Addressing the needs of traumatic brain injury
with clinical proteomics
Sean Shen1, Rachel R Ogorzalek Loo2,3, Ina-Beate Wanner4and Joseph A Loo1,2,3*
Background: Neurotrauma or injuries to the central nervous system (CNS) are a serious public health problem
worldwide. Approximately 75% of all traumatic brain injuries (TBIs) are concussions or other mild TBI (mTBI) forms.
Evaluation of concussion injury today is limited to an assessment of behavioral symptoms, often with delay and
subject to motivation. Hence, there is an urgent need for an accurate chemical measure in biofluids to serve as a
diagnostic tool for invisible brain wounds, to monitor severe patient trajectories, and to predict survival chances.
Although a number of neurotrauma marker candidates have been reported, the broad spectrum of TBI limits the
significance of small cohort studies. Specificity and sensitivity issues compound the development of a conclusive
diagnostic assay, especially for concussion patients. Thus, the neurotrauma field currently has no diagnostic biofluid
test in clinical use.
Content: We discuss the challenges of discovering new and validating identified neurotrauma marker candidates
using proteomics-based strategies, including targeting, selection strategies and the application of mass
spectrometry (MS) technologies and their potential impact to the neurotrauma field.
Summary: Many studies use TBI marker candidates based on literature reports, yet progress in genomics and
proteomics have started to provide neurotrauma protein profiles. Choosing meaningful marker candidates from
such ‘long lists’ is still pending, as only few can be taken through the process of preclinical verification and large
scale translational validation. Quantitative mass spectrometry targeting specific molecules rather than random
sampling of the whole proteome, e.g., multiple reaction monitoring (MRM), offers an efficient and effective means
to multiplex the measurement of several candidates in patient samples, thereby omitting the need for antibodies
prior to clinical assay design. Sample preparation challenges specific to TBI are addressed. A tailored selection
strategy combined with a multiplex screening approach is helping to arrive at diagnostically suitable candidates for
clinical assay development. A surrogate marker test will be instrumental for critical decisions of TBI patient care and
protection of concussion victims from repeated exposures that could result in lasting neurological deficits.
Keywords: Traumatic brain injury, Biomarker, Clinical proteomics, Mass spectrometry, Multiple reaction monitoring
A general goal of “proteomics” is to comprehend the rela-
tionship between the body’s proteins and how they change
by disease to understand human pathophysiology, and ul-
timately to provide therapeutic and diagnostic tools. The
completion of the human genome provided researchers
with the blueprint for life; proteomics offers the potential
means for analyzing the expressed genome. Proteomics at-
tempts to determine how genes function within the gen-
ome and how they communicate with each other to
(hopefully) lead to important new insights into disease
mechanisms. The potential of proteomics to advance bio-
medical research is high because the key functional com-
ponents of biochemical systems and the cellular targets of
therapeutic agents, namely proteins, are being studied.
Mapping proteomes from injured tissues, cells and bio-
fluids can potentially reveal new protein targets to explore
mechanisms of insults and to provide candidate lists for
new disease indicators or injury biomarkers as diagnostic
or prognostic tools for the clinic.
* Correspondence: firstname.lastname@example.org
1Department of Chemistry and Biochemistry, University of California-Los
Angeles, Los Angeles, CA 90095, USA
2Department of Biological Chemistry, David Geffen School of Medicine at
UCLA, University of California-Los Angeles, Los Angeles, CA 90095, USA
Full list of author information is available at the end of the article
© 2014 Shen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited.
Shen et al. Clinical Proteomics 2014, 11:11
A biomarker could be simply a molecule, such as a
protein whose presence or abundance in a biological
sample signals a disease or insult to an organ. Thus, they
are quantifiable molecules that indicate a pathophysio-
logical process. A biomarker in accessible body fluids or
tissues could greatly enhance our ability to identify pa-
tients at risk, with invisible wounds or predict outcome
of serious injury. A sensitive and specific disease or in-
jury marker such as an early protein abnormality could
provide a warning sign prior to being symptomatic, and
hence could result in more effective preventative care or
treatment options to improve outcome.
The challenges of clinical proteomics and biomarkers
The goal of clinical proteomics to discover new disease
or injury biomarkers is challenging. Beyond the number
of human genes coding for proteins, proteins are proc-
essed and modified, comprising an important dimension
of information to which present proteomic technologies
have but limited access. The total mRNA population, ac-
counting for alternate splicing, RNA editing, and use of
alternate promoters could contain 250,000 transcripts,
while various protein modifications could increase the
size of the human proteome to over 500,000 members
. Cellular proteins and their post-translational modifi-
cations (PTMs) change with the cell cycle, environmen-
tal conditions, developmental stage, and metabolic state.
Independent of these variables, biomarkers should reliably
detect changes in health status, a specific disease, or indi-
cate whether an insult like a toxic exposure or trauma has
occurred. Clearly, we need proteomic approaches that
advance beyond identifying proteins to elucidating their
co- and post-translational modifications, to following the
dynamics of those modifications, and to linking those
modifications to specific diseases or cellular responses to
an insult that inflicted an organ.
