J Neuropathol Exp Neurol
10
Copyright Ó 2008 by the American Association of Neuropathologists, Inc.
REVIEW ARTICLE
Proteomics of Human Neurodegenerative Diseases
Jing Zhang, MD, PhD, C. Dirk Keene, MD, PhD, Catherine Pan,
Kathleen S. Montine, PhD, and Thomas J. Montine, MD, PhD
Abstract
The technology, experimental approaches, and bioinformatics that
support proteomic research are evolving rapidly. The application of
these new capabilities to the study of neurodegenerative diseases is
providing insight into the biochemical pathogenesis of neurodegen-
eration as well as fueling major efforts in biomarker discovery.
Here, we review the fundamentals of commonly used proteomic ap-
proaches and the outcomes of these investigations with autopsy and
cerebrospinal fluid samples from patients with neurodegenerative
diseases.
Key Words: Alzheimer disease, Cerebrospinal fluid, Mass spec-
trometry, Neurodegeneration, Parkinson disease, Proteomics.
INTRODUCTION
Proteomic technologies provide powerful means to sys-
tematically profile the peptide or protein constituents of com-
plex mixtures and, in some instances, to use these data to
impute protein identity, amount, or both. Although comple-
mentary to genomics, there are important differences between
gene expression profiling and protein profiling. Perhaps most
important is that it is now possible to interrogate essentially
the entire transcriptome, athough there is no comparably com-
prehensive approach to the proteins encoded therein. This may
be viewed by some as sufficient justification to concentrate
on the transcriptome; however, the quantitative concordance
between message and corresponding protein is surprisingly
poor (1Y3). Moreover, the complex and rich range of chem-
ical modifications that occur to proteins after translation are
largely transparent to genomics but are coming into pro-
gressively greater focus in proteomics. Another important
consideration in the context of this review is the relative sta-
bility of protein versus messenger RNA in samples of brain
obtained from autopsy.
Human neurodegenerative diseases range from rare to
common illnesses. Indeed, some such as Alzheimer disease
From the Department of Pathology, University of Washington, Seattle,
Washington.
Send correspondence and reprint requests to: Thomas J. Montine, MD, PhD,
Department of Pathology, University of Washington, Harborview
Medical Center, Box 359791, Seattle, WA 98104; E-mail: tmontine@
u.washington.edu
This work was supported by Grants AG05136 and AG029808 from the
National Institutes of Health, by the Michael J. Fox Foundation for
Parkinson’s Research, by the C.-M. Shaw endowment, and by the Nancy
and Buster Alvord Endowment.
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Vol. 67, No.
October 2008
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(AD) and Parkinson disease (PD) pose serious public health
challenges that will increase in the coming decades. Many
discoveries have been made in the genetic causes and risk
factors for several neurodegenerative diseases (4); however,
there are relatively few proteomic studies of neurodegener-
ative diseases to date. The major foci of these investigations
have been analysis of protein from autopsy samples in studies
of pathogenesis and from cerebrospinal fluid (CSF) in the
pursuit of biomarkers. It is important to be aware of some
limitations of these initial proteomic studies of human neu-
rodegenerative diseases. Many are limited to comparisons
between 1 diseased group and controls and therefore cannot
address the important issue of disease specificity. This lim-
itation has been approached by some more recent inves-
tigations reviewed later. Another limitation has been a focus
on changes observed during clinically diagnosed stages of
disease, whereas some recent studies have begun to explore
alterations that accompany preclinical disease stages.
FUNDAMENTALS OF PROTEOMICS
Shared Features of Different Proteomic
Techniques
Although it is difficult to summarize such a dynamic and
rapidly changing scientific area, the various approaches to
protein profiling share common features. These include
sample preparation, protein or peptide separation, mass spec-
trometry (MS), and bioinformatic data processing, as well as
subsequent confirmation and validation. For the sake of clar-
ity, we will use validation to mean replication of findings by
alternative method(s) in the same samples used in profiling
and confirmation to mean replication of findings in inde-
pendent samples.
Sample preparation is a major source of variation in the
outcomes of proteomic experiments. Again, proteomics dif-
fers from genomics in that it is not yet possible to analyze
virtually all proteins in a cell, tissue, or body fluid compre-
hensively, especially in samples with wide ranges of protein
concentration. Consequently, the first step in all proteomic ex-
periments is preparation of a subproteome, or a subset of pro-
teins, that is defined by the physical or chemical means used
to isolate it from all proteins in tissue or body fluid. Examples
to follow have generated subproteomes using gradient de-
tergent extractions to enrich for hydrophobic or membrane-
bound proteins, affinity purification, immunoprecipitation,
and laser capture microdissection (Fig. 1). Typical sub-
proteomes are still complex protein mixtures that require
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Zhang et al J Neuropathol Exp Neurol  Volume 67, Number 10, October 2008
FIGURE 1. Diagram of the steps in typical proteomic experi-
ments that use liquid chromatography (LC) or 2-dimensional
gel electrophoresis (2-DE) followed by tandem mass spectrom-
etry (MS-MS). Protein identification (ID) is imputed by bioin-
formatic tools that compare tandem MS data with protein or
genomic databases.
further separation. Classically, proteins are separated by 2-
dimensional gel electrophoresis (2-DE), and spots detected
on the gel by a variety of means are excised and digested
to peptides, typically with trypsin. Alternatively, the protein
mixture in the subproteome is first digested to peptides, and
these are then separated by single or multidimensional liquid
chromatography (LC).
Mass spectrometry is an elegant technique for detecting
the mass of charged species. There are many excellent reviews
devoted to MS. Here we summarize the basics. The first step in
MS is to ionize and vaporize the analytes (i.e. proteins or their
[typically] tryptic peptide fragments) without significant frag-
mentation. This can be achieved by a variety of methods: 2
commonly used approaches are matrix-assisted laser desorp-
tion ionization (MALDI) and electrospray ionization (ESI).
Matrix-assisted laser desorption ionization mixes the analyte
with 1 of several different molecules that facilitate ionization
and vaporization. Generating peptide ions from ESI is com-
plex and is thought to involve a process called Coulombic
fission. The peptide or protein ions are then introduced into
the mass analyzer. Again, there are different types including
ion trap, triple quadrupole, time-of flight (TOF), and Fourier
transform ion cyclotron that differ in their mechanisms of ion
separation, mass accuracy, and resolution. It is critical to ap-
preciate that some, but not all, common proteomic techniques
use tandem MS (MS-MS) to analyze the daughter or fragment
ions of the parent ions whose mass was determined in first MS
dimension. In these collision-induced dissociation tandem MS
experiments, the energy applied greatly favors fragmentation
of the peptide (amide) bond over amino acid side chains in the
parent ion, thereby generating an ensemble of daughter ions,
called b ions and y ions, from which the amino acid sequence
can be deduced (Fig. 2). Detectors and multipliers generate
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the signal that is typically plotted as the total ion chromato-
gram for the first MS dimension, with the x axis being time
and the y axis being relative abundance, and mass-to-change
ratio (m/z) chromatogram for the daughter ions.
