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Clin Chem Lab Med 2007;45(4):437–449
2007 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2007.106
Review
Electrophoretic separations of cerebrospinal fluid proteins in
clinical investigations
Simona D’Aguanno1,2, Piero Del Boccio3,4,
Sergio Bernardini1,2, Enzo Ballone3,4, Carmine
Di Ilio3,4, Giorgio Federici1,2,5 and Andrea
Urbani3,4,*
1
Dipartimento di Medicina di Laboratorio, Policlinico
di Tor Vergata, Università di Roma Tor Vergata,
Rome, Italy
2
IRCCS-Fondazione S. Lucia, Rome, Italy
3
Centro Studi sull’Invecchiamento (Ce.S.I.),
Fondazione Università ‘‘G. D’Annunzio’’, Chieti, Italy
4
Dipartimento di Scienze Biomediche, Università
‘‘G. D’Annunzio’’ di Chieti e Pescara, Chieti, Italy
5
IRCCS Bambino Gesù, Laboratorio di Proteomica,
Rome, Italy
Abstract
The cerebrospinal fluid (CSF) is a key sample in the
research for novel molecular biomarkers of neurode-
generative disorders. CSF represents a repertoire of
neuro-secreted, biosynthesised and metabolised
molecular products of the central nervous system
(CNS). Diffusion of macromolecules from the periph-
eral circulatory system to the CSF is highly regulated
by the blood-brain barrier, which prevents uncon-
trolled distribution of proteins in the CNS. The devel-
opment of reproducible high resolution separations of
proteins in 2-D electrophoresis methods by the
advent of immobilised pH gradient has opened the
route to multivariate holistic protein pattern investi-
gation of CSF into neurodegenerative disorders.
Moreover, the introduction of pre-fractionation tech-
niques such as free flow electrophoresis is currently
increasing the dynamic depth of proteome analysis.
Alzheimer’s disease (AD) and other forms of demen-
tia, demyelinating diseases, Parkinson’s disease (PD),
and Creutzfeldt-Jakob disease (CJD) have been eval-
uated for biomarker discovery by CSF investigation in
multiple studies. However, the statistical design of
these clinical cross-sectional investigations remains a
limited factor given the strong statistical power
required for complex multivariate analysis. These ini-
tial evidences are of particular interest in dissecting
specific molecular mechanisms. The development of
fast and economic profiling of CSF by linear matrix
assisted laser desorption ionisation time-of-flight
*Corresponding author: Prof. Andrea Urbani, Laboratorio di
Biochimica Analitica e Proteomica, Centro Studi
sull’Invecchiamento (Ce.S.I.), Università ‘‘G. D’Annunzio’’ di
Chieti e Pescara, Via Colle dell’Ara, 66013 Chieti, Italy
Phone: q39-0871-541580, Fax: q39-0871-541598,
E-mail: a.urbani@unich.it
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2006/400
mass spectrometry (MALDI-TOF-MS) is providing a
new ancillary technology to assess sample quality
and pre-analytical requirements. In the following we
take into account all these issues in the CSF proteo-
mics investigation, especially highlighting the possi-
ble application in the development of clinical
molecular biomarkers.
Clin Chem Lab Med 2007;45:437–49.
Keywords: cerebrospinal fluid; 2-D electrophoresis;
dementia; proteomics; matrix assisted laser desorp-
tion ionisation time-of-flight mass spectrometry
(MALDI-TOF-MS); neurodegeneration.
Introduction
Cerebrospinal fluid (CSF) is one of the central nervous
system (CNS) tissues and a precious sample in the
biomarkers discovery of neurodegenerative disor-
ders. CSF is a clear fluid that functions to cushion and
protect the brain from changes in blood pressure and
trauma. In addition to this morphological function,
CSF transports neuro-secreted, biosynthesised and
metabolised cellular products within the CNS (1). This
fluid is mainly produced by the choroids plexus within
the ventricles of the brain. It circulates throughout the
ventricular system passing outside of the brain and
spinal cord by the subarachnoid space. CSF is not in
direct contact with the peripheral circulatory system
due to a highly regulated blood-brain barrier that pre-
vents passive diffusion of macromolecules (1). CSF
protein repertoire is derived both from neuronal cell
secretory activity and by active transport via pinocy-
tosis across the blood-brain barrier. Disruption of this
barrier in pathologic conditions allows macromole-
cules to enter uncontrolled into the CNS. The direct
contact of the brain interstitial fluid with the CSF
allows biochemical changes taking place in the cel-
lular compartment to be reflected in this medium.
Moreover, protein concentrations between the lum-
bar and ventricular compartments have been reported
to be homogeneous as shown by analysis of the
transthyretin (TTR) (2). This would suggest the
approximation that the CSF has an isotropic molecu-
lar composition throughout the different regions of
the CNS.
CSF is clinically accessible by the standard lumbar
puncture technique, which enables ante-mortem anal-
ysis of neurodegenerative processes. This procedure
allows investigating patients during the disease tra-
jectory with a much less invasive and more readily
obtainable sample than the brain biopsy. Spinal tap
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438 D’Aguanno et al.: Separations of CSF proteins
is a safe procedure when performed by specialised
personnel (3), and complications are mainly due to
the onset of a post-lumbar puncture headache. How-
ever, this discomfort is less common in the geriatric
population, especially in the presence of cognitive
impairment (4). These features have made CSF a
potentially powerful source of molecular biomarkers
for the diagnosis and response to treatment, as well
as for providing information on pathological process-
es underlying a number of CNS disorders, including
Alzheimer’s disease (AD), frontotemporal dementia
(FTD), dementia with Lewy bodies, multiple sclerosis,
Creutzfeldt-Jakob disease (CJD), Parkinson’s disease
(PD), traumatic brain injury (TBI) and cancer.
Traditional routine CSF evaluation employs basic
evidences such as opening liquid pressure and the
supernatant colour. Cell counts and culturing follow-
ing differential microscopic examinations are often
employed in inflammatory diseases. Molecular mark-
er investigations include a few protein analyses,
immunoblotting methods and polymerase chain reac-
tion (Table 1). However, all these laboratory tests are
often missing low abundant proteins or post transla-
tional modifications which might be associated with
the neurodegenerative process. More sensitive anal-
ysis such as the 2-D electrophoresis technique, cast
into a proteomic framework, might represent a useful
strategy to shed new light on neurodegenerative
processes. These investigations might eventually lead
to the development of a novel laboratory test for clin-
ical examinations.
Basic cerebrospinal fluid test in primary care
Basic laboratory tests are based on the comparison
of few fundamental parameters: colour, liquid pres-
sure, protein concentration and cell composition.
In non-pathologic condition CSF is a crystal clear
liquid. However, as few as 200 white blood cells
(WBCs) per mm3 or 400 red blood cells (RBCs) per
mm3 will cause CSF to appear turbid. Xanthochromia
is a yellow, orange, brown or pink discolouration of
the CSF, most often caused by the lysis of RBCs
resulting in haemoglobin breakdown to oxyhaemo-
globin, methaemoglobin, and bilirubin (5, 6).
Normal fluid opening pressure ranges from 10 to
100 mm H2O in young children, from 60 to 200 mm
H2O after 8 years of age, and up to 250 mm H2O in
obese patients. Intracranial hypotension is defined as
an opening pressure of less than 60 mm H2O. Open-
Table 1 Routine analysis of CSF in primary care searching different pathologies.
Routine CSF analysis
Colour
Pressure
White blood cell count
PCR
Protein level
ELISA and/or monoclonal antibody assays
Electrophoresis
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ing pressures above 250 mm H2O are diagnostic of
intracranial hypertension, that is present in many
pathologic states, including meningitis, intracranial
haemorrhage, and tumours (5).
Normal CSF may contain up to 5 WBCs per mm3 in
adults and 20 WBCs per mm3 in newborns. Eighty-
seven percent of patients with bacterial meningitis
will have a WBC count higher than 1000 per mm3,
while 99% will have more than 100 per mm3. Having
less than 100 WBCs per mm3 is more common in
patients with viral meningitis (5, 6). Elevated WBC
counts may also occur in intracerebral haemorrhage
conditions, in the presence of malignancy, and in a
variety of inflammatory conditions. The WBC count