Despite all of the significant advances in technologies
in proteomics since its inception in the mid-1990s, with
the development of more sensitive mass spectrometry
detectors and more selective and specific strategies for
sample processing and handling, no clinically validated
disease biomarker has been discovered by proteomics to
Meeting the challenge with targeted screens, focused
selection strategies, and clinical validation
What are the major factors that hindered finding robust
disease and injury biomarkers and how can these be
overcome? The complexity of clinical samples them-
selves is a significant limiting factor. Plasma and serum,
i.e., blood, have been biofluids of choice for measuring
levels of proteins and other biomolecules for clinical
testing, as they can be sampled noninvasively. Plasma is
a protein-rich information source containing what blood
circulation has encountered on its journey throughout
the body and tissue perfusion. The tremendous analyt-
ical challenge of the large number of plasma proteins
lays in their unbalanced abundance: albumin constitutes
over 50% of the plasma proteins (at 30–50 mg/mL) and
the most abundant 22 proteins in plasma represent ap-
proximately 99% of the total protein content in plasma
leaving the majority of proteins at very low abundance.
The estimated dynamic range of protein concentrations in
human plasma may be up to 12 orders of magnitude .
Disease or insults trigger acute events, secondary and
chronic sequelae, including inflammation, wound healing,
and adaptive changes that the compromised body under-
goes in response to the unhealthy state. In an effort to
identify original disease causes or injury factors a simple
experimental model can facilitate a targeted screen cir-
cumventing secondary, less disease-specific events. As
such, scientific experimental model design follows con-
trolled strategies for reproducibility and simplicity that
can facilitate the initial discovery by limiting candidate
markers to those proteins that are related to a disease ori-
gin or injury cause [4,5]. One common proteomics work-
flow involves a 2-dimensional separation prior to protein
identification to reduce sample complexity (Figure 1). Pro-
teins can be sorted by charge (isoelectric point) and size
using two-dimensional polyacrylamide gel electrophoresis
(2D-PAGE) and can be enzymatically digested within the
gel matrix. Despite being developed over 3 decades ago
[6,7], 2D-PAGE remains one of the most powerful separ-
ation techniques for proteomic workflows and was instru-
mental in early protein biomarker research. Following
separation, gels are stained and differentially expressed
protein spots excised, enzymatically digested with trypsin,
and identified by MS requiring only sufficiently accurate
mass measurements (low part-per million range) per-
formed on one or two tryptic peptides to identify silver-
stained protein spots .
A second strategy advocates first enzymatically (e.g., with
trypsin) or chemically cleaving (“breaking”) a complex
mixture of cellular proteins, and then “sorting” the pep-
tides by one or more steps of chromatography. MS ana-
lyzes the recovered fragments as in the previous approach,
and software matches the fragments to the proteins from
which they are derived. Examples of this experimental ap-
proach include multidimensional protein identification
technology (MudPIT) that couples two or more dimen-
sions of chromatographic separations, e.g., strong cation
exchange (SCX) with reversed-phase chromatography
[9,10]. While the outlined approaches have been instru-
mental in biomarker discovery research, the extensive
sample preparation and time required in gel fractionation
and long HPLC LC-MS/MS analyses make discovery pro-
teomics feasible for only limited numbers of samples per
project [11,12]. A simplified disease or injury model using
Shen et al. Clinical Proteomics 2014, 11:11
Page 2 of 13
a controlled experimental design may help to relieve a
proteomic screen from confounding complexities of clin-
ical samples [4,13-15].
A straightforward selection of suitable marker candi-
dates from the ‘long list’ of identified injury or disease spe-
cifically changed proteins should arrive at a manageable
‘short list’ of possible disease marker candidates. A tailored
selection strategy will consider injury cause, marker candi-
dates with the necessary reporting power for the cause as
well as organ specificity and exclusion of proteins nor-
mally present in healthy plasma and tissues.
The subsequent validation of selected disease or injury
markers from a group of candidates may occur stepwise
starting with a preclinical smaller cohort of patients and
controls, allowing to test for normality . Following ini-
tial confirmation, a larger subject cohort can be enrolled
in clinical trials allowing for receiver operating charac-
teristic curve analyses that will establish the basis for
biomarker suitability in the clinic . Currently, the
majority of biomarker validation studies have been
(ELISA). This highly sensitive method is limited for use
early in the verification process, as antibody pairs have to
be optimized for specificity and sensitivity for each marker
separately. As mass spectrometry measurements improve
in sensitivity to match immunoassay detection limits (pg/
mL), a targeted and quantitative mass spectrometry ap-
plication can provide multiplex capacity and absolute
specificity by gas-phase sequence determination, mak-
ing it an ideal alternative for assessing validity of se-
lected marker candidates.
