). RNA splicing and response to biotic stimulus modules
FTD 12 correlate with TDP-43 pathology. Upregulation of microglial and astrocytic module in C9orf72 ALS
Control 10 compared with non-C9orf72 ALS
Laferriere Brain, sarkosyl insoluble aggregates ALS 6 LC-MS/MS Spectral counting 51 proteins enriched in sarkosyl insoluble fraction in ALS, 23 in ALS but not FTD subtypes
2019 FTD-TDP A 6
[11] FTD-TDP B 3
FTD-TDP C 6
Feneberg Brain and spinal cord urea soluble ALS 16 LC-MS/MS Identification of semi-tryptic 11 tryptic TDP-43 peptides using in-solution digestion. 5 truncated peptides identified in low molecular
2020 fractions and -chymotryptic weight TDP-43 bands. Ratio of C:N terminal peptides specific for ALS tissue
[20] peptides
Control 8 MRM Absolute quantification
PD 8 with heavy peptide
AD 8
Vassileff Brain extracellular vesicles ALS 10 LC-MS/MS MS1 LFQ 16 differentially expressed protein identified involved in RNA binding, intercellular communication
2020 Control 5
[15]
Spinal cord
Strong Spinal cord ALS 1 LC-MS/MS Identification of 24 neurofilament heavy chain phosphopeptides, suggesting similar phosphorylation in ALS
2001 Control 2 and controls
[24]
Ekegren Spinal cord (including laser capture ALS 6 FT-ICR - Preliminary study, 26 proteins identified
2006 microdissected motor neurons) Control 6
[31]
Hanrieder Spinal cord ALS 4 MALDI - Decreased truncated ubiquitin in ALS
2013 Control 3 imaging
[30]
Liu 2013 Spinal cord (urea) ALS 4 LC-MS/MS 2D gel spot fluorescence Differential spots identified as glial fibrillary acidic protein, neurofilament light chain and vimentin; altered
[27] (pooled) volume intensity acetylation of GFAP identified, particularly in insoluble fraction from ALS cord
Control 4 differences
(pooled)
Engelen- Spinal cord ventral and dorsal horn ALS 10 LC-MS/MS 2D gel spot fluorescence 19 proteins altered in ventral and 2 dorsal horn. Altered regulation of mitochondrial, metabolic, and protein
Lee Control 10 volume intensity metabolism proteins
2017 differences
[26]
Iridoy Spinal cord (urea) ALS 9 LC-MS/MS MS1 LFQ 281 proteins with differential abundance in ALS, overlapping with 31 in FTD including downregulation of
EXPERT REVIEW OF PROTEOMICS

2018 FTD 8 cystatin C, neurofilament heavy and medium chains, tubulin alpha 4A chain, optineurin, vesicle-associated
[16] Control 8 membrane protein-associated protein B, SOD1; upregulation of valosin containing protein, transthyretin;
pathway analysis identified mitochondrial and metabolic pathway derrangements
(Continued )
979
 980
A. G. THOMPSON ET AL.

Table 1. (Continued).
Reference Tissue type Participants Instrument Quantitation labeling Main findings
Volkening Spinal cord ALS 6 (3 - SDS-PAGE of TDP-43 Spinal cord TDP-43 interactors include proteins involved in RNA processing, cytoskeletal proteins, RAB
2018 sporadic, 3 immunoprecipitation proteins. Limited overlap with yeast-2-hybrid screen. Different sets of TDP-43 interactors noted between
[29] familial) sporadic and SOD1 ALS cases compared with FUS and C9orf72.
Control 3
Oeckl Spinal cord, CSF ALS 8 LC-MS/MS MS1 LFQ 139 downregulated and 153 upregulated proteins including RNA splicing proteins, neurofilaments
2019 (C9orf72 4)
[28] Control 7
Muscle
Conti 2013 Muscle ALS 17 LC-MS/MS 2D gel spot fluorescence Upregulation of myosin binding protein H in ALS
[17] intensity
Motor
neuropathy
15
Healthy
control 11
Elf 2014 Muscle ALS 10 LC-MS/MS Stable-isotope dimethyl 8 downregulated and 3 upregulated proteins in ALS, relating to metabolism, glycolysis
[18] Control 12 labeling
 EXPERT REVIEW OF PROTEOMICS 981

Table 2. Endogenous TDP-43 truncation peptides identified in tissue proteomic studies in ALS. All peptides show the expected trypsin cleavage site after amino
acids lysine (K) or arginine (R) or chymotrypsin cleavage site after amino acids phenylalanine (F), tryptophan (W), and tyrosine (Y) and one nonspecific cleavage site
suggesting truncation. kDa – kilodaltons.
Study Molecular weight band (kDa) Peptide sequence Amino acid residues Truncation site
Feneberg 2021 [20] Chymotrypsin 23 RGVRLVEGILHAPDAGWGNLVY 52–83 N-terminal
23 VQVKKDLKTGHSKGF 132–147 N-terminal
23 AFVTFADDQIAQSLCGEDLIIKGISVH 226–256 C-terminal
26–28 ISNAEPKHNSNRQLERSGRF 256–275 C-terminal
26–28 SNRQLERSGRF 265–275 C-terminal
Nonaka 2009 [21] Trypsin 23 MDVFIPKPFR 219–227 N-terminal
23 EDLIIK 247–251 N-terminal
Kametani 2016 [19] Trypsin 15–20 NYPKDNK 75–84 N-terminal
30–35 LGLPWK 109–113 N-terminal
30–35 LMVQVK 131–136 N-terminal
23–25 GDVMDVFIPKPFR 215–226 N-terminal
- FGGNPGGFGNQGGFGN 276–291 C-terminal
23–25 PGGFGNQGGFGNSR 280–293 N-terminal
Chymotrypsin 45 LVEGILHAPDAGW 56–68 N-terminal
15–20 NYPKDNKRKM 76–85 N-terminal
23–25 KLPNSKQSQDEPL 176–188 N-terminal
- GSASNAGSGSGFNGG 386–400 C-terminal