The next step is synthesis of the wealth of mass spec-
tral data obtained. A major step forward in the evolution of
proteomics was the development of bioinformatic tools to
interpret the ever increasing rich data sets generated from
advancing technology. Here, the pivotal event was the devel-
opment of search engines, such as SEQUEST and MASCOT,
which correlate actual tandem mass spectra with predicted
tandem mass spectra based on species-specific amino acid
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sequences from protein or genomic databases (5, 6). It is
important to appreciate that these different programs do not
perform in exactly the same way and can produce different
peptide matches and levels of confidence. Moreover, the da-
tabases currently searched are incomplete and imperfect. It is
for this reason that all results require validation.
Specifics of Different Proteomic Techniques
Classically, experiments isolate a subproteome of in-
terest, separate the protein by 2-DE, excise the spot of interest
from the stained gel, digest the proteins contained in each
spot with trypsin, and then perform tandem MS with MALDI
TOF-TOF. This powerful approach is still extensively used,
although 2-DE is labor intensive and has a relatively low dy-
namic range. Some of these issues have been alleviated
partially by advances in technology, especially the use of fluo-
rescent dyes. Proteins in a control sample can be labeled with
1 fluorescent dye and proteins in an experimental sample la-
beled with a different fluorescent dye. The samples are then
mixed, separated by 2-DE, and the relative fluorescent inten-
sities are measured within the spot of interest before digestion
and tandem MS. In this way, analysis of fluorescent intensity
provides relative quantification, and tandem MS provides
protein identification for the spot of interest in the gel.
One response to the time and labor devoted to spot
picking was to develop multidimensional LC systems for the
separation of peptides that are coupled to tandem MS. In a
typical experiment, the subproteome is first digested to pep-
tides, and these are separated by LC and then directly ionized
by ESI followed by tandem MS. Alternatively, the separated
peptides are spotted directly onto a MALDI plate followed by
tandem MS. An advantage of these approaches is that they
can be much more automated than the 2-DE method; how-
ever, this comes with a price. The major one is that without
the gel and fluorescent dyes, there are no quantitative data.
Indeed, it is critical to appreciate that the size of the resulting
peptide ion peaks does not necessarily bear any quantitative
resemblance to the amount of protein in the subproteome; the
major sources of variance are ionization efficiency and de-
tector sensitivity. Several groups have ingeniously addressed
this shortcoming of high-throughput proteomics by adapting
a classical MS technique: the stable isotope dilution assay. In
general, stable isotope labeling coupled with MS is more
quantitatively accurate than are comparisons of spot densities
on 2-DE. All of these techniques require the introduction of
a stable isotope label, usually deuterium or 13C into samples
that are then mixed and analyzed simultaneously by MS. The
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FIGURE 2. Typical collision-induced dissociation tandem mass spectrometry data that show total ion chromatogram (time vs
relative abundance) in the upper panel. The selected ion marked by the red line in the total ion chromatogram is subjected to
further fragmentation to yield characteristic daughter ions in the lower mass-to-change ratio (m/z) chromatogram.

isotopically labeled peptide serves as a mass signature for
sample source and as the foundation for estimation of relative
abundance, and in some instances, the absolute concentration
of the ionized peptides. There are several approaches to the
introduction of isotope labels to proteins or peptides; among
these are isotope-coded affinity tags (7) and isobaric tags for
relative and absolute quantification (iTRAQ) (8). Isotope-
coded affinity tag was one of the earlier approaches and was
limited to labeling cysteinyl thiolates with light (protonated)
or heavy (deuterated) probes (Fig. 3). Isobaric tags for rela-
tive and absolute quantification now permits differential iso-
topic labeling of ?-amino lysyl groups with up to 8 different
probes. A bioinformatics approach similar to that previously
described is used for imputation of protein identity from
peptide ion data. Specialized computer software is needed
to integrate isotopic data from multiple peptides to estimate
protein concentrations. Newer methods for quantification
without isotope labels are an area of active investigation (9).
A third approach used in proteomics of neurodegener-
ative diseases is surface-enhanced laser desorption/ionization
(SELDI), also called SELDI-TOF. Surface-enhanced laser de-
sorption/ionization is a variant of MALDI that is commonly
used in biomarker discovery. There are few very attractive
features of SELDI (10). First, proteins or peptides can be
separated or enriched through a variety of plates or chips with
immobilized biochemically active materials that preferen-
tially bind proteins based on hydrophobicity, charge interac-
tions, chelation, and other characteristics. In addition, SELDI
plates can be activated with immobilized organics that can
covalently bind antibodies, other proteins such as receptors,
and even nucleic acids. After the subproteome has been in-
cubated with the desired plate, the poorly bound proteins or
peptides are washed away, thereby acting like affinity chro-
matography. The versatility of agents that can be used at this
step is a clear strength of SELDI. Another advantage is that
SELDI is relatively simple and quick compared with other
proteomic techniques. A disadvantage of SELDI as it is com-
monly used is that only a single MS dimension is used and
there is, therefore, no attempt to impute protein identity.
Rather, the typical experiment deals with finding those ions
that have optimal characteristics and then using other tech-
niques to identify the protein from which the selected ions
are derived. Finally, given the nature of the SELDI experi-
ment, it typically is not coupled with stable-isotope dilution
techniques; therefore, reproducibility and quantification are
problematic (11).
These are not the only approaches to MS. One example,
an elegant technique of particular interest to pathologists, is
tissue-based MS that can profile proteins in situ from different
regions within a tissue specimen (12). The methods previ-
ously described, however, are those that have been used to
date to investigate human neurodegenerative diseases.

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FIGURE 3. Two subproteomes were generated from human CSF: one was labeled with protonated isotope-coded affinity tags
(ICAT) reagent and the other with deuterated ICAT reagent. They were then combined before tandem mass spectrometry. The
mass-to-change ratio (m/z) chromatogram shows the relative abundance of ions conjugated to the light (protonated) versus
heavy (deuterated) ICAT reagent.

Identifying Protein Modifications
One approach to evaluating protein modifications is in
preparation of the subproteome. For example, some investi-
gators have focused on only those spots from 2-DE that have
reactivity for protein carbonyls (vide infra). Alternatively, an
antibody that recognizes only a certain posttranslational mod-
ification may be adhered to a SELDI plate. Another approach
has been to analyze tandem MS data for apparent mass shifts
not predicted by the peptide sequence. There are several com-
puter programs that perform such searches. Two contrasting
approaches are SEQUEST and MASCOT versus P-MOD and
InsPecT. The SEQUEST and MASCOT search for specified
mass shifts on specified amino acids, for example +16 on M
for methionine sulfoxide, across all peptide ions detected. In
contrast, P-MOD and InsPectT search the same spectral data
in a different manner (13, 14). Here, one provides the pro-
grams with the amino acid sequence of a protein of interest,
and they list all unexpected masses and a calculated level of
confidence.
PROTEOMIC STUDIES OF
NEURODEGENERATIVE DISEASES FROM
AUTOPSY BRAIN
In this section, we review proteomic studies of autopsy
samples from patients with neurodegenerative diseases. Al-
though there are several excellent proteomic investigations of
corresponding animal models, we limited our comments to
investigations of human tissue and grouped them by the
method of subproteome preparation.