by enzyme-linked immunosorbentassay
The need for markers of Traumatic Brain Injury (TBI)
Neurotrauma to the central nervous system (CNS) is a
serious public health problem in the US; among US ci-
vilians, TBI is most common in infants and toddlers, ad-
olescents and the elderly . The US National Institute
of Neurological Disorders and Stroke estimates that 2.5-
6.5 million Americans have had one or multiple TBIs
Candidate Biomarker Discovery
Candidate Biomarker Verification
Peptide SelectionFragmentation Fragment Selection
Detection of Candidate Markers
Quantitation with SIS peptides
Detection of Candidate Markers
by Immunoassays (e.g. Sandwich ELISA)
Standard Curve Quantitation
Candidate Protein Biomarkers
Verified Candidate Markers for Clinical Validation
(Non-trauma and trauma)
Figure 1 Candidate biomarker discovery and verification workflow. Bottom-up proteomics strategies, such as shotgun proteomics
(multidimensional LC-MS/MS) and 2D-PAGE/MS, can be applied to identify putative candidate markers (left). Candidate protein markers can be
subsequently verified and confirmed by targeted proteomics using standard ELISA methods or multiple reaction monitoring (MRM)-MS (right).
MRM-MS offers the advantages of an antibody-independent platform with capabilities for multiplexing.
Shen et al. Clinical Proteomics 2014, 11:11
Page 3 of 13
. In the US military there were over 212,000 service
men and women diagnosed with some form of TBI be-
tween January 2000-May 2011, roughly accounting for
one-third of all injured US soldiers, making TBI the sig-
nature injury of the wars in Iraq and Afghanistan com-
pared to past wars . TBI contributes to over one-
third of all injury-related deaths, yet 75-90% of all brain
trauma cases are considered to be mild TBI (mTBI),
many without visible wounds that often are undiagnosed
. Better diagnostic tools are needed to detect head in-
juries, especially mTBI, to confirm and to monitor the se-
verity of TBI in order to determine the best course of
action acutely and later post-injury. The neurotrauma
field has currently still no chemical diagnostic marker in
clinical use. Here we will outline briefly the spectrum of
TBI and give examples where a surrogate chemical
marker assay for TBI would be of great benefit to pa-
tients, high risk populations, their families and doctors.
Head injuries can be classified into penetrating and
non-penetrating TBI. Penetrating TBI involves physical
compromise of the skull by an external object resulting
in specific, focused injury most commonly characterized
by hemorrhages and lesions. Non-penetrating TBI, is
much more difficult to assess, as injuries may not be vis-
ible or located precisely. Closed head injuries are caused
by rapid acceleration and deceleration of the brain
within the skull and inflict shear and deformation forces
on gray matter tissue and white matter tracts . Each
trauma patient is a unique injury case with individual
complexity, thus the field distinguishes mainly between
severe and mild TBI (mTBI) as opposite ends of a clinical
spectrum of manifestations. Evaluating and predicting out-
come in severe TBI is often problematic, especially for pa-
tients without visible wounds such as infants.
Diagnostic neurotrauma tools include imaging tech-
niques, neurocognitive examinations, and for severe TBI
patients, the determination of post-traumatic amnesia,
but they provide only estimates of the dynamically evolv-
ing injury process. Functional MRI (fMRI) and the de-
tection of regional blood flow changes (e.g., PET scans)
are not always available, cannot be obtained in critically
ill patients, and are not definitive. Radiological brain
scans on infants and toddlers are widely considered
problematic because the radiation dose endangers the
developing brain. Absence of imaging in the pediatric
clinical praxis prevents distinguishing brain injury from
frequent intestinal flu or even infant irritability .
Non-accidental head injury, or “Shaken Baby” syndrome,
caused by rotation-acceleration strains on the brain in
the still loosely connected infant skull causes bleeding
and swelling that can lead to catastrophic intracranial
damage and can severely impair normal brain develop-
ment (and can even lead to death) . Undiagnosed
victims may be sent back to continued abuse. On the
other hand, imaging does not distinguish inflicted head
injury from non-traumatic bleeding, originating from a
trauma independent condition – a situation in which
legal authorities, parents and care-givers would greatly
benefit from an assay for brain trauma-specific chemi-
cals [24,25]. Mechanical impacts traumatizing the brain
obviously need to be clinically differentiated from
trauma in other organs or from other, non-traumatic
brain injuries like stroke, ischemia, bleeding diseases,
poisoning, epilepsy or chronic degenerative diseases for
proper treatment and activities in the operating room
and the courtrooms . Monitoring daily progression
of a severe TBI patient by repeated imaging can be quite
impractical, considering life supporting intensive care in-
strumentation. A fluid derived chemical marker for com-
promised brain cell viability will be a useful added
measure of the patients evolving status and could aid in
For the vast majority of mTBI/concussion patients,
there are no objective diagnostic or prognostic tools
. A ready diagnostic tool at point of care acutely
after TBI is needed especially for high-risk individuals
(e.g., athletes, military personnel). An objective and unam-
biguous trauma biochemical assay would be valuable for
legal authorities in forensic cases that currently rely on
neuropsychological testing that lacks premorbid base rates
and is subject to malingering and subjective interpretation.