Three studies have used proteomic analysis to identify and TDP-43 FTD subtypes. A recent example used LC-MS/MS
endogenously truncated TDP-43 peptides by identifying semi- analysis of brain tissue of patients with ALS (as well as FTD
tryptic or semi-chymotryptic N- and C-terminal TDP-43 pep­ subtypes) identified over 50 proteins enriched in the sarko­
tides (i.e. TDP-43 specific peptides with one non-enzymatically syl-insoluble fractions of ALS brain, including a subset of 23
digested terminus) in urea-soluble fractions from post mortem co-aggregating proteins that differentiated ALS from FTD
tissue of patients with ALS or FTD by in-gel digestion of lower subtypes, such as the presence of Profilin 1, 26S proteaso­
molecular weight bands [19–21]. Most endogenous truncation mal subunit D2, and Tubulin alpha 4 A chain, among
sites were found on the N-terminus of peptides, suggesting others [11].
the enrichment of C-terminal TDP-43 fragments, but also Network analysis of prefrontal cortex of ALS, ALS-FTD, and
C-terminal truncations were found, broadening the knowledge FTD patients using weighted gene co-correlation network
of pathological processing of TDP-43 (Table 2). analysis (WGCNA) has been used to identify coordinated
Measurement of the ratio of C- to N-terminal peptides changes within the protein network in ALS and FTD [25].
using targeted proteomics appears relatively specific for ALS This indicated relatively minor changes in pure ALS cases (as
compared with other neurodegenerative diseases, though would be expected given the lack of involvement of wider
abnormal truncation of TDP-43 is also found in Alzheimer’s frontal lobe areas in pure ALS), though a protein co-expression
disease cases accompanied by TDP-43 aggregation [22]. module associated with immune system functioning was upre­
Although alterations in the C:N terminal peptide ratio are gulated in ALS. Further work to define disruptions at the
not found in ALS spinal cord, concurring with immunoblot proteome level would benefit from the inclusion of additional
findings [20], measurement of C:N terminal ratio appears to CNS tissue regions salient to ALS (such as the brainstem,
be a promising approach for biomarker development. A recent thalamus and spinal cord) in order to broaden the under­
approach using aptamer enrichment prior to quantification to standing of regional and mechanistic differences in these
increase the yield of TDP-43 peptides from post mortem tissue overlapping conditions.
might improve the sensitivity of targeted analysis of trunca­ A small number of studies have used proteomics to exam­
tion peptides in biofluids, where such TDP-43 peptides are of ine spinal cord tissue. Recent studies have identified dysregu­
much lower abundance [23]. lation of mitochondrial proteins and those involved in
Proteomic analysis of post mortem tissue has also allowed carbohydrate metabolism mRNA splicing and of the neurofila­
the identification of sites of post-translational modification of ment compartment as well as altered acetylation of glial fibril­
proteins involved in ALS, specifically phosphorylation, acetyla­ lary acidic protein (GFAP) [26–28]. Spinal cord lysates from
tion, and ubiquitination of TDP-43 [19]. Although a robust sporadic ALS patients and those carrying a SOD1, FUS, and
finding in tissue samples, it has so far not been possible to C9orf72 variants have been used to explore TDP-43 interacting
reliably recapitulate disease-specific TDP-43 phosphorylation proteins, highlighting the role of TDP-43 in RNA processing
in biofluids, limiting its application as a biomarker. Disease- and translation, as well as suggesting greater overlap between
specific phosphorylation sites in neurofilament heavy chain TDP-43 interactors in FUS and C9orf72 cases and TDP-43 inter­
have also been sought in ALS, though phosphorylation actors in SOD1 and sporadic ALS cases than between these
appears similar in ALS patients and controls [24]. pairings [29].
Looking beyond disease-associated protein inclusions, A small preliminary study employed matrix-assisted laser
unbiased analysis of post mortem brain and spinal cord desorption-ionization (MALDI) imaging to explore spatial
tissue has been employed to explore broader alterations in alterations in protein expression; due to technical limitations,
the protein network occurring in ALS. Several studies have only a small number of proteins were identified, though
incorporated analysis of brain tissue from patients with ALS decreased levels of a truncated ubiquitin were observed in
982 A. G. THOMPSON ET AL.

the ALS group [30]. Preliminary exploration of the feasibility of proteomic techniques. Cytoskeletal proteins, particularly the
using laser capture microdissection of motor neurons from intermediate filament protein Vimentin, as well as GFAP and
post mortem tissue has been explored as a means to study neurofilament proteins, have been consistently identified in
the human motor neuron-specific proteome, though this has native spinal cord detergent-insoluble fractions and whole
not been applied successfully in comparative study [31]. spinal cord lysate in SOD1 mouse models [40–42]. Other pro­
Another relevant tissue with limited ALS proteomic studies teins co-aggregating in SOD1 inclusions in the SOD1G93A
is muscle. While gene expression studies have used muscle mouse model include proteins involved in glycolysis and mito­
samples, only a few studies have focused on proteomics in chondrial pathways and chaperones [40], pathways overlap­
ALS muscle (Table 1). Given the intensified collection of post­ ping with those of proteins identified as co-aggregating with
mortem muscle tissue within ALS biorepository efforts, future TDP-43 in ALS brain tissue [11].
studies to explore the proteome in different muscle tissue
types are warranted and may provide new mechanistic
3.2. Posttranslational modifications
insights into muscle degeneration that occurs during ALS
and potential new blood-based biomarkers released by Abnormal ubiquitination and phosphorylation of aggregated
muscle. proteins is a core pathological feature of ALS. Proteomic ana­
lysis has provided a platform to study different posttransla­
tional modifications in disease models, specifically focusing on
3. Proteomic analysis of disease models in ALS
TDP-43 and FUS in ALS, illustrating interplay between ubiqui­
Proteomic techniques have featured in a vast number of tylation, phosphorylation, and acetylation, as well as the
studies of cellular and animal models of ALS, contributing to importance of posttranslational modifications in the physiolo­
the major hypotheses of ALS pathogenesis. gical behavior of proteins implicated in ALS [43–45]. It has also
revealed a role of less common modifications such as citrulli­
nation in maintaining the physiological function of FUS and
3.1. Alterations in protein–protein interactions in ALS
TDP-43 and inhibit aggregation, eventually through
Defining disease-related alterations in protein–protein interac­ a decrease of binding to proteins relevant for stress granule
tion networks is an essential aspect of understanding the formation [46].
pathophysiological processes that lead to ALS, which has Posttranslational modification has also been studied using
relied heavily upon proteomics. These studies have focused proteomics of SOD1 aggregates from G93A and G37R mouse
primarily on TDP-43, extending to other ALS-associated gene models, though significant modifications were not identified
mutations, using immunoprecipitation coupled with mass [41], at odds with earlier and more recent work demonstrating
spectrometry. ubiquitination and conjugation of short ubiquitin modifier
In addition to the aforementioned post mortem tissue study proteins (SUMOylation) to aggregated SOD1 protein [47,48],
of the TDP-43 interactome [29], tissue culture models have likely reflecting the sensitivity of the mass spectrometry tech­
examined the effect of ALS-causing A315T and M337V TDP-43 niques employed. More recently, non-enzymatic deamidation
mutations using primary neuronal cultures [32] and cell lines of asparagine to aspartic acid within a proteasomally cleaved
[33,34] in physiological conditions as well as following oxida­ SOD1 peptide has been identified in the CSF of the SOD1G93A
tive stress, RNA depletion, and DNA damage [35,36]. In addi­ rat model, with a corresponding deamidated peptide detected
tion to highlighting the multifaceted interactions of TDP-43 in the CSF of human carriers (symptomatic and asymptomatic)
with splicing and translation machinery, mitochondrial pro­ of ALS-causing SOD1 variants [49]. Such deamidation has been
teins, and proteins involved in the stress response, this work shown to accelerate protein fibrillization [50], providing
has indicated that the TDP-43 interactome is condition- a potential link to ALS pathogenesis of this common post-
dependent and altered in the presence of TDP-43 mutations, translational modification.
with effects on the cellular stress response, translation, and
exosome biogenesis pathways [32,33]. Analysis of the FUS
3.3. Proteomics in models of C9orf72 ALS
interactome has highlighted major overlap in the pathway
annotations of FUS and TDP-43 interactors around RNA meta­ Current leading hypotheses as to how hexanucleotide
bolism and splicing, stress granules, exosomes, and mitochon­ repeat expansion in an intronic region of C9orf72 leads to
drial proteins [37,38], as well as its involvement in protein neuronal loss and TDP-43 accumulation center on three
degradation pathways [39].b potentially synergistic mechanisms: loss of function of
Proteomic approaches have studied the interactions of C9orf72 protein due to haploinsufficiency, toxicity due to
other proteins implicated in ALS. Systematic analysis using sense and antisense repeat RNA transcription products of
immunoprecipitation of Ubiquilin 2, FUS, Ataxin 2, C9orf72, the GGGGCC repeat region, and toxicity due dipeptide
TDP-43, and Optineurin in N2a cells demonstrated common repeat proteins formed through repeat-associated non-AUG
interactors and overlapping functional annotations, particu­ translation [51]. Recent work has explored mechanisms of
larly in relation to DNA and RNA binding, ribosomal proteins, toxicity of C9orf72 hexanucleotide repeat expansion using
and eukaryotic initiation factors for TDP-43, FUS, and Ataxin 2 proteomics, including the interactome of dipeptide repeat
interactors and protein homeostatic roles for Ubiquilin 2 and proteins, with notably consistent enrichment of ribosomal
Optineurin interactors [35]. The protein constituents and inter­ proteins across studies, as well as RNA splicing and mito­
actors of SOD1 aggregates have also been probed using chondrial proteins and proteins involved in autophagy and
 EXPERT REVIEW OF PROTEOMICS 983