Homogenates of Human Brain Regions
Four studies have used 2-DE followed by tandem MS
to investigate detergent-extractable proteins in brain regions
of patients with AD, and in 1 case, patients with Down syn-
drome. One study identified 37 proteins with altered levels in
AD (15); 11 of these had increased levels in AD and were
identified by investigators by tandem MS. No validation was
performed. Using a similar approach but not the same ex-
traction protocol, another study identified 15 proteins, the
levels of which differed in different AD brain regions (16).
Again, no validation was performed. Agreement between
these 2 similar studies is low; only increased levels of glyc-
eraldehyde 3-phosphate dehydrogenase (GAPDH) and syn-
aptotagmin I in AD were confirmed. A third similar study
identified significantly decreased levels of mitochondrial com-
plex III core protein 1 (17). This finding was not validated. A
fourth study of frontal cortex focused on 9 antioxidant pro-
teins and identified significantly increased peroxiredoxin 2
from AD patients compared with controls; parallel studies
showed that peroxiredoxin 3 levels were decreased in frontal
cortex from Down syndrome and Pick disease but not AD
patients (18).
Laser Capture Microdissection of
Hallmark Structures
There have been 3 reports of proteomic characteri-
zation of hallmark structures of neurodegenerative diseases
(i.e. amyloid plaques and neurofibrillary tangles [NFTs] from
AD and Lewy bodies [LB] from patients with dementia with
LBs [DLB]) that were enriched by laser capture microdis-
section from human brain samples. Amyloid plaques were
localized by thioflavin-S staining, microdissected, and then
extracted with detergent; NFTs and LBs were identified by
immunohistochemistry (tau-2 for NFTs, >-synuclein [SNCA]
for LBs) and solubilized with formic acid; all were then
separated with LC followed by MS-MS. A total of 488 pro-
teins were identified from laser capture microdissection of
amyloid plaques; several of these were validated by immu-
nohistochemistry. Of these, 26 proteins were enriched in
captured amyloid plaques compared with areas without am-
yloid. Of particular interest was the identification of dynein
heavy chain in both human postmortem amyloid plaques
as well as in plaques obtained from APPswe/PS1$E9 trans-
genic mice (19).
Seventy-two proteins were identified in NFTs obtained
by laser capture microdissection by 2 or more unique pep-
tides, of which 63 had no previously known association with

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J Neuropathol Exp Neurol  Volume 67, Number 10, October 2008
NFTs (20). One of these proteins, GAPDH, localized to
NFTs by immunohistochemistry, was validated in the
detergent-insoluble fraction from hippocampus of patients
with AD and was confirmed by coimmunoprecipitation with
PHF-tau. Coincident with this publication was the observa-
tion by others that polymorphisms of GAPD may be genetic
risk factors for late-onset AD (21). This finding has been cor-
roborated by others, although the overall effect may be small
(22). Others have subsequently shown that GAPDH localizes
to LBs (23), can be oxidized and aggregated by exposure to
AA peptides (24), and has increased expression in the hip-
pocampus of AD patients compared with controls (25).
Traditional biochemical or histochemical methods
previously identified approximately 70 molecular compo-
nents of LBs in both the brainstem and cerebral cortex (26);
SNCA has been identified as one of the major constituents. A
recent study used laser capture microdissection of SNCA-
immunoreactive cerebral cortical LBs from patients with
DLB coupled with LC/ESI/MS/MS. This platform identified
156 protein with 2 or more unique peptides; of these, 17 had
previously been associated with cerebral cortical or brainstem
LBs (27). One particular protein, heat shock cognate 71 was
previously associated only with brainstem LBs.
Insoluble Proteins in Cerebral Cortex
Accumulation of insoluble proteins is a defining char-
acteristic of several neurodegenerative diseases, including
AD and PD (28, 29). Indeed, it was the identification of
detergent-insoluble protein such as AA and paired helical
filament tau that ushered in the modern era of neurodegener-
ative disease research (30, 31). Conversion to insoluble forms
disrupts protein function, can lead to further cellular damage,
and may precede the formation of the hallmark inclusions
(32). To gain insight into disease pathogenesis, we have
focused modern proteomic techniques on this class of
pathological protein (33). We used LC-MS-MS and identified
in the detergent-insoluble fraction of late-onset AD temporal
cortex samples 125 proteins by 2 or more unique peptides
that included several proteins critical to AA production, com-
ponents of synaptic scaffolding, and products of genes linked
to an increased risk of late-onset AD. Each of 15 candidates
were validated by Western blot including GAPDH (33), and
the levels of several are altered in hippocampi of AD patients
compared with controls (25). AA, tau, and 7 of 8 other newly
identified detergent-insoluble proteins were confirmed to be
increased in temporal cortex by Western blot, not only from
patients with late-onset AD but from patients with AD as-
sociated with mutations in PSEN1 and PSEN2. All of these
except tau were elevated in individuals with mild cognitive
impairment, whereas none except AA were elevated in aged
APPswe mice. These results extend the amyloid hypothesis
for AD to include widespread protein insolubility that is not
exclusive to AA, even early in the course of AD before the
onset of dementia.
We have used iTRAQ labeling combined with MALDI-
TOF-TOF to analyze the detergent-insoluble proteome in
frontal cortex from 4 groups of individuals: patients with late-
onset AD and their controls from mainland United States and
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Proteomics in Neurodegeneration
Chamorros from Guam who had Parkinson-dementia complex
or their controls (Fig. 4). In addition to the expected increase
in abnormal frontal cortical AA peptides, tau, ubiquitin, and
apolipoprotein E (apoE) in AD, and tau in Parkinson-
dementia complex, we identified SNCA as a major detergent-
insoluble protein in Parkinson-dementia complex but not AD
despite a lack of LBs in corresponding tissue samples (34).
The proteomic findings were confirmed by ELISA in frontal
and temporal cortices in which SNCA levels in Parkinson-
dementia complex patient samples were quantitatively similar
to patients with DLB and echo earlier findings of abnormal
SNCA immunohistochemical findings in some patients with
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Parkinson-dementia complex (35Y38).
Modified Proteins
Substantial antibody-based evidence supports accumu-
lation of proteins that are pathologically modified by oxi-
dative or nitrative reactions in several neurodegenerative
diseases, including AD. One limitation of these earlier studies
has been the difficulties in identification of the modified pro-
teins. Proteomic techniques provide approaches to investigate
this facet of AD pathogenesis.
Several elegant studies have combined immuno-
chemical detection of protein carbonyls (a form of protein
oxidation) in 2-DE followed by tandem MS for protein iden-
tification from soluble extracts of AD cerebrum. Proteins path-
ologically oxidized in AD include glutamine synthase, creatine
kinase BB, UCHL1, >-enolase, and DRP-2/CRMP2 (39, 40).
At least 3 of these oxidized proteins validated earlier results,
including those on UCHL1-validated earlier results (41).