Thus, for high-risk groups, for mild and severe TBI cases
as well as for all pediatric neurotrauma patients, there is
an urgent need for an accurate, unambiguous chemical
measure indicating that a significant impact to the brain
Moreover, a second hit to a concussed, vulnerable brain
can, in rare cases, have a catastrophic outcome with per-
manent brain damage or even death (known as the second
impact syndrome) . Several repeated concussions over
time can in later years cumulate in irreversible brain dam-
age with devastating psychological and cognitive decline, a
pathological condition now defined as chronic traumatic
encephalopathy (CTE) [29,30]. Military personnel and vet-
erans with mild TBI often suffer from post-traumatic
stress disorder (PTSD) after being exposed to blast waves
from explosive devices [31,32].
Certain areas of the brain may be more susceptible to
concussive trauma. A recent study investigated longitu-
dinal changes in global and regional brain volume in pa-
tients one year after mTBI and correlated such changes
with clinical and neurocognitive metrics. Magnetic res-
onance imaging data showed measureable global brain
atrophy, larger than that in control subjects one year
after mTBI. Atrophy was found in specific regions of the
cingulate cortex independent of the site of initial trauma.
The cingulate cortex’s role in rational cognitive func-
tions such as empathy, impulse control, and emotion
Shen et al. Clinical Proteomics 2014, 11:11
Page 4 of 13
correlate strongly with the patient’s observed clinical
symptoms of increased depression and anxiety .
These finding are supported by an independent study of
National Football League players and referees using posi-
tron emission tomography (PET) with a tau specific
tracer that showed higher densities of tau tangles in re-
gions of the brain involved in a nearby region (caudate
nucleus) that is also associated with learning, memory,
emotion, and language comprehension. The deposition
of tau tangles is consistent with those observed in CTE
autopsy patients .
Current evaluation of concussion is basically an assess-
ment of neurocognitive deficits, often not immediate
and requires extensive neuropsychological testing that is
subject to motivational confounds, while critical care
treatment decisions have to be made immediately by
emergency clinical personnel and surgeons. Severity
classification of TBI patients relies on assessing the level
of consciousness, commonly with the Glasgow Coma
Scale (GCS), which is an insensitive measure. Testing re-
lies on verbal communication, and proper motor control
and eye function, which are often impaired after TBI.
Brain function-altering substances such as drugs, alco-
hol, pain medication, sedatives, or even induced coma as
part of emergency and intensive care routine obviously
compromise the use of memory recall and the GCS. Al-
though predictors of TBI exist, such as the Standardized
Assessment of Concussion test, these tools offer little
insight into the pathology of the disease beyond deter-
mining whether a concussion has occurred or not. Be-
cause of this lack of insight into TBI, licensed health
care providers of concussive sports injury are conserva-
tive in their approach to player safety after injury with
the hope that coordination between sideline and clinical
practitioners will aid in improving our understanding of
the extent of impairment for various types of sports re-
lated concussions .
Current potential TBI biomarker candidates
An ideal biomarker should be both specific to head
trauma as well as sufficiently sensitive to be measured and
quantified reproducibly in patient blood or other periph-
eral or proximal fluid samples (such as CSF) by an assay
of choice. These markers should be acutely released into
the fluids following injury and show a distinct temporal
signal pattern. The identification of a unique TBI bio-
marker(s) or surrogate brain cell injury markers that meet
these criteria would provide physicians with an objective
method for early diagnosis of brain injury and enable early
assessment of severity, intervention, and monitoring dis-
ease progression . Multiple neurotrauma signature
markers would allow for correlation analyses with im-
proved statistical power using multivariant logistic re-
gression or similar analyses . Finding candidate TBI
markers is pursued typically by these strategies: (1) Clas-
sical deduction chooses proteins with literature reported
association to brain injury or its secondary events like in-
flammation, axonal degeneration or reactive astrogliosis.
(2) Hypothesis driven animal trauma model studies report
changes in specific proteins using available antibodies or
pathway tailored kits . (3) Discovery of trauma associ-
ated proteins using a proteomic screen of samples derived
from animal injury models or small patient cohorts [39-43].
Surprisingly, few screens address the impact of mechanical
trauma on brain cells, i.e., cell death [13,14,44]. After briefly
summarizing currently investigated candidate TBI markers,
we will evaluate challenges and alternatives in identifying
Part of the pathology of CNS injury is characterized by
secondary effects, including the inflammatory response
to TBI. Cytokines are key mediators in the process of
(neuro)inflammation  and increased concentrations
of these compounds have been associated with severe
CNS injury as well as post-traumatic hypoxia [46,47].
For example, elevated levels of interleukin-10 (IL-10), an
anti-inflammatory cytokine, was measured in low pg/mL
levels in CSF and low-mid pg/mL levels in serum
(Table 1) and correlated with severe TBI determined by
the GCS [46,48-50]. Higher Il-10 serum levels illustrate
the systemic nature of an inflammatory response. Such
responses are systemic in nature and not specific to TBI,
but occur with any insult, hence inflammatory markers
are not ‘pointing to brain injury’.