seen in normal adult CSF is comprised of approxi-
mately 70% lymphocytes and 30% monocytes. Occa-
sionally, a solitary eosinophil or polymorpho-
nucleocyte (PMN) will be seen in normal CSF (6). The
majority of patients with Guillain-Barré syndrome (a
chronic inflammatory demyeliating neuropathy of the
peripheral nervous system) will have 10 or fewer
monocytes per mm3 and a minority of patients will
have 11–50 monocytes per mm3 (5). Up to 50 mono-
cytes per mm3 are seen in about 25% of patients with
multiple sclerosis (6). The cellular content of CSF
must be taken into account regarding the pre-analyt-
ical phase in sample preparation. Centrifugation step
of this sample should be employed prior to aliquoting
and freezing in order to avoid cell rupture and con-
sequent proteome variation.
Cell cultures is the gold analysis for diagnosing bac-
terial meningitis (2). Viral meningitis is mainly caused
by an enterovirus; culture for herpes simplex virus
has a sensitivity of 80%–90% (5). Polymerase chain
reaction (PCR) has been a great advance in the differ-
ential diagnosis of meningitis. PCR has high sensitiv-
ity and specificity for many infections of the CNS, it
is fast, and can be done with small volumes of CSF.
PCR has been especially useful in the diagnosis of
viral meningitis (7). PCR of the CSF has a sensitivity
of 95%–100% for herpes simplex virus type 1, Epstein-
Barr virus, and enterovirus (5).
As regards protein in basic CSF laboratory tests, the
total protein concentration reference values range in
a normal human being from 0.2 mg/mL to 0.8
mg/mL (5). The most abundant proteins in human
CSF are albumin and immunoglobulins (Igs) that con-
stitute more than 50% and 15% of the total protein
mass, respectively (8). Among the other abundant
polypeptide components of human CSF are: apolipo-
Disease
Hyperbilirubinaemia, meningeal melanomatosis
Meningitis, intracranial haemorrage, tumours
Bacterial and viral meningitis
Viral meningitis
Infections, intracranial haemorrhages, multiple sclerosis,
Guillain Barré syndrome, malignancies, endocrine
abnormalities
Alzheimer’s disease (hyperphosphorylation of Tau protein)
Multiple sclerosis (oligoclonal IgG bands)
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proteins, a1-antitrypsin, TTR, retinol-binding protein
(RBP), prostaglandin D2 synthase, cystatin C, plasmin-
ogen, gelsolin, b2-microglobulin, haemopexin, fibrin-
ogen, ubiquitin, complement components/factors,
kallikrein-6, a1b-glycoprotein, a2-HS glycoprotein, b-
amyloid (A b) fragment (1).
High levels of Igs, specifically IgG class, are found
in 95% of multiple sclerosis patients (9), however,
these oligoclonal bands may be produced in many
other conditions such as CNS infection and paraneo-
plastic syndromes. The presence of two or more oli-
goclonal bands is considered a positive test for
multiple sclerosis (a neurologic disorder resulting in
the demyelination of neurons in the CNS), which is in
fact an autoimmune disorder. More specific immu-
nochemical methods have been employed in both
CNS tumours and in dementias. Identification of auto-
antibodies, anti-Yo and anti-Hu, in the CSF are diag-
nostic for certain breast, lung and ovarian tumours
(10). Dorn et al. (11) reported the case of a paraneo-
plastic cerebral degeneration (PCD) in a female
patient with breast cancer and the indication of anti-
Yo antibodies in the CSF and serum.
The levels of a number of cytokines are markedly
increased in the CSF of PD patients compared with
healthy individuals. PD is characterised by the degen-
eration of dopaminergic neurons probably caused by
apoptosis, which is known to be controlled by cyto-
kines. Among the cytokines shown to be elevated in
the CSF of PD patients are tumour necrosis factor-a
(TNF-a), IL-1b, -2, -4, -6, transforming growth factor
(TGF)-a, -b1, and b2, suggesting that nonsteroidal
anti-inflammatory drugs may be useful in limiting the
loss of dopaminergic neurons (7).
Measurement of b-amyloid peptide 1–42, total and
hyperphosphorylated Tau protein in CSF is becoming
a valuable diagnostic tool for AD in recent years (1).
The employment of these molecular markers based
on immunochemistry technology, such as ELISA or
xMAP, is currently providing the latest frontier in the
differential discrimination of dementias and early
diagnosis. The multiparametric bead-based assays for
the quantisation of these protein levels represents the
first successful application of a multifactorial analysis
in the diagnosis of neurodegenerative disorders (12).
These studies have highlighted the strong potential
impact of multivariate evaluation on diagnosis incre-
menting the interest in the development of novel
tools for the proteome analysis of CSF in order to
identify potential protein biomarkers in different neu-
rological disorders. The introduction of protein micro-
and nano-scale analytical set up has led to the
employment of 2-D gel electrophoresis (2-DE) and
mass spectrometry (MS) methods for the CSF
investigation.
Mapping human cerebrospinal fluid by
electrophoresis
The first 2-DE map of the CSF was published in 1980
(13). Since that time, protein spot detection and iden-
tification technologies have improved and updated
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D’Aguanno et al.: Separations of CSF proteins 439
CSF maps have been published (8, 14–17). The
improvement in protein detection has turned this
method not only into a more sensitive and quantita-
tively dynamic tool but also into a tool compatible
with subsequent MS and bio-informatic analysis.
The results published in recent years are summa-
rised in Table 2. A representative 2-DE map of a single
individual CSF sample affected by multiple sclerosis
obtained in our laboratories is shown in Figure 1.
After acetone precipitation and reswelling in sample
buffer (8), 30 mg of CSF were loaded on pH 3-10NL
IPG strips. The second dimension was performed on
a 9%–16% acrylamide gradient. After silver staining
about 300 spots could be visualised.
The first attempts to obtain an annotated CSF map
were performed by immunostaining after 2-DE blots,
partial amino acid sequencing by Edman degradation
and comparison with other 2-DE maps (13). Sickmann
et al. (14) tried to define the standard protein com-
position of CSF using a CSF pool consisting of sam-
ples from 25 different patients without any symptoms
of neurological diseases or blood-brain barrier mal-
functions. Analytical and micropreparative 2-DE gels
were employed to obtain an overview on a broad
range of first-dimension pH values of 3–10. Combin-
ing two overlapping 2-DE gels (pI 4–7 and pI 6–11)
they obtained more extended CSF maps with a total
of 480 defined spots. They applied different approach-
es for protein identification, such as peptide mass fin-
gerprints using matrix assisted laser desorption
ionisation-mass spectrometry (MALDI-MS), the frag-
mentation of peptide ions using MALDI-post source
decay (PSD) or a strategy based on a mHPLC separa-
tion of the digest mixture followed by an MS/MS
analysis.
Following this, Yuan et al. (15) tested several sam-
ple preparation methods in order to obtain a higher
quality 2-DE pattern. Their efforts were addressed to
remove salts by protein precipitation with either ace-
tone or trichloroacetic acid/acetone, or sample treat-
ment with a Bio-Spin column. More spots were
visualised on the 2-DE gel of human CSF, and a rel-
atively high protein recovery was obtained when a
Bio-Spin column was used to process a human CSF
sample. Sixty-one protein spots, obtained from 2-DE
gels with a pH range of either 3–10 or 4–7, were iden-
tified by MALDI-MS and PSD-MS. These 61 protein
spots represent 22 proteins. In particular, six of those
proteins were not annotated in any previously pub-
lished 2-DE maps (Table 3).
Finehout et al. (16) reported the creation of a
detailed map, based on state-of-the-art 2-DE and MS
technology, of the ante-mortem CSF proteome from
a single individual. In particular they used a large for-
mat 2-DE gel (18 cm=16 cm=1.5 mm), a fluorescent
stain (SYPRO Ruby) with fluorescence laser scanning,
and matrix assisted laser desorption ionisation time-
of-flight (MALDI-TOF)-MS for protein identification.
High amounts of samples were loaded on pH 3–10
IPG strips to explore the range of the lower concen-
tration protein. The work compared CSF from a
hydrocephalus patient wa condition characterised by
an increase in the volume of CSF associated with dila-
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440 D’Aguanno et al.: Separations of CSF proteins