A similar approach coupled immunologic detection of
nitrotyrosine residues with tandem MS to identify >-enolase,
triosephosphate isomerase, and neuropolypeptide h3 as tar-
gets for this type of pathological modification in AD cerebral
cortex (40, 42). Moreover, the authors extended their inves-
tigations to autopsy samples from individuals who died with
FIGURE 4. Experimental approach for isobaric tags for
relative and absolute quantification labeling of 4 samples.
MS, mass spectrometry.
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Zhang et al J Neuropathol Exp Neurol  Volume 67, Number 10, October 2008
amnestic mild cognitive impairment (MCI), a clinically de-
fined condition that largely represents prodromal AD. They
showed that modified proteins in amnestic MCI partially
overlap with those identified in patients with dementia from
AD (43, 44).
One group used affinity-purification with the MC1
monoclonal antibody to isolate a soluble fraction of PHF-tau
from AD brain (45). In addition to identifying a large number
of phosphorylated sites, they found that soluble PHF-tau is
ubiquitin conjugated within its microtubule-binding domain
at residues Lys-254, Lys-311, and Lys-353. Polyubiquitin
conjugates formed mostly at Lys-48 of ubiquitin, suggesting
a failure of the ubiquitin-proteasome system.
Another study investigated methionine oxidation to its
sulfoxide in neuron-specific A-III tubulin and AA peptides
(46). We used P-MOD to identify and map posttranslational
modifications using tandem MS data from detergent-insoluble
protein fractions from AD temporal cortex. Our results con-
firmed the direct observations of others by identifying me-
thionine 35 sulfoxides in AA peptides and numerous sites of
tau phosphorylation. P-MOD mapped several abundant
methionine sulfoxides to neuron-enriched A-III tubulin but
not >-III tubulin, its heterodimeric partner. These findings
point to oxidative modification of A-III tubulin as a potential
contributor to the neuronal cytoskeletal disruption in AD.
Lewy Body Disorders
Lewy body disorders are a recent consensus classifica-
tion of diseases that share the histopathologic hallmark of LB
formation (47). This includes several neurodegenerative
diseases, the most common of which are PD and DLB. We
previously described the proteomic analysis of laser capture
microdissected LBs from cerebral cortex of patients with
DLB. Basso et al (48) examined protein levels in the sub-
stantia nigra pars compacta from 4 patients who died of PD
and 4 controls using 2-DE followed by MALDI-TOF-TOF.
They identified 44 proteins in their subproteome extracted
with urea and detergent. Nine of these showed abundant
changes between the 2 groups; several were mitochondrial
and reduced oxygen species-scavenging proteins. A recent
study compared 3 subcellular fractions of frontal cortex from
4 groups: control individuals and patients with PD who had
brainstem, limbic, or frontal cortex LB formation (49). Each
sample was labeled with specific iTRAQ reagent before anal-
ysis by MALDI TOF/TOF. A total of 1,864 nonredundant
proteins were identified; 199 of these displayed significant
changes in relative abundance between PD groups and con-
trol. One particular mitochondrial protein, mortalin, was fur-
ther validated by Western blot and found to be decreased
in all PD groups as well as in a cellular model of PD (50).
Mortalin seems to have several activities, including molec-
ular chaperoning, mitochondrial import, and energy gener-
ation, and response to oxidative stress (51).
Other Neurodegenerative Diseases
Although there are several excellent studies of murine
and cellular models of amyotrophic lateral sclerosis (ALS),
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Huntington disease, and prion diseases, we are unaware of any
that has examined human brain or spinal cord from patients
with these diseases.
PROTEOMIC STUDIES OF HUMAN CSF
Cerebrospinal fluid is widely considered an imperfect
but relatively accessible portal into the neurochemical changes
that accompany neurological diseases (52Y57). Over the past
few years, several groups have used proteomics to charac-
terize the human CSF proteome and to profile CSF bio-
markers related to a few major neurodegenerative diseases,
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such as AD, PD, and ALS (58Y67). Together, these studies
have generated lists of protein candidates associated with
each disease that are altered in relative abundance within
CSF. Major discrepancies have also been noted among dif-
ferent groups of investigators that are likely related to several
factors especially preparation of the subproteome.
CSF Sample Collection and Storage
Human CSF derives from choroid plexus and the
extracellular fluid of the brain and spinal cord (68); the vol-
ume of approximately 140 to 150 mL in an adult is renewed
every 6 to 8 hours (69, 70). It has a rostrocaudal protein
concentration gradient that is about 2.5-fold (71). Moreover,
CSF production rate varies throughout the day and is altered
by aging and some medications (72, 73). Thus, CSF provides
a temporally limited window into the neurochemical activity
of the CNS that is confounded by regionally varying mixture
with a transudate of plasma, circadian variation, changes with
age, and alteration by medications. Great care must obviously
be taken, therefore, to match fractions of CSF obtained from
individuals for study.
In addition to physiological variables, contamination
with blood during lumbar puncture is another critical factor.
The CSF protein content is approximately 1/200th that of
blood, yet the protein profile overlaps extensively with blood
(74Y76). As a result, even minute contamination with blood
can have large effects on the concentrations of CSF proteins.
Several methods are available to test for trace contamination
of blood in CSF samples, such as red blood cell density and
measuring concentrations of exclusive blood proteins in CSF;
one such protein is apoB (77). Because it is extremely dif-
ficult to obtain CSF samples without some blood contami-
nation, we consider CSF with more than 10 red blood cells
per microliter or an apoB serum: CSF concentration ratio less
than 6,000 unacceptably contaminated by blood (58, 61, 77).
Using relative apoB concentration is particularly helpful with
samples that have been previously sedimented.
Although a seemingly trivial and straightforward pro-
cedure, CSF sample storage can significantly influence
sample integrity. Protein degradation can occur, particularly
when samples are kept at j20-C or have undergone multiple
freeze-thaw cycles; this may be especially problematic for
cystatin C (52, 78). Another unsettled issue related to CSF
storage is the addition of protease inhibitors (79, 80). Cur-
rently, no recommendation has been made on this issue by
the Human Proteome Organization.
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J Neuropathol Exp Neurol  Volume 67, Number 10, October 2008
Characterization of CSF Proteome
Several laboratories are using tandem MS to define the
human CSF proteome. Early experiments used CSF and 2-DE
and identified a few hundred proteins (81Y84). The number
of identified CSF proteins has increased exponentially since
the application of LC-based proteomics (58, 61Y63, 85Y87).
Indeed, with improvements in chromatography and MS, the
human CSF proteome has been expanded to more than 2,000
proteins (88).
CSF Proteins in Neurodegenerative Diseases
Characterization of the human CSF proteome is 1 step
toward identifying protein profiles that can aide in diagnosis
and monitoring progression of neurodegenerative diseases.
Several groups are pursuing this goal using a variety of
platforms.
Surface-Enhanced Laser Desorption/Ionization
Several SELDI-based studies have focused on AD.