With their elongated axonal and dendritic processes,
neurons are exposed to shear forces associated with the
whiplash trauma of a concussion. Acute plasma mem-
brane permeablility, or mechanoporation, compromises
cell integrity and is linked to diffuse axonal injury in re-
sponse to a mechanical impact [71-73].
Tau protein is a member of microtubule-associated
proteins involved in maintaining cytoskeletal structure
and axonal transport. It is expressed by CNS neurons
and oligodendrocytes and found primarily in axons .
Traditionally used in the diagnosis of Alzheimer’s dis-
ease, elevated levels of Tau in CSF and serum have been
linked to CNS insults like TBI and stroke [62,75]. CSF
and serum studies of TBI patients have measured ele-
vated Tau protein concentrations in the 1000 ng/mL
range in young adult TBI patients, whereas it is three or-
ders of magnitude lower in neonates with brain insults
[59,65,66]. Because of Tau’s chronic accumulation after
various CNS insults, it seems less useful as an acute
head trauma marker.
Shen et al. Clinical Proteomics 2014, 11:11
Page 5 of 13
Mylein basic protein (MBP) is released with myelin
debris that accumulates with axonal damage in the injured
brain or spinal cord. MBP is one of three proteins com-
prising the myelin sheath essential for axonal impulse
conduction . MBP markers have shown promise in the
appraisal of TBI with serum levels in the low-mid ng/mL
range [77,78]. Similar to GFAP (vide infra), studies have
demonstrated degradation of MBP isoforms as a result of
Neuron Specific Enolase (NSE), Microtubule-associated
protein 2 (MAP-2) and ubiquitin C-term hydrolase L1
(UCH-L1) all display differential expression patterns in
TBI patients. NSE, a glycolytic enzyme isoform of neu-
rons, has been documented to increase following head
trauma , but has a slow elimination process, making it
difficult to distinguish between primary and secondary in-
juries . Additionally, NSE is released during the
process of hemolysis, making it difficult to pin down the
source of injury .
Microtubule-associated protein 2 (MAP-2) is a cytoskeletal-
associated protein localized to dendrites of neurons that
is believed to function in the growth and maturation of
dendrites as well as cytoskeletal organization . Previ-
ous studies have demonstrated that MAP-2 is absent from
damaged regions of the brain and that serum levels in-
crease early after injury . Mondello et al. assessed the
long-term release of MAP-2 in blood 6 months post
trauma by ELISA immunoassay and found that severe TBI
patients had significantly higher serums levels of MAP-2
compared to normal non-TBI patients. TBI patients in a
vegetative state, as assessed by the GCS, however, showed
no increase in serum MAP-2 versus controls. This suggests
that MAP-2 could provide insight into the mechanism of
neuronal remodeling as well as discriminate between
patients with deficits in consciousness and increased risk
of unfavorable outcomes .
Ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been
identified in a cell death culture assay and is verified by
ELISA to be significantly increased in TBI patients .
Neurodegenerative marker UCH-L1 fluid levels are also
elevated in ischemia, vasospasm, infarction, and carbon
monoxide poisoning [86-88]. UCH-L1 is a proteolytically
stable, abundant neuronal protein [69,70,85,89]. Future
studies will show whether these proteins would be present
in mTBI subjects without significant brain cell death.
Trauma specific breakdown products of neuronal and glial
Spectrin breakdown products (SBDPs) have been identi-
fied as potential TBI biomarkers in rat CSF fluid .
αII-Spectrin is the submembraneous cortical cytoskel-
eton of neurons and astroglia, sharing 50-59% homology
with the abundant erythroid α-spectrin [44,91]. Cell-
death associated spectrin fragments of molecular weight
150 kDa (SBDP150) and two N-terminal fragments at
145 kDa (SBDP145) and 120 kDa (SBDP120) cleaved by
calpain and caspase-3 have been identified in a cell death
culture model [87,92,93]. Using a sandwich ELISA meth-
odology, Mondello et al. showed both SBDP145 and
SBDP120 increased in patients post-TBI, with SBDP145
present immediately post-trauma and SBDP120 most
accurately measured 24 hr post-injury. SBDP CSF
levels greater than specific thresholds were shown to
correlate with poor outcome and mortality and the
temporal expression of SBDP for non-surviving pa-
tients differed from that of surviving patients. Thus, if
cross-reactivity and breakdown specificity is controlled,
Table 1 Candidate marker biofluid concentrationsa
Process or source,
Concentration in TBI biofluids (ng/mL)
IL-10 Inflammation 0.001-0.060 (children, )0.050-0150 
0.002-0.005 (adult, )
S100B Astroglia 1.0-15.0  0.01-0.70 [46,52,53]
GFAP Astroglia 9.0-22.0 0.14-15.0 
NFL/NFH/P-NFH Axon0.13-2.5 and 49–562 [56,57]NA
MBP Axon/oligodendrocytesNA 0.50-100.0 [46,58]
Axon Tau: 0.035-5.72 (neonate, )0.91- 5.1 [60,61]
1,519.6 – 2,308 (adult, [62-66])
Aβ 1–42: 1.17 (adult, 
NSE Neuron10-30 10-20 
UCH-L1Neuron 20-300 1.0-15 
α-spectrin-II BDP Neuron+astroglia 0.0-100  NA
MAP-2Neuron NA0.04-0.06 
aSelected examples of reported TBI markers for which some concentrations were found; NA – not available.