Table 2 A short report of the latest 2-DE maps.

Reference Sample IEF SDS-PAGE Staining Total spots Identified

preparation (number) spots

Sickmann et al. Pooled CSF 100 mg of sample 12% S, 480 65

(2000) (14) from 25 different for analytical gel (I) C (24 different
individuals (18 cm IPG strip protein)
3–10 NL);
Concentration 500 mg for
using 5 kDa cut- micropreparative
off filters gel (18 cm IPG
strip 4–7, 6–11)1
Yuan et al. CSF aliquots 75–85 mg of CSF; 12–14% S NR 61
(2002) (15) from 3 different 18 cm IPG strip (G) (22 different
individuals, 3-10 L; 4–73 protein)
desalted by
precipitation
with acetone,
TCA/acetone or
using Bio spin
column2
Finehout et al. CSF aliquot 100 mg of CSF; 12–15% SR 1030* 600
(2004) (16) from single 18 cm IPG strip (G) (82 different
individual precipitated 3–10 NL4 protein)
with 70% ethanol
Ogata et al. Concentration 100 mg of 12% S, 707 55
(2005) (17) of pooled CSF depleted CSF; (I) SR (12 new protein)
using 10 kDa cut-off 18 cm IPG strips
filter before HPLC 3–10 NL4
multiple affinity
removal column
Yuan et al. Prefractionation Each CSF 11% S NR 62
(2005) (8) of pooled CSF fraction loaded (I)
protein by SPE on 18 cm IPG
strips 3–10 NL4
Hu et al. Concentration 50 mg of 10% D 2100 -
(2005) (18) of 1.5–2 mL of depleted CSF; (I)
CSF from single 24 cm IPG strips
individual 10 kDa 3–10 NL3
cut-off filter before
multiple affinity
removal column
1
Multiphor II (Pharmacia, Uppsala, Sweden); 2Biorad, Hercules, USA; 3Ettan IPGphor IEF system (Amersham Pharmacia Bio-
tech); 4PROTEN IEF (Biorad); SDS-PAGE, sodium dodecylsulphate-polyacrylamide gel electrophoresis; S, silver staining; SR,
SYPRO Ruby; C, Coomassie G-250; D, DIGE; I, isocratic acrylamide gel; G, gradient acrylamide; *different spots contain albu-
min; NR, not reported.