proteasomal systems detected in the interactomes of the A recent study examining the Caprin-1 proteome in stress
toxic arginine-containing dipeptides [52–56]. Protein inter­ granules identified a new hnRNP, SNRNP200, that was also
actors of repeat RNA transcripts have also been identified localized to cytoplasmic aggregates in ALS spinal cord [74].
using proteomics, with enrichment (perhaps unsurprisingly)
of proteins involved in RNA metabolism and containing RNA
recognition motifs [57]. 4. Proteomics of biofluids and the search for ALS
Mass spectrometry has also delineated the interactome of biomarkers
C9orf72 protein, demonstrating enrichment for autophagy Biofluid-based biomarkers present several potential opportu­
proteins, cytoskeletal components as well as ubiquilin 2 and nities in ALS. Although for the most part, the diagnosis of ALS
heterogeneous ribonucleoprotein A1 and A2/B1, proteins is not difficult to achieve in the specialist clinic, sensitive and
implicated in ALS through rare genetic mutations and roles specific biomarkers have long held promise as a means to
in proteostasis, RNA processing, and stress granules; and sepa­ resolve diagnostically challenging ALS cases or enable earlier
rately enrichment of mitochondrial proteins and chaperones, nonspecialist diagnosis [75]. Identifying useful biomarkers that
providing convergence on TDP-43-associated disease altera­ fulfil this promise, however, has proven a major challenge. The
tions [35,58–60]. axonal cytoskeletal neurofilament proteins neurofilament light
chain (NFL) and phosphorylated neurofilament heavy chain
3.4. Subcellular compartment alterations (pNFH) have long led on this front [76,77]. Although showing
promising specificity and sensitivity in retrospective and pro­
Proteomic analysis of subcellular fractions has been applied spective analysis [78–81], the fact that they are nonspecific
to cellular and ALS disease models based on SOD1 mutation markers of axonal degeneration (i.e. are not ALS-specific) and
and overexpression, indicating significant alterations in the show relatively modest rises in slower-progressing, harder-to-
proteome of cell lines, spinal cord and brain tissue of diagnose cases, has hampered their translation into clinical
rodents overexpressing wild-type and mutant SOD1, relating use [82]. ALS has also so far been resistant to combinatorial
to multiple pathways including mitochondria, metabolism, diagnostic approaches such as CSF Abeta/Tau ratio in
and protein degradation and overlapping with proteomic Alzheimer’s disease [83] or recently developed protein aggre­
evidence from human tissue in sporadic ALS [26,61–65]. gation-based assays such as RT-QuIC in prion diseases and
Alterations in the nucleocytoplasmic distribution of TDP-43 more recently synucleinopathies [84,85].
are a key histopathological feature of ALS, and nuclear pore A more important role for ALS biomarkers lies in the mea­
complex dysfunction has been observed in ALS models, surement of underlying disease activity and target engage­
particularly relating to C9orf72 ALS [66]. Comparative pro­ ment in ALS, to support development of novel therapeutics.
teomic analysis of nuclear and cytoplasmic fractions from Drug trials in ALS currently rely on clinical outcome measures,
a HEK293 C9orf72 hexanucleotide repeat model indicates primarily functional decline as measured by the revised ALS
a shift in the distribution of proteins involved in RNA meta­ functional rating scale (ALSFRS-R), a 48-point score that
bolism and translation toward the cytoplasm [67]. declines through the course of the disease [86]. Clinical sta­
Alterations in the nucleocytoplasmic distribution of RNA ging systems, decline in respiratory function, muscle strength
processing and translation proteins are also observed fol­ measures, and survival are also frequently employed [86]. All
lowing TDP-43 knock-down [68], while RNA transport path­ of these measures accrue slowly over time, and most are
way alterations have been demonstrated with confounded by subjectivity or effort and consequently require
overexpression of mutant SOD1, though these alterations prolonged follow-up periods with large numbers of partici­
are opposed to those observed in the C9orf72 model [69]. pants to measure change. Driven by the desire to detect
effects while maintaining relatively small sample size and
trial duration, these measures also promote a tendency to
3.5. Stress granules
limit trial eligibility to those in early disease stages or with
Proteomic analysis of stress granule cores – membraneless aggressive disease, in whom change is detectable over a short
organelles comprising RNA and protein that form by timescale; consequently, this may limit the generalizability and
liquid–liquid phase separation in response to stress [70] – eventually access of patients with less aggressive disease to
indicates a major role for ALS-associated RNA-binding pro­ effective treatment [87].
teins including TDP-43, FUS, and other heterogeneous Biomarkers represent an opportunity to provide sensitive,
ribonucleoproteins (hnRNPs) in stress granule physiology objective, rapidly changing measures that could reduce trial
[71]; stress granule cores are proposed to act as a nidus for duration and sample sizes, hastening the therapeutic devel­
TDP-43 aggregation [72]; time-series proteomic analysis of opment process while reducing costs and broadening trial
stress granule disassembly indicates the importance of inclusion and providing highly valuable information about
altered SUMOylation in delaying stress granule disassembly the underlying frequent failure of preclinically promising
in a Drosophila C9orf72 ALS model [73]. Accordingly, drugs in clinical trials [88]. A number of studies have been
dipeptide repeat proteins have also separately been performed to evaluate specific proteins as pharmacody­
found to interact with stress granule proteins [54]. namic or prognostic biomarkers in ALS model systems or
984 A. G. THOMPSON ET AL.