Shen et al. Clinical Proteomics 2014, 11:11
Page 6 of 13
SBDPs in CSF may aid to predict the severity of injury
and mortality .
Astroglia are the most abundant cells in the human
cerebral cortex  and respond to insult by becoming
reactive, a process that involves gene expression, mor-
phological changes, proliferation, and the formation of a
glial scar around lesions [95-99]. However, astrocytes are
also trauma victims as they are especially vulnerable to
acidosis, pressure elevation, and hypoxic/ischemic dam-
age, known co-morbidities of TBI [100-103]. Human as-
trocytes display very long thin processes that cross
through several laminae from the pia to the ventricular
walls, so called interlaminar processes and are hence
vulnerable to shear and deformation forces similar to
those that cause diffuse axonal injury in white matter
tracks [104,105]. Two of the most well studied TBI
marker candidates are S100β and glial fibrillary acidic
protein (GFAP), both glial proteins. S100β is a calcium
binding protein that is predominantly produced by as-
trocytes within the CNS. Because S100β is also produced
in a variety of non-CNS cells (e.g., lymphocytes, bone
marrow, adipocytes , and glia of peripheral nerves), brain
specificity is its problem . It has, however, been re-
ported that the few extracranial sources of S100β are
short lived compared to S100β from cerebral lesions
[107,108]. Elevated S100β concentrations have been
measured in the ng/mL and pg/mL range in TBI patient
CSF and serum, respectively [51,53]. Despite the imme-
diate spike in S100β levels, it has been found that S100β
measurements taken 24 hours post TBI offer the most
prognostic value for patient outcome due to initial inter-
ference from external S100β . S100β is released into
the perivascular space immediately following blood brain
barrier (BBB) compromise and may serve as a BBB-
permeability marker . Additionally, higher levels of
S100β have been correlated to patients suffering from
post traumatic hypoxia, demonstrating the interrelation
between secondary effects and amplified biomarker ex-
GFAP is an intermediate filament that is highly
enriched in CNS astroglia, but is also expressed in
Schwann cells and olfactory ensheathing glia of periph-
eral nerves [110-112]. GFAP levels are persistently ele-
vated after severe TBI in CSF and serum, relate to poor
outcome, and are predictive for mortality [54,55,113].
Serum levels of GFAP show high variability or no eleva-
tion after mTBI, yet reports are confounded by varying
delimitation of ‘mild’ as to include more moderate cases
with lesions and positive imaging signals or not. Thus
the discriminative power of GFAP as a mild neuro-
trauma biomarker is conflicted [56,114]. Measured CSF
levels of several biomarkers in boxers acutely after one
or repeated blows to the head as well as after 14 days,
revealed elevated levels of GFAP with large variations
among the boxers suffering a concussion . GFAP
breakdown products are found after TBI and are being
explored as insult-specific markers [114-116].
Strategies for addressing the challenges in identifying
and validating new TBI biomarkers
For a brain cell specific protein to be a trauma marker,
either it should be selectively expressed in response to
the trauma and then discharged into fluids, or cytosolic
proteins released solely from injured neurons and glia
with compromised membrane integrity or dying brain
cells [73,117]. A suitable study design to identify fluid-
derived trauma specific proteins would employ a tar-
geted proteomic screen on a defined trauma model. Ex-
perimental animal injury models were developed with
the effort to mimic human TBI as closely as possible
while underlying cellular and molecular mechanisms of
acute trauma are still scantly investigated. The predominant
criterion is to recapitulate the clinical manifestation of
TBI over studies using simplified reproducible trauma
models with the goal to determine primary cellular injury
consequences [4,5]. Most commonly used injury models
include focal injuries with the animal’s head in a fixed pos-
ition, like fluid percussion and controlled cortical impact,
which produce a focal contusion with hematoma and
hemorrhage while the dura remains intact [118,119]. Also
used is Marmarou’s weight drop model where distributed
forces cause diffuse injury with the animal’s head unre-
strained in a helmet and the brain is therefore subjected
to rotational forces as well [120,121]. Blast injury models
historically use shock tubes and larger animals, but have
been adapted recently to rodents as well as investigated
for milder blast effects from explosion exposures in the
Developing biochemical markers of TBI by proteomics
and mass spectrometry
Proteomic studies of injured brain or spinal cord tissue
are being done in these injury models and are providing
lists of protein changes that are difficult to interpret due
to the complexity of events at and around a dynamically
changing lesion site and variations between models
[39,40,42]. Injury zones are not reproducibly defined from
lab to lab as histopathological analyses have for long not
followed standardized analysis and reporting criteria .