tion of the cerebral ventricles (6)x with two sample
controls. According to these authors the spot pattern
shows a comparable protein content so that the CSF
map created using this hydrocephalus sample may be
applicable to maps of other CSF samples. This hydro-
cephalus map contains 600 resolved spots. Of the 82
proteins identified by MS, 25 have not appeared in
previously published 2-DE CSF maps, and 11 have not
been previously reported to exist in CSF (Table 3). A
webpage containing protein identification and scoring
information is available at http://www.leelab.org/
csfmap.
It is interesting to note that, because of the high
percent of albumin in CSF, they found different mixed
spots of albumin and other proteins.
The presence of very abundant proteins such as
albumin and IgG limits the total number of CSF pro-
teins that can be analysed with a 2-DE based
approach. In fact, one of the issues that limits the
analysis of the proteome in CSF is due to the large
dynamic range of protein concentrations in this sam-
ple of fluids. These might span over twelve orders of
magnitude between the highest and lowest expressed
proteins (19). For successful biomarker and therapeu-
tic target discoveries, it is advantageous to be able to
identify differentially expressed proteins globally and
to assess the level of post-translational modifications
for both high and low-abundance proteins. Removal
of abundant proteins can improve the number of pro-
teins to be identified by reducing the dynamic range
of protein levels in the biological fluid to better match
that of the downstream analytical platform (20). For
this purpose several state-of-the-art and commercial
methods are available. Currently the most widely
used albumin removal technique is the Cibracon Blue
chromatography; however, this method is non-specif-
ic, and it is known to bind numerous other proteins
(21). Fractionation with an organic solvent can also

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D’Aguanno et al.: Separations of CSF proteins 441

Figure 1 2-DE slab gel of CSF protein from a subject affected with multiple sclerosis. 30 mg of sample were loaded on pH
3–10 IPG strips. The second dimension was performed on a 9%–16% acrylamide gradient. After silver staining, about 300
spots could be visualised. Spots containing a1-glycoprotein, a1-antitrypsin apolipoprotein (apo) A-IV, Gc-globulin, albumin,
C3a, serotransferrin, IgG heavy chains, IgG light chains, haptoglobin b chain, apoJ, apoA-1, serum retinol binding protein
(SRBP), TTR are labelled with a continuous line.

reduce the dynamic range of CSF by separating pro-
teins by their molecular weights (22). Yuan et al. (8)
took advantage of the different hydrophobic proper-
ties of CSF proteins and a reversed-phase solid-phase
extraction (SPE) cartridge was used to prefractionate
three individual CSF samples into three separate frac-
tions prior to 2-DE resolution of the proteome. A por-
tion of the high-abundance CSF proteins were
removed from two (eluted with 35% and 50% aceto-
nitrile) of the three fractions. Some traces of CSF pro-
teins were preferentially enriched in the two fractions,
and many proteins were detected in 2-DE gels of the
fractions. Among the novel proteins identified, 62 pro-
tein spots that represent 42 proteins were characte-
rised. Ogata et al. (17) evaluated avian IgY antibody
microbead spin filters and prepacked HPLC multiple
affinity columns for removal of six abundant proteins
(albumin, transferrin, IgG, IgA, IgM, and fibrinogen
with microbeads, and albumin, transferrin, IgG, IgA,
antitrypsin and haptoglobin with HPLC column) from
pooled CSF samples of 25 healthy individuals. Follow-
ing the protein removal, 2-DE gels were compared
also for phospho- and glycoprotein content using spe-
cific fluorescent stains. The column format removed
the major proteins more effectively and approximate-
ly 50% more spots were visualised when compared
to the 2-DE gel of CSF without protein depletion. Fifty
proteins were identified from 66 spots, and among

Table 3 New proteins identified in CSF samples in recent years.

References New identified protein

Yuan et al. (2002) (15)
Finehout et al. (2004) (16)
Ogata et al. (2005) (17)
*Different isoforms.
PRO2619, pigment epithelium-derived factor, albumin homolog, kallikrein-6 precursor,
DJ717I23.1, and AMBP protein precursor
ApoCIII, b-1,3-N-acetylglucosaminyltransferase 1, cathepsin L, collagen a1(I) chain, comple-
ment factor H contactin 1 isoform 2, cyclin D1, nidogen 2, phenolsulphating sulphotransferase
1, proprotein convertase subtilisin
Plasma protease CI inhibitor precursor*, 72 kDa type IV collagenase precursor, vitamin K-
dependent protein S precursor, complement component C9 precursor*, serum paraoxon-
ase/arylesterase 1, cathepsin L precursor

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442 D’Aguanno et al.: Separations of CSF proteins

them 12 proteins (24%) have not been annotated in
previously published 2-DE gels (Table 3). Phospho-
and glycoprotein identified by this approach are sum-
marised in Table 4, where these results are compared
with the information available in the Swiss-prot data-
base. Hu et al. (18) analysed six individual CSF sam-
ples by multi-affinity depletion, two-dimensional
difference gel electrophoresis (2-D DIGE) and tandem
mass spectrometry. The 2-D DIGE technique dramat-
ically enhances the resolving power of 2-DE and mini-
mises the significant variation in the presence of
protein spots that generally occurs between different
gels (even from identical samples) by enabling mul-
tiple samples to be analysed on the same gel (23).
Before performing the first dimension on 24-cm pH
3–10 NL IPG strips, each sample is labelled with one
of the three spectrally distinguishable fluorescent
dyes (i.e., Cy2, Cy3, and Cy5) used in 2-D DIGE. Load-
ing 50 mg of each labelled sample is possible to visua-
lise about 2100 resolved spots.
The depletion of the most abundant proteins from
biological fluid samples (such as plasma, saliva,
urine) might be followed by further fractionation steps
to possibly further enrich the percentage of low-con-
centrated protein component. Recently, several stud-
ies (24, 25) described a new approach to proteomic
analysis which uses preparative isoelectric focusing
(IEF) by free flow electrophoresis (FFE) for a first-

Table 4 Phospho- and glycoprotein identified by Ogata et al. (17) compared with the information available in the Swiss-prot
database.