Carrette and colleagues (52) have used SELDI to com-
pare AD patients with controls and have identified several
unique ion peaks that can differentiate disease from con-
trols groups. Several of these putative AD biomarkers have
been subsequently purified and identified as cystatin C, A2-
microglobulin isoforms, to name a few. Notably, however,
cystatin C is not only prone to freeze/thawing artifacts (78),
but also seems to be nonspecific to AD as it also is altered in
patients with Creutzfeldt-Jakob disease (89), multiple scle-
rosis (90), and chronic pain (91). One study used SELDI to
investigate CSF from patients with AD or frontotemporal
dementia versus controls; some peaks were discriminating,
but no proteins were identified (92). Another study used
SELDI analysis of CSF from 113 patients with MCI who
were followed for up to 6 years and 28 controls who were
followed for 3 years after lumbar puncture (93). These in-
vestigators discovered a panel of 17 candidate biomarkers
that could distinguish between patients with stable MCI and
patients with MCI who progressed to AD; however, the iden-
tity of most of these proteins is not yet known.
Two studies have reported SELDI-based identification
of CSF biomarker candidates for ALS. One study identified
30 ion peaks with statistically significant differences between
controls and patients with ALS. Transthyretin and cystatin
C were identified and validated to be decreased in ALS CSF,
whereas the carboxy-terminal fragment of the neuroendo-
crine protein 7B2 was increased in CSF from ALS patients
(94). In another study, 3 candidates were identified by SELDI
as being significantly lower in CSF from patients with ALS
(n = 36) compared with controls (n = 21). These findings were
validated in a separate set of samples, and 2 candidates were
identified as cystatin C and a peptic fragment of neuro-
secretory protien VGF (66).
We are aware of 1 study that used SELDI to investigate
CSF from patients with frontotemporal dementia (n = 16)
versus controls (n = 12). Ten ion peaks with good signal-
to-noise ratio and resolution were significantly differentially
expressed in frontotemporal dementia; 5 were increased
and 5 were decreased. Five of the ion peaks were purified
and identified by tandem MS as a fragment of neurosecretory
Ó 2008 American Association of Neuropathologists, Inc.
Copyright @ 2008 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
Proteomics in Neurodegeneration
protien VGF, transthyretin (TTR), conjugated TTR, cystatin
C, and a fragment of chromogranin B (95). Another group
also has noted the potential for the relative concentrations of
conjugated TTR in CSF to discriminate between AD and
controls (96).
Two-Dimensional Gel Electrophoresis
Tandem MS
Five major studies have used 2-DE coupled with tan-
dem MS in the search for CSF biomarkers for AD. Castano
et al (97) examined pooled CSF samples obtained from neu-
ropathologically confirmed AD and nondemented control
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subjects (n = 43 for both groups) and identified 5 differ-
entially expressed proteins in AD versus controls. Davidsson
et al (59) identified 6 proteins and their isoforms that were
significantly altered in CSF of AD (n = 15) patients versus
controls (n = 12). Hu et al (65) discovered 11 proteins or
isomers that are altered significantly in AD patients (n = 2)
versus controls (n = 4). Finehout et al (64) reported a panel of
23 spots (21 that have been identified) that could be used to
differentiate AD (n = 34) from non-AD (n = 9 normal
controls and n = 24 other diseases) with high sensitivity and
specificity. Puchades et al (60) compared samples from AD
patients and normal control subjects (n = 7 for both groups)
revealing 9 proteins to be significantly altered in AD. Only
10 proteins were identified in 2 or more of these studies.
These include albumin precursor, apoA1, apoE, >-1A gly-
coprotein, >-1 antitrypsin (or precursor), A-2 microglobulin,
complement component 3, prostaglandin D synthase, retinal
binding protein, and TTR.
Liquid Chromatography-Tandem MS
A third approach is multidimensional LC followed by
tandem MS. One group used this technology coupled with
isotope-coded affinity tags labeling to investigate specific
changes in the proteome associated with aging (61) or AD
(62) and identified approximately 400 CSF proteins, many
of which had altered relative abundance in AD. The same
groups subsequently used iTRAQ labeling to compare the
CSF proteome of patients with AD, PD, DLB, and age-
matched controls. More than 1,500 CSF proteins were iden-
tified; of those, 136, 72, and 101 proteins seemed to be
uniquely associated with AD, PD, and DLB, respectively
(58). Although these studies validated several of their
findings, only a very small fraction of candidate biomarkers
has been confirmed across platforms, underscoring the need
for testing the use of proteomics-discovered candidates by al-
ternative means in large-scale clinical studies.
Multianalyte Profile
We extended our iTRAQ proteomics discoveries by
developing a multianalyte profile (MAP) for the 8 best-
performing biomarker candidates and then conducted an in-
dependent clinical study using CSF from 95 control subjects,
48 patients with probable AD, and 40 patients with probable
PD. Our 8-member MAP agreed with expert diagnosis for
95% of controls, 95% of probable PD, and 75% of probable
AD. This MAP (in decreasing order of contribution to clas-
sification) consisted of tau, brain-derived neurotrophic factor,
929
Zhang et al J Neuropathol Exp Neurol  Volume 67, Number 10, October 2008
interleukin 8, AA42, A2-microglobulin, vitamin D-binding pro-
tein, apoAII, and apoE. This first large-scale clinical appli-
cation of a proteomic-discovered MAP suggests a panel of
8 CSF proteins that are highly effective at identifying PD but
only modestly effective at identifying AD (98). The MAPs
for other candidate CSF biomarkers are pending. Finally,
MAPs are not limited to antibody-based platforms. For ex-
ample, MS-based MAPs for biomarker confirmation or vali-
dation are under active investigation (99). This new platform
stands out for its precision without a requirement for
antibodies.
CONCLUSIONS
The technology, experimental approaches, and bioinfor-
matics that support proteomic research continue to undergo
rapid evolution. The application of these new capabilities to
the study of neurodegenerative diseases is providing insights
into the biochemical pathogenesis of neurodegeneration as
well as fueling major efforts in the discovery of biomarkers.
Experience gained so far combined with expected advances in
the near future will lead to greater throughput, accuracy, and
reproducibility. Recent studies previously cited and additional
future studies will focus on the critically important issues of
disease specificity and changes in the proteome that occur
during latent and prodromal stages of disease. Ultimately, the
convergence of validated and confirmed data from multiple
laboratories will establish richly informative consensus sub-
proteomes for human brain regions and CSF in health and
disease.
REFERENCES
1. Ideker T, Thorsson V, Ranish JA, et al. Integrated genomic and pro-
teomic analyses of a systematically perturbed metabolic network.