patient-derived samples [89–93]. Recent ALS clinical trials CSF pools from ALS patients and controls [117,118], with
have explored the use of protein biomarkers as pharmaco­ subsequent matrix-assisted laser desorption-ionization mass
dynamic biomarkers of treatment effect or as inclusion cri­ spectrometry or tandem MS identification of differentially
teria and then monitoring of treatment effect during the abundant protein spots including upregulation of Alpha-
trial [94,95]. 1-antitrypsin precursor and Zn-alpha-2-glycoprotein, both
The application of proteomic technologies to cerebrospinal demonstrating sometimes contradictory alterations in other
fluid (CSF) and blood from ALS patients has been a staple of proteomic and immunoassay studies [119–121].
efforts to identify potential ALS biomarkers over the last two More recent studies have employed LC-MS/MS of individual
decades. Most proteomic studies have used CSF, due to its or pooled CSF samples, with preanalytical abundant protein
proximity to the CNS cells affected by ALS [96]. The relatively depletion or prefractionation techniques to drive additional
low content of highly abundant proteins compared to serum and proteomic depth and in some cases isobaric labeling to
plasma, reducing the need for depletion methods or separation enhance quantitative accuracy [28,108].
approaches, and the lower risk of detecting signals of secondary A major and consistent feature of recent LC-MS/MS proteo­
systemic metabolic alterations related to the disease (for exam­ mic datasets has been the upregulation of a set of related glial
ple malnutrition due to swallowing difficulties) are additional proteins involved in innate immunity, the chitinase proteins, in
advantages of studying CSF compared with serum or plasma, ALS. The first recognition of coherent alterations in chitinase
though much of the CSF proteome is in fact blood-derived [97]. proteins used LC-MS/MS of pooled CSF samples from ALS
The obvious disadvantage is, of course, the relatively invasive patients and controls, identifying a striking upregulation of
approach to CSF sampling when compared to blood. the active chitinase Chitotriosidase 1 (CHIT1) alongside eleva­
Most frequently, bottom-up shotgun proteomic tion of the two related inactive chitinase proteins Chitinase
approaches have been employed, though over this time 3-like protein 1 (CHI3L1 or YKL-40) and Chitinase 3-like protein
much of the breadth of proteomic technology has been 2 (CHI3L2 or YKL-39) [122].
applied at some point to the study of ALS. Reproducibility Though the authors were unable to orthogonally validate
has been an issue; despite alterations in over 500 proteins CHI3L1, subsequent independent proteomic, immunoassay-
detected over the course of CSF proteomic experiments, only based, and enzyme activity studies have consistently indicated
a handful have been demonstrated in two or more studies and marked elevation of CHIT1 and, to a lesser extent, CHI3L1 and
even fewer have survived external orthogonal validation tech­ CHI3L2, in CSF (but not blood) from patients with ALS
niques [98]. Proteomic studies of human biofluid samples in [28,108,114,123–128].
ALS are summarized in Table 3. Emerging literature in ALS indicates that CHIT1 is primar­
The first mass spectrometric study of CSF in ALS used Fourier ily produced by microglia [98]; intrathecal injection of CHIT1
transform ion cyclotron resonance (FT-ICR) of tryptically leads to microglial activation, astrogliosis, and loss of motor
digested CSF samples from a small cohort of ALS patients and neurons in rodents [129]. CHIT1 levels correlate with the
healthy controls to produce a classifier based on the resulting rate of functional decline in ALS, a proxy for the aggressive­
spectrograms [107]. Although this early foray into proteomics in ness of disease as well as neurofilament levels [114,126–
ALS identified no individual biomarker candidates, it represents 128]. CHI3L1, on the other hand, is produced by a subset of
the first mass spectrometric analysis of ALS patient CSF and activated astrocytes and correlates more closely with the
foretold the later use of multiple proteomic features as the burden of upper motor neuron pathology and cognitive
basis for classification algorithms; similar machine learning impairment in ALS [114,128]; correspondingly, while CHIT1
approaches have been utilized in more recent studies [108]. levels are markedly increased in ALS but more modestly so
Subsequent early proteomic biomarker studies in ALS moved in FTD, CHI3L1 levels show more modest elevation in ALS
toward surface-enhanced laser desorption-ionization TOF and more pronounced elevation in FTD [123]. CHI3L1 is less
(SELDI-TOF) mass spectrometry analysis for top-down proteo­ closely associated with disease progression, but is similarly
mics of CSF from ALS patients [109–111]. Between these three correlated with neurofilament levels when compared with
studies, some overlap was observed with lower levels of Cystatin- CHIT1; both CHIT1 and CHI3L1 have shown inconsistent
C detected in all three (and validated using CSF immunoblot) as associations with survival as well as inconsistent small long­
well as decreases in Transthyretin in two studies. As the major itudinal increases [114,124,127,128].
constituent of lower motor neuron Bunina body inclusions spe­ Overall, CHIT1 and CHI3L1 represent a recent major
cific to ALS, Cystatin-C was of particular interest as a biomarker; success for proteomic biomarker discovery in ALS.
external validation however has subsequently proved contra­ Although they do not outperform neurofilaments in terms
dictory [112,113]. Additionally, in the most recent SELDI-TOF of prognostic or classifier performance, they represent dif­
study [111], incorporating samples from 100 ALS patients and ferent dimensions of the underlying disease process – spe­