Accession number Protein ID Modification Residues involved

Q14515 SPARC-like protein 1 *GN *N169, *N176, *N196,
precursor p *N280, *N412, *N476
P04004 Vitronectin precursor *GN *N86, *N169, *N242
p
P08253 Collagenase precursor *GN *N573, *N642
p
P01042 Kininogen precursor GN N169, N205, N294;
GO T401, T533, T542, T546,
p T557, T571, S577, T593, T628
P01019 Angiotensinogen precursor GN N47, N170, N304, N328
p
P02765 a2-HS-glycoprotein p S138, S330
precursor P N156, N176,
GN T256, T270, S346
GO
P00751 Complement factor B precursor p N122, N142, N285, N378;
GN N291
GGlc
P43652 Afamin precursor g *N33, *N109, N402, *N488
GN
*GN
P02790 Haemopexin precursor g T24, N64,N187, N240, N246, N453
GN
GO
P04217 a1B-glycoprotein g N44, N179, N363, N371
precursor GN
P02748 Complement component g W48, W51, *N277, N415
C9 precursor GC
*GN
GN
P08697 a2-antiplasmin g *N196, *N295, *N309, *N316
precursor *GN
P01011 a1-antichymotrypsin g N96, N106, N127
precursor GN
P01008 Antithrombin-III precursor g N128, N167, N187, N224
GN
P02763 a1-acid glycoprotein 1 g N33, N56, N72, N93, N103
precursor GN
P41222 Prostaglandin-H2 g N51, N78
D-isomerase precursor GN
p, phosphorylated protein according to Ogata et al. (17); g, glycosylated protein according to Ogata et al. (17); P, phospho-
rylated protein reported by ExPASy; *GN, potential N-linked (GlcNAc...) according to ExPASy; GN, N-linked (Glc-
NAc...)glycosylation reported by ExPASy; GO, O-linked (GalNAc...) glycosylation reported by ExPASy; GGlc, N-linked (Glc)
glycation reported by ExPASy; GC, C-linked (Man) reported by ExPASy.

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 Article in press - uncorrected proof
dimension fractionation of complex peptide mixtures.
FFE has been found to have advantages in both
improved sample recovery, probably due to the
absence of gel media or membranous material, and
higher sample loading proteins. Because of these
advantages, FFE has been widely used for pre-frac-
tionation of samples. Sample peptides could be dis-
solved in FFE separation buffer and fractionated by
IEF into a 96-well microlitre plate according to the pI.
Each fraction could be separated by 0.02–0.10 pH
units depending on the pH gradient created. Imme-
diately after FFE separation, the pH of each FFE frac-
tion could be measured using a microelectrode. By
using non-denaturating IEF solutions, proteins could
be fractionated in their native state allowing precom-
plexed proteins and peptides to remain in their non-
covalent bound form during fractionation. The
addition of RP-HPLC in the second dimension could
present a significant advantage because of its
increased loading capacity, as well as the additional
advantages such as the high recovery of low Mr pro-
teins and high-resolution protein separation. Recent-
ly, Moritz et al. (25) used FFE/RP-HPLC-MS in a
preliminary analysis for the isolation and identifica-
tion of several low-abundance proteins present in
blood, such as human L-selectin (17 ng/mL) and
fibrinogen (24 ng/mL). Instead of RP-HPLC, each frac-
tion collected after FFE could be loaded on an immo-
biline drystrip of an appropriate zoom pH range
gradient followed by a classical 2-DE slab gel, other-
wise each fraction could be loaded on a 1-D gel and
then analysed by LC-MS or analysed directly by LC-
MS.
Alternatively to 2-DE or FFE, high resolution in pep-
tide separation could be performed in a single step
using capillary electrophoresis (CE) (26). In the case
of CE, the analytes do not merely follow the liquid
flow (as in HPLC technique), but their migration is, to
a large extent, determined by the electric field
strength. This creates a very simple separation prin-
ciple based on charge, which facilitates a homoge-
nous separation and contributes to the power of CE.
Due to a demand for alternative separation technol-
ogies and the attractive advantages of CE in compar-
ison with HPLC (27), a number of efforts have been
made to achieve stable CE-MS coupling (28). A severe
limitation of CE is the inability to analyse higher-
molecular weight proteins, since these tend to precip-
itate under the conditions generally used for
separation (28). However, there is presently extended
evidence that the smaller-molecular weight peptides
in body fluids are highly complex holding a plethora
of information that can be exploited for biomarker dis-
covery. Wittke et al. (29) used CE in a CSF investiga-
tion. They used a CE-system equipped with a 90 cm,
50 mm ID bare fused-silica capillary (Beckman Coulter,
Fullerton, CA, USA). The running buffer consisted of
20% v/v acetonitrile and 0.25 M formic acid in HPLC-
grade water. The samples were injected by applying
a positive pressure of 1–6 psi, which resulted in a
sample plug of approximately 50–300 nL, and the sep-
aration was performed for 60 min. During the entire
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D’Aguanno et al.: Separations of CSF proteins 443
run the capillary temperature was held constant at
358C. The MS analysis was performed in positive elec-
trospray mode with an ESITOF sprayer-kit from Agi-
lent Technologies (Agilent Technologies Inc., Santa
Clara, CA, USA), using a Micro-TOF-MS (Bruker-Dal-
tonics, Bremen, Germany). By this approach they
resolved, as defined by their accurate molecular mass
and migration time, about 450 different proteins and
polypeptides with molecular masses from 0.8 to
15 kDa. Unfortunately, proteins such as albumin and
tranferrin, interfere with this CE-MS setup by precip-
itating under the low pH and organic solvent condi-
tions employed. Thus, Wittke et al. (29) decided to
remove all larger proteins using ultrafiltration. While
this approach may remove proteins that might hold
disease-specific information, it also enables the repro-
ducible analysis of the smaller-molecular weight pep-
tides, which might contain a significant amount of
information.
Combining 2-DE maps with mass spectrometry
profiles
Direct MS approach, in particular linear matrix assist-
ed laser desorption-ionisation time-of-flight mass
spectrometry (MALDI-TOF-MS), is an ancillary tech-
nology to the more extensive 2-DE investigations,
offering the possibility both to perform high-level
quality control experiments on protein samples (30)
and to accurately investigate the low-molecular
weight protein region (31, 32). In neuroscience the
standard tests for clinical analysis of proteins are
immunoassay techniques (33). The advantage of
these is the high sensitivity but it loses some impor-
tant characteristics that can be found in proteomic
investigations such as the ability to determine post-
translational modification and altered epitope in pro-
teins involved in a pathogenic event. Therefore
MALDI-TOF-MS is considered a promising technique
for clinical applications, moreover, time and cost of
analysis for each sample are particularly low, and the
sample preparation procedures eventually might han-
dle dozens of samples in a single acquisition run.
Recently, MALDI-TOF-MS and surface-enhanced laser
desorption/ionisation (SELDI)-TOF-MS were employ-
ed by several groups to investigate and detect poten-
tial novel disease biomarkers in different biological
matrices (34). Although this direct MS approach car-
ries out a limited window of the total proteome, a
number of studies have emerged investigating
possible alteration of the CSF proteins in neurological
diseases as useful indicators of pathological
abnormalities.
Westman et al. (35) described the use of direct
MALDI-TOF-MS in a study of CSF proteins. They ana-
lysed proteins in CSF without pre-purification steps,
obtaining good resolution and high sensitivity
(100 fmol protein) in the m/z range investigated
(10,000–20,000 Th). Such a mass resolution enables,
in fact, the discrimination between the hydroxy-
lated and non-hydroxylated forms of cystatin C at
 Article in press - uncorrected proof
444 D’Aguanno et al.: Separations of CSF proteins