Science 2001;292:929Y34
2. Johnson MD, Yu LR, Conrads TP, et al. Proteome analysis of DNA
damage-induced neuronal death using high throughput mass spectrom-
etry. J Biol Chem 2004;279:26685Y97
3. Griffin TJ, Gygi SP, Ideker T, et al. Complementary profiling of gene
expression at the transcriptome and proteome levels in Saccharomyces
cerevisiae. Mol Cell Proteomics 2002;1:323Y33
4. Tsuang DW, Bird TD. Genetics of dementia. Med Clin North Am 2002;
86:591Y614
5. Eng JK, McCormack AL, Yates JR. An approach to correlate tandem
mass-spectral data of peptides with amino-acid-sequences in a protein
database. J Am Soc Mass Spectrom 1994;5:976Y89
6. Fenyo D, Beavis RC. A method for assessing the statistical significance
of mass spectrometry-based protein identifications using general scoring
schemes. Anal Chem 2003;75:768Y74
7. Gygi SP, Rist B, Gerber SA, et al. Quantitative analysis of complex
protein mixtures using isotope-coded affinity tags. Nat Biotechnol 1999;
17:994Y99
8. Ross PL, Huang YN, Marchese JN, et al. Multiplexed protein quan-
titation in Saccharomyces cerevisiae using amine-reactive isobaric tag-
ging reagents. Mol Cell Proteomics 2004;3:1154Y69
9. Haqqani AS, Kelly JF, Stanimirovic DB. Quantitative protein profiling
by mass spectrometry using label-free proteomics. In: Starkey M,
Elaswarapu R, eds. Genomics Protocols, vol. 439. Totowa, NJ: Humana
Press, 2008;241Y56
10. Dijkstra M, Vonk RJ, Jansen RC. SELDI-TOF mass spectra: A view on
sources of variation. J Chromatogr B Analyt Technol Biomed Life Sci
2007;847:12Y23
11. Albrethsen J. Reproducibility in protein profiling by MALDI-TOF mass
spectrometry. Clin Chem 2007;53:852Y58
930
Copyright @ 2008 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
12. Cornett DS, Reyzer ML, Chaurand P, et al. MALDI imaging mass
spectrometry: Molecular snapshots of biochemical systems. Nat Methods
2007;4:828Y33
13. Tanner S, Shu H, Frank A, et al. InsPecT: Identification of posttransla-
tionally modified peptides from tandem mass spectra. Anal Chem 2005;
77:4626Y39
14. Hansen BT, Davey SW, Ham AJ, et al. P-Mod: An algorithm and
software to map modifications to peptide sequences using tandem MS
data. J Proteome Res 2005;4:358Y68
15. Schonberger SJ, Edgar PF, Kydd R, et al. Proteomic analysis of the brain
in Alzheimer’s disease: Molecular phenotype of a complex disease
process. Proteomics 2001;1:1519Y28
16. Shiozaki A, Tsuji T, Kohno R, et al. Proteome analysis of brain proteins
in Alzheimer’s disease: Subproteomics following sequentially extracted
protein preparation. J Alzheimers Dis 2004;6:257Y68
Downloaded from https://academic.oup.com/jnen/article-abstract/67/10/923/2916851 by guest on 13 May 2019
17. Kim SH, Vlkolinsky R, Cairns N, et al. Decreased levels of complex III
core protein 1 and complex V beta chain in brains from patients with
Alzheimer’s disease and Down syndrome. Cell Mol Life Sci 2000;57:
1810Y16
18. Krapfenbauer K, Engidawork E, Cairns N, et al. Aberrant expression of
peroxiredoxin subtypes in neurodegenerative disorders. Brain Res 2003;
967:152Y60
19. Liao L, Cheng D, Wang J, et al. Proteomic characterization of
postmortem amyloid plaques isolated by laser capture microdissection.
J Biol Chem 2004;279:37061Y68
20. Wang Q, Woltjer RL, Cimino PJ, et al. Proteomic analysis of
neurofibrillary tangles in Alzheimer disease identifies GAPDH as a
detergent-insoluble paired helical filament tau binding protein. FASEB J
2005;19:869Y71
21. Li Y, Nowotny P, Holmans P, et al. Association of late-onset
Alzheimer’s disease with genetic variation in multiple members of the
GAPD gene family. Proc Natl Acad Sci U S A 2004;101:15688Y93
22. Lin PI, Martin ER, Bronson PG, et al. Exploring the association of
glyceraldehyde-3-phosphate dehydrogenase gene and Alzheimer disease.
Neurology 2006;67:64Y68
23. Olah J, Tokesi N, Vincze O, et al. Interaction of TPPP/p25 protein with
glyceraldehyde-3-phosphate dehydrogenase and their co-localization in
Lewy bodies. FEBS Lett 2006;580:5807Y14
24. Cumming RC, Schubert D. Amyloid-beta induces disulfide bonding and
aggregation of GAPDH in Alzheimer’s disease. FASEB J 2005;19:
2060Y62
25. Sultana R, Boyd-Kimball D, Cai J, et al. Proteomics analysis of the
Alzheimer’s disease hippocampal proteome. J Alzheimers Dis 2007;11:
153Y64
26. Wakabayashi K, Tanji K, Mori F, et al. The Lewy body in Parkinson’s
disease: Molecules implicated in the formation and degradation of
alpha-synuclein aggregates. Neuropathology 2007;27:494Y506
27. Leverenz JB, Umar I, Wang Q, et al. Proteomic identification of novel
proteins in cortical Lewy bodies. Brain Pathol (Zurich, Switzerland)
2007;17:139Y45
28. Baba M, Nakajo S, Tu PH, et al. Aggregation of alpha-synuclein in
Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy
bodies. Am J Pathol 1998;152:879Y84
29. Trojanowski JQ, Lee VM. Brain degeneration linked to Bfatal at-
tractions[ of proteins in Alzheimer’s disease and related disorders. J
Alzheimers Dis 2001;3:117Y19
30. Glenner GG, Wong CW. Alzheimer’s disease: Initial report of the
purification and characterization of a novel cerebrovascular amyloid
protein. Biochem Biophy Res Comm 1984;120:885Y90
31. Lee VM, Balin BJ, Otvos L Jr, et al. A68: A major subunit of paired
helical filaments and derivatized forms of normal Tau. Science 1991;
251:675Y78
32. Skovronsky DM, Lee VM, Trojanowski JQ. Neurodegenerative dis-
eases: New concepts of pathogenesis and their therapeutic implications.
Ann Rev Pathol 2006;1:151Y70
33. Woltjer RL, Cimino PJ, Boutte AM, et al. Proteomic determination of
widespread detergent-insolubility including AA but not tau early in the
pathogenesis of Alzheimer’s disease. FASEB J 2005;19:1923Y25
34. Yang W, Woltjer RL, Sokal I, et al. Quantitative proteomics identifies
surfactant-resistant alpha-synuclein in cerebral cortex of Parkinsonism-
dementia complex of Guam but not Alzheimer’s disease or progressive
supranuclear palsy. Am J Pathol 2007;171:993Y1002
Ó 2008 American Association of Neuropathologists, Inc.