141 controls, levels of the acute phase protein C-reactive protein cifically microglial and astrocytic activity – that represent
(CRP) were found to be elevated in ALS with confirmatory pathways potentially amenable to disease-modifying treat­
enzyme-linked immunosorbent assay (ELISA); although elevated ments [114]. Chitinase proteins are therefore well-placed to
serum CRP has been associated with worse prognosis in ALS measure treatment response in these areas, though it
patients, a recent ELISA validation of CSF CRP levels did not should be noted that common CHIT1 and CHI3L1 poly­
confirm this finding [114–116]. morphisms leading to alterations in expression are recog­
Two CSF proteomic studies in ALS incorporated 2D gel nized (though do not appear to slow the progression of
electrophoresis to identify differentially abundant proteins in ALS) [123,130].
Table 3. Proteomic studies of human biofluid samples in ALS. ALS – amyotrophic lateral sclerosis; CSF – cerebrospinal fluid; EV – extracellular vesicle; FT-ICR – Fourier transform ion cyclotron resistance; iTRAQ – isobaric tags for
relative and absolute quantitation; LC-MS/MS – liquid chromatography tandem mass spectrometry; MALDI – matrix-assisted laser desorption-ionization; PBMC – peripheral blood mononuclear cells; SELDI – surface-enhanced
laser desorption-ionization; SWATH – sequential window acquisition of all theoretical spectra; TMT – tandem mass tag; TOF – time-of-flight.
Reference Fluid Participants Instrument Quantitation/labeling Main findings
Cerebrospinal fluid
Ramstrom CSF ALS 12 FT-ICR - Construction of machine learning classifier
2004 [107] Control 10
Ranganathan CSF ALS 23 SELDI-TOF Peak selection based on 30 peaks differentially abundant p < 0.01 including Transthyretin, Cystatin C, and
2005 [109] Disease control 19 machine learning Neuroendocrine protein 7B2 precursor
Healthy control 12 classifier
Pasinetti 2006 CSF ALS 36 SELDI-TOF Peak intensity 3 peaks differentially abundant including Cystatin C and Neurosecretory protein VGF
[110] Healthy control 21
Ranganathan CSF ALS 20 SELDI-TOF Peak intensity 27 peak differences between ALS living and post mortem CSF including hemoglobin
2007 [99] Healthy control 16 subunits. Decreased cystatin C and transthyretin in post mortem and living CSF from
ALS post mortem 17 ALS patients compared with controls. NB some samples overlapped with earlier study.
Control post mortem 16 (8 AD, 8 non-neurological)
Brettschneider CSF ALS 18 (9 rapidly progressing, 9 slowly progressing) MALDI-TOF 2D gel electrophoresis, Higher CSF Fetuin A and Transthyretin in rapidly progressing ALS patients
2008 [100] differential intensity
Ryberg 2010 CSF ALS 100 (85 sporadic, 15 familial) SELDI-TOF - 33 peaks differing in intensity between ALS and controls including C-reactive protein,
[111] Disease control (Alzheimer’s 53, multiple sclerosis Cystatin C, and an isoform of Transthyretin
18, other neurological diseases 29)
Healthy control 41
Von Neuhoff CSF ALS 35 MALDI-TOF - Peaks selected using random forest classifier of MALDI-TOF data. Top peaks for
2012 [120] Disease control 23 (other neurological disorders) LC-MS/MS classification included Cystatin C precursor, Chromogranin A, and Neurosecretory
protein VGF precursor
Collins 2012 CSF ALS 11 pools (9 sporadic, 2 familial) LC-MS/MS Increased RNA-binding protein motif 45, RNA-binding protein 40, and nucleolar RNA
[101] Healthy control 8 pools helicase 2 in ALS
Disease control 6 pools (2 multiple sclerosis, 2
Alzheimer’s, 1 upper motor neuron disease, 1
lower motor neuron disease)
Mendonca CSF ALS 10 (pooled) MALDI-TOF 2D gel electrophoresis, 17 proteins differentially abundant including Matrin 3 and 40S ribosomal protein S10
2012 [118] Disease control 10 (pooled) differential intensity
Varghese 2013 CSF ALS 10 (pooled) LC-MS/MS Isobaric tags (2-plex) Chitotriosidase 1, Chitinase 3-like protein 1, Chitinase 3-like protein 2, and Osteopontin
[122] Controls 10 (pooled) upregulated in ALS. ELISA validation confirmed elevation of all but Chitinase 3-like
protein 1
Collins 2015 CSF ALS 90 (9 pools) LC-MS/MS Spectral counting 123 differentially abundant proteins between ALS and controls including increased
[108] Healthy control 95 (8 pools sporadic ALS, 2 pools Neurofilament medium polypeptide, Chitinase 3-like protein 2, and complement C3;
familial ALS) decreased abundance including cystatin C and Ephrin type-A receptor 4. Pathway
Disease controls (20 multiple sclerosis, 20 analysis indicated alterations in proteins involved in axonogenesis and axon
Alzheimer’s disease, 10 lower motor neuron regeneration, synapse organization, inflammation, and extracellular matrix regulation
diseases, 10 upper motor neuron diseases)
Chen 2016 CSF ALS 35 (pooled) LC-MS/MS Isobaric tags (2-plex) 35 proteins altered based on iTRAQ ratio >1.2 or <0.8 (single pool from each group
[121] Healthy control 10 (pooled) compared) in ALS group including downregulation of Alpha-1 antitrypsin, upregulation
of Glutamate receptor 4, Apolipoprotein AII
Thompson CSF ALS 43 (longitudinal) LC-MS/MS MS1 label-free Upregulation of Chitotriosidase 1, Chitinase 3-like protein 1, and Chitinase 3-like protein 2
2018 [124] PLS 6 in ALS compared with healthy control, mimics, and Parkinson’s disease. Upregulation
Healthy control 20 of Chitotriosidase 1, Chitinase 3-like protein 2 compared with PLS. Correlation of
Disease control (Parkinson’s disease 20, ALS mimics proteins with pNFH and disease progression rate observed. Longitudinal increases in
12) Chitinase 3-like protein 1 observed
Bereman 2018 CSF, ALS 33 LC-MS/MS MS1 label-free 118 proteins with altered abundance in CSF, involved in proteolysis, signal transduction,
[102] blood Healthy control 30 nervous system development, and cholesterol binding and including increased
EXPERT REVIEW OF PROTEOMICS