m/zs13,344 and 13,361 Th, respectively. The ability
of MALDI-TOF to carry out good analytical perform-
ances in a mass range 1000–20,000 Da makes this
technique a complementary tool in the investigation
of CSF proteome covering a little window in the total
proteome but with high analytical quality. In fact, it is
difficult to obtain high quality 2-DE gel below 5000 Da
(36).
Although it is possible to analyse a crude sample
by MALDI-TOF-MS, many extraction strategies are
used to pre-purify proteins and peptides before anal-
ysis, improving analytical performances. A number of
reports describe the use of the SELDI platform which
is based on functionalised probes that possess spe-
cific chromatographic or affinity-capture properties
(37). These modified probes allow selective enrich-
ment of specific types of samples from biological flu-
ids with reduced time needed for sample preparation
steps. However, the MS data acquired by these tech-
nique are particularly poor, especially in resolution
power (30). In addition, several off-line enrichment-
purification methods are employed to couple with
MALDI-TOF-MS platform, such as microSPE extrac-
tion, that consist of a simple microcolumn purification
and sample preparation technique (38). The frit-less
miniaturised columns can be custom-made with var-
ious functionality and selectivity, including reversed-
phase (C4, C8, C18), IMAC, graphite, ion exchange,
and other resins (39–41). A modification of the micro-
column method is the use of the magnetic bead discs
constituted of a network of inert polytetrafluoroeth-
ylene (Teflon) where a number of chromatographic
materials are embedded (42). The choice of different
MALDI matrices offers the possibility to produce spe-
cific spectra profiles depending on the ionisation
properties of the analytes of interest. The more com-
mon organic molecules employed as a matrix for
sample ionisation include: sinapinic acid, DHB, CHCA,
etc. (43, 44). Some of the most abundant proteins in
human CSF are proteins with low molecular weight
such as TTR (1000 fmol/mL), cystatin C (400 fmol/mL),
and b2-microglobulin (90 fmol/mL), at 13,761, 13,343
and 11,729 Da, respectively (6). In fact, in the 5–20 kDa
mass range they are the principal signals present in
the MALDI-TOF spectra. Figure 2 shows a classical
MALDI-TOF-MS profile of CSF of a healthy control
subject in the 5–20 kDa range, using sinapinic acid as
matrix, TFA as ionic coupling and ZipTip C4 as a solid
phase extraction. Under these experimental condi-
tions, there are about 20 signals in the spectrum. The
signals in the low m/z region of the spectrum corre-
spond to the double charged most abundant proteins
(Mq2Hq) and other unknown polypeptides. The sig-
nal at m/zcalcs11,728 Th for (MqH)q was assigned to
b2-microglobulin, a non-covalently bound part of the
class I major histocompatibility complex (MCH) (35,
45). Mass accuracy after external calibration usually
rises by "1 Da for the peaks of free b2-microglobulin
with a typical peak resolution higher than 1000
FWHM. The two observed peaks at m/zcalcs13,344
and 13,360 Th for (MqH)q shown in panel I of Figure
2 have been previously assigned to the two species
of cystatin C in CSF, the non-hydroxylated forms and
the hydroxylated Pro3 species, respectively (46, 47).
This modification leads to a q16 Da increment in the
protein molecular mass which might match the
expected Dmsq15 Da related to the only known alle-
lic variant of cystatin C (Leu68Gln). However, this poly-

Figure 2 Linear MALDI-TOF-MS positive ion mode profile of CSF protein from a healthy control subject in the 5–20 kDa
range. Sinapinic acid was employed as desorption matrix, TFA as ionic coupling and ZipTip C4 as a solid phase extraction
support. The zoomed signals of cystatin C are shown in panel I at m/zcalcs13,344 Th and 13,360 Th for (MqH)q. Panel II
shows the TTR region of the spectrum with the free TTR single-charged ion at m/zcalcs13,761 Th and other disulphide oxidative
post-translational forms.

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D’Aguanno et al.: Separations of CSF proteins 445

morphism is correlated with a rare hereditary form of
amyloid angiopathy (HCCAA), which leads to cerebral
haemorrhage in the carrier patients (48). Multiple sig-
nals in panel II of Figure 2, were assigned with the
free TTR single-charged ion at m/zcalcs13,761 Th (46)
and other disulphide conjugate forms of TTR in which
cysteine residue at position 10 is forming a mixed S-
S bridge with free thiols present in biological fluids,
as reported by Nakanishi et al. (49). In this previous
work the oxidative post-translational form of TTR
at m/zcalcs13,879 Th was assigned with TTRq120
(cysteine-cysteine disulphide) and the m/zcalcs13,936
Th with TTRq177 (cysteinyl-glycinyl-cysteine disul-
phide).
CSF MS pattern-profiling will undoubtedly attain a
prominent and lasting position in the future in the
diagnosis of neurological diseases. However, today
the pattern-profiling proteomics methodology may
not be quite reproducible, because several lines of
evidence indicate that there are a number of clinical
and analytical chemistry factors that are major
sources of variability and bias (50) (CSF sample col-
lection, preparation, storage and handling, sample
extraction, etc.). In a previous work published by Car-
rette et al. (30), it was highlighted that a possible N-
terminal amino acid degradation of cystatin C in CSF
occurred when the sample was stored for 3 months
at y208C, whereas this cleavage did not occur when
stored at y808C. This truncation occurred in all the
CSF samples analysed irrespective of the underlying
neurological status, indicating a storage-related phe-
nomenon rather than a physiological or pathological
processing of the protein. It determines a modifica-
tion in the CSF protein profiling showing the appear-
ance of a new form of the protein at 12,536 Th (loss
of the first 8 amino acids) that can be carried out by
an artefact result. It may alter the level of the intact
protein and could be confused with a biomarker of the
specific disease. Some other alteration processes can
occur such as oxidation«etc., therefore the funda-
mental step for the proteomics study is the optimal
pre-analytical storage condition of any sample. Future
perspectives focus on standardising sample prepara-
tion and preservation, and reliable methods validation
procedure in order to evaluate the significance of pro-
tein-profiling proteomics in the clinical practice. Direct
linear MALDI-TOF-MS approach could become a use-
ful tool for this biological validation procedure. It may
be a rapid investigation for checking the possible
sample alterations by quality control signals, before a
more extensive 2-DE analysis.
Biomarker discovery in CSF
The introduction of such a large platform of molecular
investigations, based on the electrophoretic separa-
tion of CSF proteins, has already produced a number
of valuable evidences in the biomarker discovery
field. Some of the most interesting works are reported
in Table 5.
Dementias
In the area of neurodegenerative disorders proteomic
technologies in order to explore novel molecular bio-
markers have been particularly applied in the
research of differential diagnosis of dementias. These
efforts have been particularly focused on the defini-
tion of possible early diagnosis markers of AD. This
is the main cause of senile dementia and its degen-