J Neuropathol Exp Neurol  Volume 67, Number 10, October 2008
35. Winton MJ, Joyce S, Zhukareva V, et al. Characterization of tau
pathologies in gray and white matter of Guam parkinsonism-dementia
complex. Acta Neuropathol (Berl) 2006;111:401Y12
36. Sebeo J, Hof PR, Perl DP. Occurrence of alpha-synuclein pathology in
the cerebellum of Guamanian patients with parkinsonism-dementia
complex. Acta Neuropathol 2004;107:497Y503
37. Forman MS, Schmidt ML, Kasturi S, et al. Tau and alpha-synuclein
pathology in amygdala of Parkinsonism-dementia complex patients of
Guam. Am J Pathol 2002;160:1725Y31
38. Yamazaki M, Arai Y, Baba M, et al. Alpha-synuclein inclusions in
amygdala in the brains of patients with the parkinsonism-dementia
complex of Guam. J Neuropathol Exp Neurol 2000;59:585Y91
39. Castegna A, Aksenov M, Aksenova M, et al. Proteomic identification of
oxidatively modified proteins in Alzheimer’s disease brain. Part I:
Creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal
hydrolase L-1. Free Radic Biol Med 2002;33:562Y71
40. Castegna A, Aksenov M, Thongboonkerd V, et al. Proteomic identi-
fication of oxidatively modified proteins in Alzheimer’s disease brain.
Part II: Dihydropyrimidinase-related protein 2, alpha-enolase and heat
shock cognate 71. J Neurochem 2002;82:1524Y32
41. Choi J, Levey AI, Weintraub ST, et al. Oxidative modifications and
down-regulation of ubiquitin carboxyl-terminal hydrolase L1 associated
with idiopathic Parkinson’s and Alzheimer’s diseases. J Biol Chem
2004;279:13256Y64
42. Butterfield DA, Sultana R. Identification of 3-Nitrotyrosine-modified
brain proteins by redox proteomics. Methods Enzymol 2008;440:
295Y308
43. Sultana R, Reed T, Perluigi M, et al. Proteomic identification of nitrated
brain proteins in amnestic mild cognitive impairment: A regional study. J
Cell Mol Med 2007;11:839Y51
44. Butterfield DA, Reed TT, Perluigi M, et al. Elevated levels of
3-nitrotyrosine in brain from subjects with amnestic mild cognitive
impairment: Implications for the role of nitration in the progression of
Alzheimer’s disease. Brain Res 2007;1148:243Y48
45. Cripps D, Thomas SN, Jeng Y, et al. Alzheimer disease-specific con-
formation of hyperphosphorylated paired helical filament-Tau is poly-
ubiquitinated through Lys-48, Lys-11, and Lys-6 ubiquitin conjugation.
J Biol Chem 2006;281:10825Y38
46. Boutte AM, Woltjer RL, Zimmerman LJ, et al. Selectively increased
oxidative modifications mapped to detergent-insoluble forms of AA and
beta-III tubulin in Alzheimer’s disease. FASEB J 2006;20:1473Y83
47. Lippa CF, Duda JE, Grossman M, et al. DLB and PDD boundary issues:
Diagnosis, treatment, molecular pathology, and biomarkers. Neurology
2007;68:812Y19
48. Basso M, Giraudo S, Corpillo D, et al. Proteome analysis of human
substantia nigra in Parkinson’s disease. Proteomics 2004;4:3943Y52
49. Shi M, Jin J, Wang Y, et al. Mortalin: A protein associated with
progression of Parkinson disease? J Neuropathol Exp Neurol 2008;67:
117Y24
50. Jin J, Hulette C, Wang Y, et al. Proteomic identification of a stress
protein, mortalin/mthsp70/GRP75: Relevance to Parkinson disease. Mol
Cell Proteomics 2006;5:1193Y204
51. Kaul SC, Deocaris CC, Wadhwa R. Three faces of mortalin: A
housekeeper, guardian and killer. Exp Gerontol 2007;42:263Y74
52. Carrette O, Demalte I, Scherl A, et al. A panel of cerebrospinal fluid
potential biomarkers for the diagnosis of Alzheimer’s disease. Proteo-
mics 2003;3:1486Y94
53. Blennow K. CSF biomarkers for Alzheimer’s disease: Use in early
diagnosis and evaluation of drug treatment. Expert Rev Mol Diagn 2005;
5:661Y72
54. Davidsson P, Sjogren M. The use of proteomics in biomarker discovery
in neurodegenerative diseases. Dis Markers 2005;21:81Y92
55. D’Ascenzo M, Relkin NR, Lee KH. Alzheimer’s disease cerebrospinal
fluid biomarker discovery: A proteomics approach. Curr Opin Mol Ther
2005;7:557Y64
56. Galasko D. Biomarkers for Alzheimer’s diseaseVclinical needs and
application. J Alzheimers Dis 2005;8:339Y46
57. Olsson A, Vanderstichele H, Andreasen N, et al. Simultaneous measure-
ment of {beta}-amyloid(1Y42), total tau, and phosphorylated tau
(Thr181) in cerebrospinal fluid by the xMAP technology. Clin Chem
2005;51:336Y45
58. Abdi F, Quinn JF, Jankovic J, et al. Detection of biomarkers with a
Ó 2008 American Association of Neuropathologists, Inc.
Copyright @ 2008 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
Proteomics in Neurodegeneration
multiplex quantitative proteomic platform in cerebrospinal fluid of
patients with neurodegenerative disorders. J Alzheimers Dis 2006;9:
293Y348
59. Davidsson P, Westman-Brinkmalm A, Nilsson CL, et al. Proteome
analysis of cerebrospinal fluid proteins in Alzheimer patients. Neuro-
report 2002;13:611Y15
60. Puchades M, Hansson SF, Nilsson CL, et al. Proteomic studies of
potential cerebrospinal fluid protein markers for Alzheimer’s disease.
Brain Res Mol Brain Res 2003;118:140Y46
61. Zhang J, Goodlett DR, Peskind ER, et al. Quantitative proteomic
analysis of age-related changes in human cerebrospinal fluid. Neurobiol
Aging 2005;26:207Y27
62. Zhang J, Goodlett DR, Quinn JF, et al. Quantitative proteomics of
cerebrospinal fluid from patients with Alzheimer disease. J Alzheimers
Dis 2005;7:125Y33
Downloaded from https://academic.oup.com/jnen/article-abstract/67/10/923/2916851 by guest on 13 May 2019