Chitinase 3-like proteins 1 and 2. 20 altered plasma proteins, including upregulation of

Apolipoprotein B-100
(Continued )
985
 986

Table 3. (Continued).
Reference Fluid Participants Instrument Quantitation/labeling Main findings
Oeckl 2020 CSF ALS 26 (12 sporadic, 14 familial) LC-MS/MS Isobaric tags (4-plex) 29 upregulated proteins in ALS compared with control including chitinase proteins,
[28] Asymptomatic gene carriers 14 (8 C9orf72, 5 SOD1, 1 MRM validation neurofilament proteins, apolipoproteins; Ubiquitin carboxyl-terminal hydrolase
TARDBP) isozyme L1, Microtubule Associated Protein 2, and Transmembrane glycoprotein NMB
Healthy control 16 upregulated with MRM validation; 3 downregulated histone proteins. No differentially
abundant proteins between gene carriers and healthy controls. 16 proteins showed
correlation with predicted symptom onset in gene carriers
Zhu 2020 [103] CSF ALS 21 MRM Absolute quantitation Elevation of Ubiquitin carboxyl-terminal hydrolase isozyme L1 and Transmembrane
A. G. THOMPSON ET AL.

Parkinson’s disease 21 with heavy peptides glycoprotein NMB in ALS CSF.

Healthy control 25
Barschke 2020 CSF C9orf72 ALS 16 LC-MS/MS Isobaric tags (4-plex) 236 proteins differentially regulated comparing C9 ALS and C9 FTD including
[131] C9orf72 FTD 8 MRM validation Neurofilament medium polypeptide, Chitotriosidase 1; elevation of Ubiquitin carboxyl-
Asymptomatic C9orf72 carrier 11 terminal hydrolase isozyme L1 in ALS compared with FTD and reduction in Neuronal
pentraxin receptor in FTD compared with gene carriers
Mellinger 2020 CSF ALS 15 (longitudinal) LC-MS/MS DIA Mixed model analysis of peptide assoication with ALSFRS-R change using LASSO variable
[104] selection; peptides selected including Albumin, Transthyretin, Reelin.
Thompson CSF ALS 41 (longitudinal) LC-MS/MS MS1 label-free Upregulation of immune protein module and downregulation of axonal protein module
2021 [153] network Parkinson’s disease 19 WGCNA of proteomic in ALS; alterations in regulation of TDP-43 interactors and proteins involved in gene
Healthy control 20 data from earlier expression
study
Cerebrospinal fluid extracellular vesicles
Feneberg 2014 CSF EV ALS 9 MRM MRM of TDP-43-Flotilin No significant differences in TDP-43-Flotilin ratio in CSF Evs in ALS and FTD compared
[135] FTD 4 ratio in CSF EVs with controls
Control 8
Thompson CSF EV ALS 12 (4 C9orf72) LC-MS/MS MS1 label-free Downregulation of proteasomal subunits and the proeasome-like protein Bleomycin
2020 [136] Healthy control 4 hydrolase in ALS
Hiyashi 2020 CSF EV ALS 3 LC-MS/MS MS1 label-free 3 proteins with increased abundance in ALS CSF EVs, including Novel INHAT repressor,
[137] Disease control 3 (normal pressure hydrocephalus) Programmed cell death 6-interacting protein, 11 with decreased abundance including
Complement C1q subcomponent subunit C, Reelin, and Charged multivesicular body
protein 4a
Blood
Palma 2008 Plasma ALS 7 MALDI TOF 2D gel electrophoresis, Alterations in vitamin D binding protein isoforms in patients with familial ALS
[105] Healthy control 4 LC-ESI-TOF differential intensity
Disease control (muscular dystrophies and Charcot-
Marie-Tooth) 4
Conraux 2013 Plasma ALS 40 MALDI-TOF Support vector machine Discriminating peaks included complement C3, Integrin alpha-IIb, Platelet factor 4, and
[148] Healthy control 40 LC-MS/MS to identify most Thymosin beta-4
discriminating peaks
Haggmark Plasma ALS 367 (350 sporadic, 17 familial) Antibody array Fluorescence intensity Elevated Neurofilament medium polypeptide, Regulator of G-protein signaling 18, Solute
2014 [106] Control 101 (352 carrier family 25 member 20 in ALS plasma
antibodies/278
proteins)
Xu 2018 [149] Plasma ALS 42 LC-MS/MS SWATH-MS 30 proteins with altered abundance in ALS including lower Serum albumin,
Healthy control 18 Apolipoprotein A-II and A-IV, and higher Ficolin-3
Zubiri 2018 Plasma ALS 12 (6 rapid, 6 slow progressing) LC-MS/MS Isobaric tags (10-plex; Upregulation of 18 proteins in rapid progression group including Tubulin beta chain,
[147] PBMC ALS 5 (2 rapid, 3 slow progressing) calibrator with PBMC Apolipoprotein E, Brain acidic soluble protein 1, C4 binding protein alpha chain.
pool as calibrant) Downregulation of 10 proteins in rapid progressing group including 60S ribosomal
protein L12, an isoform of Complement C1, and an isoform of Ficolin 3. Wide ranging
pathway alterations
(Continued )
Table 3. (Continued).
Reference Fluid Participants Instrument Quantitation/labeling Main findings
Leoni 2019 Plasma ALS 30 (14 bulbar onset, 16 limb onset) LC-MS/MS Isobaric tags (10-plex) Differential expression of 554 proteins between bulbar and limb onset using TMT
[146] PBMC ALS 12 (12 bulbar onset, 12 limb onset) calibrator to enrich for brain proteins, with pathway analysis indicating multiple
pathways including complement activation, muscle contraction, and cell migration.
Analysis of PBMCs identified alterations in 132 proteins between bulbar and limb onset
ALS, with pathway alterations in platelet degranulation, interferon signaling among
others.
Katzeff 2020 Serum ALS 28 LC-MS/MS Isobaric tags (10-plex) 8 proteins increased (including Apolipoprotein L1, Complement C3), 15 decreased in FTD
[150] FTD 62 (including Complement C1S and Complement C7) compared with controls; 6 proteins
Healthy control 22 increased (including Apolipoprotein L1 and Creatine Kinase M-type) and 8 proteins
decreased (including Complement C7) in ALS compared with healthy control.
Peripheral blood mononuclear cells
Nardo 2009 PBMC ALS 10 MALDI TOF/TOF Actin, ATPase, CD41, A-actinin, Vinculin, Filamin A nitrated in ALS PBMCs
[151] Healthy control 5
Nardo 2011 PBMC ALS 94 MALDI-TOF/TOF 2D gel electrophoresis, Alterations in proteins involved in energy metabolism, protein degradation, cytoskeleton,
[152] Disease control 23 differential intensity DNA and RNA binding, inflammatory response. Included Calreticulin, TDP-43,
Healthy control 41 Cyclophylin A, Protein disulfide-isomerase and Interleukin-1 receptor-associated kinase
4
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988 A. G. THOMPSON ET AL.