Table 5 Molecular markers identified in CSF.

Neurologic disorder Potential protein markers References

Alzheimer’s disease and b-amyloid, hyperphosphorylated Tau protein, (1, 3, 12, 22, 30, 46)
very mild dementia a1b-glycoprotein, prostaglandin D2 synthase,
cystatin C and b2-microglobulin,
thioredoxin, chitinase 3-like 1

Frontotemporal Granin-like neuroendocrine precursor, (51)

dementia pigment epithelium-derived factor, retinol-
binding protein, apolipoprotein E,
haptoglobin, albumin

Multiple sclerosis Oligoclonal bands, psoriasin, NB-1, annexin (9, 52, 53)
1, EWI-2, NCP-2, semenogelins 1 and 2,
complement factor H-related protein 1/FHR-1,
procollagen C-proteinase enhancer protein,
aldolase A, N-acetyllactosaminide b-1,3-N-
acetylglucosaminyltransferase, tetranectin,
cystatin A, superoxide dismutase 3 and
glutathione peroxidase.
CRTAC-1B, tetranectin, SPARC-like protein,
autotaxin t

Breast cancer Autoantibodies anti-Yo and anti-Hu (10, 11, 54)

Creutzfeld-Jakob disease 14–3–3, cystatin C (55, 56)

Parkinson’s disease TNF-a, IL-1b, -2, -4, -6, transforming growth (7)
factor -a, -b1, and b2