63. Pan S, Wang Y, Quinn JF, et al. Identification of glycoproteins in human
cerebrospinal fluid with a complementary proteomic approach. J
Proteome Res 2006;5:2769Y79
64. Finehout EJ, Franck Z, Choe LH, et al. Cerebrospinal fluid proteomic
biomarkers for Alzheimer’s disease. Ann Neurol 2007;61:120Y29
65. Hu Y, Malone JP, Fagan AM, et al. Comparative proteomic analysis of
intra- and interindividual variation in human cerebrospinal fluid. Mol
Cell Proteomics 2005;4:2000Y09
66. Pasinetti GM, Ungar LH, Lange DJ, et al. Identification of potential CSF
biomarkers in ALS. Neurology 2006;66:1218Y22
67. Bowser R, Cudkowicz M, Kaddurah-Daouk R. Biomarkers for amyo-
trophic lateral sclerosis. Expert Rev Mol Diagn 2006;6:387Y98
68. McComb JG. Recent research into the nature of cerebrospinal fluid
formation and absorption. J Neurosurg 1983;59:369Y83
69. Han CY, Backous DD. Basic principles of cerebrospinal fluid meta-
bolism and intracranial pressure homeostasis. Otolaryngol Clin North
Am 2005;38:569Y76
70. Bergsneider M. Evolving concepts of cerebrospinal fluid physiology.
Neurosurg Clin N Am 2001;12:631Y38, vii
71. Huhmer AF, Biringer RG, Amato H, et al. Protein analysis in human
cerebrospinal fluid: Physiological aspects, current progress and future
challenges. Dis Markers 2006;22:3Y26
72. Nilsson C, Stahlberg F, Thomsen C, et al. Circadian variation in human
cerebrospinal fluid production measured by magnetic resonance imag-
ing. Am J Physiol 1992;262:R20YR24
73. Preston JE. Ageing choroid plexus-cerebrospinal fluid system. Microsc
Res Tech 2001;52:31Y37
74. Blennow K, Fredman P, Wallin A, et al. Protein analysis in cere-
brospinal fluid. II. Reference values derived from healthy individuals
18Y88 years of age. Eur Neurol 1993;33:129Y33
75. Blennow K, Fredman P, Wallin A, et al. Protein analyses in ce-
rebrospinal fluid. I. Influence of concentration gradients for proteins
on cerebrospinal fluid/serum albumin ratio. Eur Neurol 1993;33:
126Y28
76. Blennow K, Fredman P, Wallin A, et al. Protein analysis in cere-
brospinal fluid. III. Relation to blood-cerebrospinal fluid barrier function
for formulas for quantitative determination of intrathecal IgG production.
Eur Neurol 1993;33:134Y42
77. Osman I, Gaillard O, Meillet D, et al. A sensitive time-resolved
immunofluorometric assay for the measurement of apolipoprotein B
in cerebrospinal fluid. Application to multiple sclerosis and other
neurological diseases. Eur J Clin Chem Clin Biochem 1995;33:53Y58
78. Carrette O, Burkhard PR, Hughes S, et al. Truncated cystatin C in
cerebrospinal fluid: Technical [corrected] artefact or biological process?
Proteomics 2005;5:3060Y65
79. Hu S, Loo JA, Wong DT. Human body fluid proteome analysis.
Proteomics 2006;6:6326Y53
80. Hardt M, Thomas LR, Dixon SE, et al. Toward defining the human
parotid gland salivary proteome and peptidome: Identification and
characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass
spectrometry. Biochemistry 2005;44:2885Y99
81. Davidsson P, Nilsson CL. Peptide mapping of proteins in cerebrospinal
fluid utilizing a rapid preparative two-dimensional electrophoretic
procedure and matrix-assisted laser desorption/ionization mass spec-
trometry. Biochim Biophys Acta 1999;1473:391Y99
82. Raymackers J, Daniels A, De Brabandere V, et al. Identification of
two-dimensionally separated human cerebrospinal fluid proteins by
931
Zhang et al
N-terminal sequencing, matrix-assisted laser desorption/ionization–mass
spectrometry, nanoliquid chromatography-electrospray ionization-time
of flight-mass spectrometry, and tandem mass spectrometry. Electro-
phoresis 2000;21:2266Y83
83. Sickmann A, Dormeyer W, Wortelkamp S, et al. Towards a high
resolution separation of human cerebrospinal fluid. J Chromatogr B
Analyt Technol Biomed Life Sci 2002;771:167Y96
84. Finehout EJ, Franck Z, Lee KH. Towards two-dimensional electro-
phoresis mapping of the cerebrospinal fluid proteome from a single
individual. Electrophoresis 2004;25:2564Y75
85. Maccarrone G, Milfay D, Birg I, et al. Mining the human cerebrospinal
fluid proteome by immunodepletion and shotgun mass spectrometry.
Electrophoresis 2004;25:2402Y12
86. Ramstrom M, Ivonin I, Johansson A, et al. Cerebrospinal fluid
protein patterns in neurodegenerative disease revealed by liquid
chromatography-Fourier transform ion cyclotron resonance mass spec-
trometry. Proteomics 2004;4:4010Y18
87. Wenner BR, Lovell MA, Lynn BC. Proteomic analysis of human
ventricular cerebrospinal fluid from neurologically normal, elderly
subjects using two-dimensional LC-MS/MS. J Proteome Res 2004;3:
97Y103
88. Xu J, Chen J, Peskind E, et al. Characterization of proteome of human
cerebrospinal fluid. Int Rev Neurobiol 2006;73:29Y98
89. Sanchez JC, Guillaume E, Lescuyer P, et al. Cystatin C as a potential
cerebrospinal fluid marker for the diagnosis of Creutzfeldt-Jakob
disease. Proteomics 2004;4:2229Y33
90. Irani DN, Anderson C, Gundry R, et al. Cleavage of cystatin C in the
cerebrospinal fluid of patients with multiple sclerosis. Ann Neurol 2006;
59:237Y47
932
Copyright @ 2008 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol  Volume 67, Number 10, October 2008
91. Mannes AJ, Martin BM, Yang HY, et al. Cystatin C as a cerebrospinal
fluid biomarker for pain in humans. Pain 2003;102:251Y56
92. Simonsen AH, McGuire J, Podust VN, et al. A novel panel of
cerebrospinal fluid biomarkers for the differential diagnosis of Alzhei-
mer’s disease versus normal aging and frontotemporal dementia. Dement
Geriatr Cogn Disord 2007;24:434Y40
93. Simonsen AH, McGuire J, Hansson O, et al. Novel panel of
cerebrospinal fluid biomarkers for the prediction of progression to
Alzheimer dementia in patients with mild cognitive impairment. Arch
Neurol 2007;64:366Y70
94. Ranganathan S, Williams E, Ganchev P, et al. Proteomic profiling of
cerebrospinal fluid identifies biomarkers for amyotrophic lateral scle-
rosis. J Neurochem 2005;95:1461Y71
95. Ruetschi U, Zetterberg H, Podust VN, et al. Identification of CSF
biomarkers for frontotemporal dementia using SELDI-TOF. Experimen-
Downloaded from https://academic.oup.com/jnen/article-abstract/67/10/923/2916851 by guest on 13 May 2019
tal Neurology 2005;196:273Y81
96. Biroccio A, Del Boccio P, Panella M, et al. Differential post-translational
modifications of transthyretin in Alzheimer’s disease: A study of the
cerebral spinal fluid. Proteomics 2006;6:2305Y13
97. Castano EM, Roher AE, Esh CL, et al. Comparative proteomics of
cerebrospinal fluid in neuropathologically-confirmed Alzheimer’s dis-
ease and non-demented elderly subjects. Neurol Res 2006;28:155Y63
98. Zhang J, Sokal I, Peskind ER, et al. CSF multianalyte profile
distinguishes Alzheimer and Parkinson diseases. Am J Clin Pathol
2008;129:526Y29
99. Pan S, Rush J, Peskind ER, et al. Application of targeted quantitative
proteomics analysis in human cerebrospinal fluid using a liquid
chromatography matrix-assisted laser desorption/ionization time-of-
flight tandem mass spectrometer (LC MALDI TOF/TOF) platform. J
Proteome Res 2008;7:720Y30
Ó 2008 American Association of Neuropathologists, Inc.