More recent analyses have used isobaric labeling with origin and identify more robust means of extracting a relevant
prefractionation to improve proteomic depth in CSF to EV population using proteomics would be highly valuable.
quantification of almost 2000 proteins [28,131], identifying Ultimately, a combination of proteomics and transcriptomics
upregulation of the proteins Ubiquitin C-terminal hydrolase of EVs may provide the optimal ALS-specific biomarker.
L1, Mictrotubule-associated protein 2, and Glycoprotein
NMB in ALS patients in addition to neurofilament and chit­
4.2. CSF proteomics in the presymptomatic period
inase proteins, validated within-cohort using targeted pro­
teomics and subsequently using single molecule array The identification of highly penetrant ALS-causing genetic
(SIMOA), as well as comparing CSF findings with post mor­ variants, particularly C9orf72 hexanucleotide repeat expansion
tem tissue [28,131]. The comparison of protein level changes and mutations in SOD1, in upwards of 10% of ALS patients has
in CSF and tissue contributes to the understanding of the spawned a cohort of first degree relatives of ALS gene carriers
origin of alterations of the CSF proteome. The upregulation with known high risk of carrying a developing ALS [5].
of neurofilaments in CSF but lower levels in spinal cord Evidence from neurophysiological studies and measurement
tissue is in agreement with the release of neurofilaments of neurofilament and chitinase proteins in asymptomatic gene
into the extracellular space by degenerating axons. In con­ carriers suggests that significant neurodegeneration and
trast, neuroinflammatory proteins such as chitinases, microglial activation is detectable only months before symp­
Glycoprotein NMB, and Macrophage-capping protein are tom onset [125,142–144]. Studying gene carriers during the
increased in both, indicating elevated tissue expression dur­ period before onset of neurodegeneration therefore offers an
ing disease [28]. opportunity to define early events preceding neurodegenera­
tion, identify biomarkers that might predict the onset of symp­
toms, or measure presymptomatic therapeutic response,
4.1. Extracellular vesicle proteomics – a window on
thereby enabling treatment prior to the onset of symp­
intracellular processes in ALS
toms [145].
Extracellular vesicles (EVs) are 50–200 nm structures, including To date, only one proteomic study has addressed this, com­
exosomes and microvesicles, released by virtually all cells, paring 14 asymptomatic carriers of SOD1 and C9orf72 mutations
including neurons and glia of the central nervous system with controls and ALS patients using isobaric tag labeled, pre­
[132]. Alterations in EV biogenesis pathways have been iden­ fractionated LC-MS/MS approach [28]. Despite quantifying 1929
tified in cellular models of genetic ALS and implicated as proteins, no proteins with significantly differing levels between
a potential vector for the intercellular spread of toxic oligo­ gene carriers and non-carriers were identified, perhaps attribu­
mers of TDP-43 [32,133]. EVs are also an attractive target for table to the relatively small asymptomatic carrier group and the
biomarker discovery efforts due to their intracellular origin, mixture of underlying gene mutations reflecting multiple path­
potentially opening a window on mechanisms of dis­ way alterations upstream of motor neuron degeneration [28].
ease [134].
However, the low number of EVs in CSF, combined with
4.3. Blood proteomics
the contribution of multiple CNS cell types and the use of
MS-incompatible isolation techniques (such as those invol­ Relatively few studies have examined the serum, plasma, or
ving polyethylene glycol based precipitation), poses major peripheral blood mononuclear cell (PBMC) proteome in ALS.
challenges to the application of proteomic approaches Recent studies have used isobaric labeling of brain tissue
[134]. Research in this field applying mass spectrometry alongside plasma samples in order to improve the relevance
approaches to CSF EVs for biomarker discovery in ALS has of the identified proteome and circumvent the problems of
so far been very limited, including one targeted proteomic highly abundant proteins suppressing signal from more low
study measuring relative exosomal TDP-43 levels, which did abundance, potentially more interesting, proteins [146,147].
not differ between ALS patients and controls [135], and two Consistent themes indicate alterations in complement pro­
small shotgun proteomic studies, which identified teins and apolipoproteins, though reproducibility of individual
decreased proteasomal and proteasome-like proteins in findings has been lacking [146–152].
ALS [136], a pathway previously implicated through post
mortem analysis of spinal cord tissue; and increased levels
4.4. Proteomics transcending biofluid, pathology and
of the nucleolar protein Novel INHAT repressor [137], both
disease model boundaries
of which await external verification.
An alluring means to simplify access to CNS biomarkers has Modern bioinformatic and proteomic techniques offer the cap­
emerged through the analysis of CNS-derived EVs extracted ability to link alterations in the tissue, model, and biofluid pro­
from serum by immunoprecipitation of EVs carrying the neu­ teomes. Proteomic biofluid studies in ALS have generally detected
ronal lineage marker L1CAM [138]. This method has been used changes presumed to reflect downstream consequences of neu­
in targeted biomarker development approaches in Alzheimer’s rodegeneration, such as the leakage of neurofilament proteins
disease and Parkinson’s disease [139,140]. Whether EVs iso­ from damaged neurons, activation of glial cells, the effects of
lated in this way truly represent a pool of CNS origin is hotly synaptic loss, or altered extracellular matrix regulation
debated, in part due to expression of L1CAM beyond the CNS [28,108,124]. A handful of studies have attempted to bridge this
and some evidence indicating that most serum L1CAM is gap using pathway analysis [108], network analysis [153], or direct
a cleaved ectodomain [141]. Further work to delineate their comparison of post mortem tissue and biofluid proteomic changes
 EXPERT REVIEW OF PROTEOMICS 989