Tissue derived protein signals are products of a changing
composition of viable, injured, and dead cells as well as in-
filtrating non-neural cells, that complicate the interpret-
ation of proteomic studies [97,99]. An effort has been
made in recent years to standardize and compare sever-
ities of commonly used TBI animal models across centers
. Defining common data elements for collection,
Shen et al. Clinical Proteomics 2014, 11:11
Page 7 of 13
analysis protocols, and reporting of fluid samples and
histopathological defining features of injury models will
help this field in interpreting proteomic and biomarker
preclinical studies as well as clinical data collection and in-
Proteomic TBI marker projects on biofluids using ro-
dent injury models have been few due to naturally limited
available fluid amounts [42,127], but biofluid neurotrauma
marker candidates have been studied in pig blast injury
models [128-132]. Human proteomic analyses have started
from severe trauma patient’s CSF and plasma from indi-
vidual patients [41,133]. Bioinformatics analysis tools are
expected to facilitate systems level understanding of neu-
rotrauma protein changes [134,135]. While bioinformatics
tools are indispensable for classification, consensus-based
data collection, and data mining, they will not make the
bottleneck of biomarker candidate selection much easier.
Hanrieder et al. describes a workflow using matrix-
assisted laser desorption ionization time-of-flight (MALDI-
TOF) MS/MS in conjunction with off-line nano-LC sample
fractionation . In their study, ventricular CSF samples
from 3 severe TBI patients displaying different symptoms
were taken at various time points post-trauma and ana-
lyzed by nano-LC MALDI-TOF MS/MS to determine
temporal protein expression changes. CSF samples were
digested with trypsin and labeled with isobaric tags for
relative and absolute quantitation using the iTRAQ
method [137,138]. Labeled tryptic digests were then sepa-
rated on a nano-flow LC system equipped with on-line
fraction collection capable of depositing fractions directly
onto MALDI sample plates for MALDI-TOF MS/MS-
based identification and quantification. Several proteins
were increased after injury. Additionally, relative quantifi-
cation using iTRAQ labeling revealed temporal changes in
protein expression for several inflammation-related pro-
teins (e.g., serum amyloid, fibroinogen alpha chain, cerulo-
plasmin) as well as known neurotrauma-related proteins
Due to the confounding complexity of clinical TBI and
clinic-resembling animal injury models, we propose a
targeted proteomic screen using a well-characterized
in vitro cell-based trauma model as a starting point for
TBI marker candidate identification [139-144]. This will
limit protein changes to those directly related to an
acute mechanical trauma by applying an abrupt pressure
pulse inflicting shear forces and deformation onto cor-
tical brain cells in a reproducible fashion at various se-
verities . We are finding robust cellular release
patterns that correlate with cell injury and cell death of
rodent and human astrocytes matured and stretched in
a prototype of this injury model  (Levine et al.,
submitted; manuscript in preparation). A suitable selec-
tion strategy needs to be applied to any trauma-release
protein list to eliminate proteins found in healthy
human plasma and to focus on brain-specific proteins
Verifying biochemical markers for TBI by proteomics and
One analytical challenge that is unique to TBI for meas-
uring candidate biomarkers lays in the unpredictably
fluctuating protein concentrations among CSF samples
from different TBI patients (low microgram/ml to sev-
eral mg/ml range). This maybe due to as variables such
as the patient’s varying blood–brain barrier integrity,
hemorrhage, brain cell protein leakage, as well as waves
of brain cell death. This is unlike healthy CSF or plasma
with constant and physiologically controlled protein
levels allowing for sample preparation with reproducible
protein amounts . These injury specific variables
can be addressed only by relating all measurements to
raw, unprocessed sample volume regardless of depletion
and other processing steps including optimizing protein
amounts for trypsin digestion or immunoassay applica-
tions. There are also injury-related but not-trauma specific
secondary changes in protein composition in trauma
CSF that could be caused by secondary infection due to
hospitalization that could reduce protein amounts or
bacterial proteins present in the samples. Such samples
should be omitted from an initial biomarker validation
The accepted “gold standard” of single-protein measure-
ments is the ELISA immunoassay, which takes advantage
of the specificity and diversity of IgG antigen recognition.
Yet, while ELISA is well touted for its high sensitivity
(~1 pg/mL), it is not without limitations . ELISA
methods rely on antibodies for protein detection and assay
development ideally uses two antibodies against different
epitopes of the candidate TBI marker. Non-specific bind-
ing of immunoglobulins to abundant plasma proteins may
contribute to a background problem, limiting the avail-
ability of suitable highly specific antibodies ideally from
different host animals to cancel out non-specific binding.
The availability of such antibody pairs often requires de
novo generation, lengthening the assay development time.
Thus, the lack of multiplex capacity may exclude using
the ELISA platform as initial validation tool of candidate
TBI markers in patient samples .
By not relying on antibody-antigen binding, quantitative
mass spectrometry is well suited to meet the challenge of
overcoming the verification bottleneck where immunoas-
says cannot be applied. MRM-MS is quickly becoming the
preferred method of candidate biomarker verification be-
cause of the discriminating power of mass analyzers to ac-
curately measure and quantify multiple specific proteins
within a single sample set. Specific peptide fragments (via
trypsin digestion) corresponding to the candidate proteins
are selected to act as stoichiometric representatives (or
Shen et al. Clinical Proteomics 2014, 11:11
Page 8 of 13
surrogates) within a complex patient CSF or blood sample.