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446 D’Aguanno et al.: Separations of CSF proteins
erative process is supposed to initiate 20–30 years
before the clinical onset of the disease (3). However,
prior to pursuing any cross-sectional clinical investi-
gation, CSF samples should be compared between
individuals to identify patterns of proteins associated
with inter-individual variability. It is also important to
examine the variation within individuals over a short
period of time so that one can better interpret poten-
tial changes in CSF between individuals, as well as
changes within a given individual over a longer time
span. In order to study these aspects, Hu et al. (18)
analysed 12 CSF samples, composed of pairs of sam-
ples from six individuals, obtained 2 weeks apart, by
2-D DIGE. For a differential proteome analysis it is
necessary to increase reproducibility between 2-DE
gels so that the 2-D DIGE technique seems to be the
better choice to minimise variability of spot resolving.
The inclusion of an internal standard of pooled
samples on all gels allows for improved inter-gel
alignment of gel features and relative quantification
of spot volumes (23). Proteins identified by this study
that vary in abundance in two or more individuals
over a 2-week time period are prostaglandin D2 syn-
thase, TTR, apoE, chromogranin A, chromogranin B,
semaphorin L and scrapie responsive protein 1. The
same authors compared four clinical dementia rating
(CDR) scores of 0 (cognitively normal) individuals and
two CDR 0.5 (very mild dementia, believed to be clin-
ically due to AD). Eleven of the 13 spots selected were
found to represent eight different proteins. Interest-
ingly, four of these proteins have been shown to have
altered levels in AD CSF, including a1b-glycoprotein,
prostaglandin D2 synthase, cystatin C and b2-micro-
globulin (46). Consistent with previous studies, a1b-
glycoprotein is decreased in the CDR 0.5 group, while
cystatin C and b2-microglobulin are increased (57).
Prostaglandin D2 synthase was found to increase in
the CDR 0.5 group, while another study reported a
decrease of prostaglandin D2 synthase (58). They
observed an increase in thioredoxin level in the CDR
0.5 group, while Lovell et al. (59) reported a decrease
in this protein in AD brain by Western blot. Intrigu-
ingly, several isoforms of chitinase 3-like, 18 also
known as GP-39 cartilage protein (60), were found to
be increased in the CDR 0.5 group. This protein is pri-
marily produced by human chondrocytes and syno-
vial fibroblasts and has been shown to be a target
antigen in patients with rheumatoid arthritis (61). As
for AD, FTD is difficult to diagnose based on beha-
vioural tests and is often mistaken for AD or other
psychologic disorders in elderly patients. Analysis of
CSF from FTD patients and controls using 2-DE fol-
lowed by MS characterisation revealed that the levels
of granin-like neuroendocrine precursor (Pro-SAAS),
pigment epithelium-derived factor (PEDF), RBP, apoE,
haptoglobin and albumin are significantly altered in
FTD patients (51).
Carrette et al. (46) employed SELDI-TOF-MS with
strong anionic exchange protein chips, and detected
four over-expressed and one under-expressed poly-
peptide in AD CSF. These polypeptides were further
identified as cystatin C, two b2-microglobulin iso-
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forms, a VGF polypeptide and an unnamed polypep-
tide (46). All of the markers found in this investigation
have been also highlighted in the work of Wittke et al.
using a CE-MS approach (29). However, the N-termi-
nal cleavage of cystatin C, as we have previously dis-
cussed, is due to a pre-analytical phase artefact (30).
Cancer
Linear MALDI-TOF-MS was also employed by Dekker
et al. to investigate tryptic peptide profiles in total CSF
to diagnose leptomeningeal metastases in breast can-
cer patients (54). They studied CSF samples from 106
patients and 45 controls. In this study, total proteins
were first digested with trypsin and the resulting pep-
tides were measured by MALDI-TOF-MS. Then the
resulting mass profiles were processed and com-
pared by newly developed bioinformatic tools. A total
of 164 discriminating peaks were detected and it dis-
tinguishes breast cancer patients with and without
leptomeningeal metastasis with an accuracy of 77%,
sensitivity of 79%, and specificity of 76% (54). Cystatin
C was also considered a potential biomarker for the
diagnosis of CJD (55), where in four patients an ove-
rexpression of the protein was found in respect to the
control group (ns4).
Prion
The prion protein, which typically, in mono-dimen-
sional SDS-PAGE, was resolved into three major
bands (the diglycosylated form Mr ca. 34 kDa, the
mono-glycosylated, Mr ca. 30 kDa and the unglyco-
sylated species, Mr ca. 27 kDa), accompanied by some
minor truncated forms (Mr in the range 18–22 kDa).
In 2-DE maps the prion protein gives origin to strings
of some 60 spots, covering the pI 4–8 range, in the
same Mr interval, indicating a most intriguing and var-
iegated glycosylation pattern. Upon 2-DE mapping of
CSF and immunoblot analysis, Righetti et al. (56)
could identify a major spot (pI 4.8, Mr 30 kDa) fol-
lowed by some two to three minor spots (pIs 5.0–6.0,
same Mr value) of the same 14–3–3 anti-apoptotic
protein. By this test, CJD could be differentiated from
all the other degenerative dementias, which are
14–3–3 negative (in CJD, the rapid and massive brain
cell damage releases large quantities of 14–3–3 in the
CSF). Another protein that appears very promising as
a marker for CJD is cystatin C, which has been report-
ed strongly up-regulated in this pathology (56). How-
ever, the high susceptibility of this protein to
proteolysis may affect the clinical value of this marker
(30).
Multiple sclerosis
Dumont et al. (52) tried to construct a protein map of
2-DE separated CSF proteins from five patients affect-
ed by multiple sclerosis. This results in a disease pro-
teins database where there is no differential analysis
of the protein profiling among patients and normal
individuals. By means of LC-MS/MS, 65 different pro-
teins were identified from 300 spots. Fifteen proteins
 Article in press - uncorrected proof
have not been reported in CSF 2-DE studies by other
investigators: psoriasin, NB-1, annexin 1, EWI-2, NCP-
2, semenogelins (SEM)1 and 2, complement factor H-
related protein 1/FHR-1, procollagen C-proteinase
enhancer protein (PCPE), aldolase A, N-acetyllactos-
aminide b-1,3-N-acetylglucosaminyltransferase, tet-
ranectin (TETN), cystatin A, superoxide dismutase3
and glutathione peroxidase. Hammack et al. (53) per-
formed 2-DE and peptide mass fingerprinting to iden-
tify proteins from CSF pooled from three multiple
sclerosis patients and CSF pooled from three patients
with non-multiple sclerosis inflammatory central
nervous system disorders. Resolution of CSF proteins
on three pH gradients (3–10, 4–7 and 6–11) enabled
identification of a total of 430 spots in the multiple
sclerosis CSF proteome that represented 61 distinct
proteins. The gels containing MS CSF revealed 103
protein spots that were not seen on control gels. All
but four of these 103 spots were proteins known to
be present in normal human CSF. The four exceptions
were: CRTAC-1B (cartilage acidic protein), tetranectin
(a plasminogen-binding protein), SPARC-like protein
(a calcium binding cell signaling glycoprotein), and
autotaxin t (a phosphodiesterase). It remains un-
known whether these four proteins are related to the
cause and pathogenesis of the disease considered.
Sample size and statistical power
In order to perform a clinical screening as a good
base for translational research, it is fundamental to
collect enough samples to assign a value of statistical
significance to each of the numerous identified spots
in 2-DE image analysis. Effective statistical methods
should be applied to proteomic investigations in
order to establish fixed parameters in analysing data
on the base of the specific aim of the investigation
and the sample size. Hu et al. (18) performed a com-
parative proteomic analysis of six individuals belong-
ing to two groups: four cognitively normal and two
very mildly demented individuals. The authors con-
sidered a limited subset of 306 spots across six gels
(one for each patient), among which 13 spots were
found to be statistically different, according to the t-
test. The same authors claimed that these preliminary
differences need to be validated with a much larger
sample set. In particular, they calculate that if a par-
ticular marker differs in level by 50% between groups
and the standard deviation is 30% of the mean for
both groups, one could detect a significant difference
(as0.05 and a power of 0.8) with a simple size of
ns6. This is based on a classical approach for the
calculation of the sample size for clinical investiga-
tions. However, in the field of the genetic studies, dif-
ferent authors suggest a more robust statistical
approach, capable of calculating the sample size for
experiments in which the final data analysis will con-
sist of performing many one-way ANOVA analyses
and using the false discovery rate (FDR) to account
for multiple testing (62). For example, by applying the
ANOVA test to the work of Hu et al. (18), assuming 13
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D’Aguanno et al.: Separations of CSF proteins 447
significantly different spots and 293 not significantly
different, and following the methods proposed by
Pouns and Cheng (62), one can estimate that it would
be necessary to enroll 26 individuals, 13 for each
group. These numbers reflect a maximum level for
FDR of 0.05 and a statistical power near 80%. Unfor-
tunately, this calculation lacks some parameters. In
fact, we can just hypothesise that the ratio of the sum
of squared differences between each group mean and
an overall mean to within-group variance is about 1.6
for the 13 significant tests. This information would
require access to all the parameters which characte-
rise each protein spot, data which are generally not
given in proteomic articles at the present time. Nev-
ertheless, on the basis of the methods of Benjamini
and Hochberg (63), one can determine an a-value as
the p-value cut-off; at this a-value of alpha we antici-
pate that no more than 2.9% of the significant protein
spots will be false discoveries and the 80% of the true
alternatives will be declared significant. The fixed val-
ue of the power, in fact, indicates the desired propor-
tion of true alternatives to be detected as significant.
Considering these conditions as the starting point, but
assuming that the ratio of the sum of squared differ-
ences between each group mean and an overall mean
to within-group variance is 1 (a theoretical situation),
we obtain a total sample number of 19 individuals for
each group. If a study has a reduced number of the
samples (6 individuals instead of 26), the resulting
power is dramatically lowered. In fact, based on the
method that considers the FDR value and assuming a
within-group variance of about 1.6, the resulting pow-
er of the study of Hu et al. would be approximately
10%. This calculation is purely speculative since we
do not have direct access to the raw data, however,
it might suggest the terrific impact of the sample size
in proteomic investigations.
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Received October 10, 2006, accepted January 17, 2007