[28,154]. These have identified a degree of overlap between net­ proteins have been demonstrated initially in proteomic and
work-level changes in the CSF proteome with RNA processing, subsequently immunoassay studies [114,122–124,126].
cellular stress, and metabolic pathways identified in ALS models. Elevated levels of neurofilament proteins, initially identified
Detecting clear perturbations of these pathways in the biofluid in candidate-driven immunoassay studies, have also been
proteome that could find clinical use has not yet occurred. identified with increasing consistency in recent proteomic
studies, particularly Neurofilament medium polypeptide,
which has so far been neglected by target-driven studies
5. Expert opinion
[28,108,131]. Chitinase proteins, particularly Chitotriosidase 1,
Proteomic analysis has cut across the field of ALS research. It represent a major success for proteomic biomarker develop­
has redefined our understanding of the molecular histopatho­ ment and are now front-running ALS biomarkers; though they
logical hallmarks of ALS and diverted the course of scientific have not improved upon the classifier or prognostic perfor­
study accordingly [9]. As outlined herein, proteomics has also mance of neurofilament proteins, they encapsulate alternative
highlighted pathophysiological mechanisms of ALS, including dimensions of the disease process so might find use in drug
alterations in protein–protein interaction networks brought trials targeting glial mechanisms or through the eventual
about by ALS-associated genetic variants, the importance of advent of personalized treatment of ALS. Identifying ALS-
proteins implicated in ALS in the cellular stress response, and specific biomarkers, such as those based on disease-
widespread changes in nuclear and cytoplasmic proteomes. associated truncated or posttranslationally modified forms of
Recent proteomic studies have identified major biomarkers TDP-43, remains a major challenge to which targeted proteo­
capable of quantifying different dimensions of the disease mic methodologies could offer solutions in future [12].
and linked findings from disease models and post mortem The multiplex nature and absolute quantitative capabilities
tissue with alterations in the protein network in patient CSF. of targeted mass spectrometry, protein arrays, and aptamer-
Many techniques have been utilized, including a range of based proteomics would also be highly suitable for a panel
preanalytical methods, ionization and separation methods, mass approach to biochemical diagnosis of ALS, though a suitable
spectrometers, and bioinformatic approaches [155]. Within the set of proteins remains elusive [23,166,167].
field of mechanistic study, proteomics has provided highly valu­ Defining the biochemical landscape in the preclinical per­
able insights into the consequences of ALS gene mutations and iod in ALS gene carriers is a major challenge that looms large
pathways involved in ALS, though interpretation is necessarily [145]. Antisense oligonucleotide therapies targeting the com­
tempered by the types of model used, particularly in relation to mon gene mutations in ALS have reached the clinical trial
overexpression models and the use of SOD1 mutation-based arena in symptomatic patients [94]. Asymptomatic gene car­
models, which, given the pathological differences between riers probably have the most potential to benefit from these
SOD1 and other familial and sporadic ALS forms, may not be treatments, but the unpredictable age of onset, even in
a faithful reflection of upstream biological differences leading to genetic cases, high costs, and the invasiveness of intrathecal
sporadic ALS [156]. Given alterations in gene transcription, trans­ treatment are major barriers to use in this group [145].
lation, and metabolic pathways in ALS, it would be well-suited to Proteomics is ideally placed to identify biomarkers of treat­
multi-omic analysis, integrating proteomic, transcriptomic and ment response or predict symptom onset that could help to
metabolomic datasets together, which has been thus far limited remove this barrier by enabling better timing and monitoring
in ALS [157,158]. of treatment. Detecting subtle proteomic changes in this
Proteomics of pathological tissue and disease models group, though, will require major improvements in proteomic
stands to benefit from spatial proteomic techniques such as depth, quantitative accuracy, and large longitudinal cohorts.
MALDI imaging, which have so far found limited use in ALS Some of this may occur by improving the relevance of the
research [30,159,160], in order to resolve compartmentalized proteome of study, for example using analysis of CSF or
aspects of ALS pathology. Techniques to separate tissues and neuronal-derived serum EVs, or through advances in proteo­
enhance the purity of in vitro models, such as laser capture mic approaches such as data independent acquisition meth­
micro-dissection and fluorescence-activated cell sorting odologies, which have been seldom used in the ALS field to-
(FACS), offer additional means to decipher changes occurring date.
within individual cell types and their relative contribution to The goals of ALS biomarker studies are to provide insights
the disease process [161], which could in future be further into disease mechanisms and biomarkers that are useful in
enhanced by nascent single cell proteomics [162]; newer tech­ drug development and clinical trials. Continued studies that
niques such as MALDI-2 mass spectrometry promise subcellu­ incorporate biomarkers in ALS drug development programs
lar compartment resolution [159]. Newer technologies that use and clinical trials will generate the data necessary for regula­
multiplex immunofluorescence microscopy data from up to 40 tory agencies to accept biomarkers in their decision-making
different proteins could also enable spatial resolution of many processes regarding new treatments for ALS.
proteins within the same tissue sample [163].
Within the biomarker field [,], reproducibility of proteomic
discoveries has been a major problem, driven in part by the Declaration of Interests
issues of inconsistent preanalytical sample handling, the sto­
AGT is supported by the Medical Research Council and Motor Neurone
chastic nature of data dependent acquisition (DDA) proteomic Disease Association (MNDA) Lady Edith Wolfson Clinician Scientist
pipelines and the heterogeneity of the disease [164,165]. In Fellowship MR/T006927/1. PO receives research support from the Michael
the last decade, however, consistent signals in the chitinase J. Fox Foundation for Parkinson´s Research (Grant ID: MJFF-010349) and
990 A. G. THOMPSON ET AL.

Alzheimer Forschung Initiative e.V. (20059CB). RB receives research support amyotrophic lateral sclerosis Human Postmortem Tissues. Cells.
from NIH/NINDS grant NS116385, Target ALS, the Fein Foundation, and the 2020;9(7):1709.
Barrow Neurological Foundation. RB is a founder of Iron Horse Diagnostics, 16. Iridoy MO, Zubiri I, and Zelaya MV , et al. Neuroanatomical quanti­
a company focused on biomarker assays and therapeutics for ALS and other tative proteomics reveals common pathogenic biological routes
neurologic diseases. RF and BK were supported by the Chinese Academy of between amyotrophic lateral sclerosis (ALS) and frontotemporal
Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China dementia (FTD). Int J Mol Sci. 2019; 20(1):4 .
(grant number: 2018-I2M-2-002). MO is funded by ALSA. The authors report 17. Conti A, Riva N, Pesca M, et al. Increased expression of Myosin
no other conflicts of interest. MRT receives support from the MNDA. binding protein H in the skeletal muscle of amyotrophic lateral
sclerosis patients. Biochim Biophys Acta - Mol Basis Dis. 2014;1842
(1):99–106.
ORCID 18. Elf K, Shevchenko G, Nygren I, et al. Alterations in muscle proteome
of patients diagnosed with amyotrophic lateral sclerosis.
Alexander G Thompson http://orcid.org/0000-0003-1063-3277 J Proteomics. 2014;108:55–64.
Patrick Oeckl http://orcid.org/0000-0002-7652-7023
19. Kametani F, Obi T, and Shishido T, et al. Mass spectrometric analy­
Robert Bowser http://orcid.org/0000-0001-5404-7259 sis of accumulated TDP-43 in amyotrophic lateral sclerosis brains.
Markus Otto http://orcid.org/0000-0003-4273-4267
Sci Rep. 2016;6(1).
Roman Fischer http://orcid.org/0000-0002-9715-5951
• Mass spectrometric study exploring truncation and post trans­
Martin R Turner http://orcid.org/0000-0003-0267-3180 lational modification of detergent-insoluble TDP-43
aggregates
20. Feneberg E, Charles PD, and Finelli MJ, et al. Detection and quanti­
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