The mass spectrometer (usually a triple quadrupole
analyzer) is then set to scan for the precursor peptide ion,
fragment the precursor in the collision cell, and then se-
lect for a specific precursor fragment (known as a transi-
tion). Because the mass spectrometer is not expending
resources scanning through all the ions within a complex
patient sample, the signal from less abundant peptides are
no longer being masked by highly abundant ions, partially
addressing the problems with high dynamic range limita-
tions. Additionally, MRM provides a more cost-effective
alternative for quantification compared to traditional
ELISA methods by using stable isotope-labeled internal
standards of the selective candidate peptides. Using the
method of isotope dilution , isotopically labeled pep-
tides are spiked into digested CSF or blood samples and
the relative peak heights between the endogenous peptides
and isotope-labeled peptides are used to quantify selected
candidate biomarkers. This approach has been greatly
aided by the increased availability of stable isotope-labeled
standard (SIS) peptides manufactured and sold by life sci-
ence companies . MRM-MS has long been a
method of choice for detecting marker metabolites for
amino acids, organic acids, and fatty acid disorders in
newborns . The success of these quantitative
methods in candidate biomarker discovery/verification
has been well documented in a variety of samples such
as synovial fluid , CSF , and plasma .
The MRM-MS platform is ideally suited to address the
challenge of validating several marker candidates at once
(multiplexing) and measuring their levels together with
candidate TBI markers reported in the literature. This is
in large part due to advances in in pre-analysis enrich-
ment methods  as well as improvements in both
sensitivity and speed of modern mass spectrometers that
allow for detection and quantitation in the low-mid ng/mL
concentration range. Hybrid orbitrap mass spectrometers
such as the Q-Exactive have demonstrated the ability to
detect up to 10 amol of heavy SIS peptides in the presence
of 10 ng- 1 ug of yeast tryptic digest background with up
to 10 ppm mass accuracy . Coupled with the high re-
solving power of orbitrap detectors (up to 140 K for the
Q-Exactive) and fast duty cycles to collect full MS/MS
spectra, these instruments should be able to confidently
identify surrogate peptides. When comparing the low cost
of SIS peptide generation from commercial sources to the
cost of antibody generation and capacity to multiplex
more than ten within a single analytical sample, the mass
spectrometry platform is a feasible choice for TBI candi-
date marker verification for the early preclinical validation
stage. Following this initial verification, antibodies will be
generated only for the most robustly detected TBI marker
candidates for ELISA assay development for future clinical
trials and diagnostic use.
Combining a targeted screen, a focused selection strat-
egy, and a stepwise approach from preclinical validation
towards clinical translation offers a feasible pipeline for
candidate TBI marker identification and preparation for
its diagnostic use. Validation through a stepwise increas-
ing sample cohort and moving from severe TBI CSF to
matching plasma samples and then to mTBI plasma
samples will provide verification where experimental
analyses and patient samples are matched with appropri-
ate positive controls along the way.
Moreover, the emergence of targeted MS-technologies
brings promise to the development of an efficient bio-
marker discovery to verification pipeline for TBI. This
pipeline could consist of the initial application of proteo-
mics technologies in the form of comparative 2D-PAGE
and shotgun LC-MS/MS to identify and discover candi-
date biomarkers from trauma and healthy subject sam-
ples. This is followed by the development of quantitative
MRM-MS to assess the biological significance of these
markers followed by clinical validation in a larger scale.
With the possibility of multiplexing using proteomic
methods such as MRM-MS, the time required for pre-
clinical verification can be reduced as tens of marker
candidate proteins can be monitored concurrently in
clinical samples. This process will help narrow the pool
of potential surrogates from which the most specific and
easily measured candidates can be chosen for clinical
validation and assay development.
The authors declare that they have no competing interests.
SS, RROL, IBW, and JAL reviewed the relevant literature. All authors
participated in the drafting of the manuscript, and all have read and
approved the final manuscript.
Support from the US National Institutes of Health (R21 NS072606 to IBW) is
1Department of Chemistry and Biochemistry, University of California-Los
Angeles, Los Angeles, CA 90095, USA.2Department of Biological Chemistry,
David Geffen School of Medicine at UCLA, University of California-Los
Angeles, Los Angeles, CA 90095, USA.3Molecular Biology Institute, University
of California-Los Angeles, Los Angeles, CA 90095, USA.4Semel Institute for
Neuroscience and Human Behavior, David Geffen School of Medicine at
UCLA, University of California-Los Angeles, Los Angeles, CA 90095, USA.
Received: 15 April 2013 Accepted: 10 February 2014
Published: 28 March 2014
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Cite this article as: Shen et al.: Addressing the needs of traumatic brain
injury with clinical proteomics. Clinical Proteomics 2014 11:11.
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