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Cleavage of cystatin C is not associated with multiple sclerosis.

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Cleavage of Cystatin C Is
Not Associated with
Multiple Sclerosis
Piero Del Boccio, PhD,1–3 Damiana Pieragostino, BSc,1–3
Alessandra Lugaresi, MD, PhD,1,4 Maria Di Ioia, MD,1,4
Barbara Pavone, BSc,1–3 Daniela Travaglini, MD,1,4
Simona D’Aguanno, PhD,5,6
Sergio Bernardini, MD, PhD,5,6 Paolo Sacchetta, PhD,1,2
Giorgio Federici, MD, PhD,3,5,6 Carmine Di Ilio, PhD,1,2
Domenico Gambi, MD,1,4 and Andrea Urbani, PhD1–3
Recently, Irani and colleagues proposed a C-terminal cleaved
isoform cystatin C (12.5kDa) in cerebrospinal fluid as a
marker of multiple sclerosis. In this study, we demonstrate
that the 12.5kDa product of cystatin C is formed by degradation of the first eight N-terminal residues. Moreover, such
a degradation is not specific in the cerebrospinal fluid of
multiple sclerosis, but rather is given by an inappropriate
sample storage at ⫺20°C. We conclude that the use of the
12.5kDa product of cystatin C in cerebrospinal fluid might
lead to a fallacious diagnosis of multiple sclerosis. Preanalytical validation procedure is mandatory for proteomics investigations.
Ann Neurol 2007;62:201–204
Multiple sclerosis (MS) is an often disabling demyelinating disease of the central nervous system. The cause
of MS has not been fully elucidated, although genetic,
environmental, and immunological factors play a
pathogenetic role.1– 4 A definite diagnosis is still difficult to ascertain, especially at disease onset, for the lack
of reliable molecular markers.
A clinical proteomics investigation on biofluids is
currently largely pursued to tentatively identify protein
profiles that are diagnostic or prognostic indicators of
disease course.5–7
Linear matrix-assisted laser desorption ionization
time-of-flight mass spectrometry (MALDI-TOF-MS)
has already been used recently, in the version of
surface-enhanced laser desorption ionization TOF-MS,
by several groups to detect potential novel biomarkers
of several neurological disorders8 –11 in cerebrospinal
fluid (CSF) samples. However, pattern-profiling proteomics methodology may not be quite reproducible,
because a number of clinical and preanalytical chemistry factors constitute major sources of variability and
Recently, Irani and colleagues14 reported a selective
C-terminal cleavage of cystatin C with a mass signal in
the spectrum at 12.5kDa, in the CSF of a subgroup of
patients with MS and clinically isolated syndromes. In
this work, surface-enhanced laser desorption ionization
TOF-MS was used to investigate the differential protein profiling from various neurological diseases. This
cystatin C isoform was 100% specific for a subgroup of
MS and clinically isolated syndromes patients, and the
authors suggest an adaptive host response that may also
be able to differentiate these patients’ diagnoses from
other inflammatory diseases.
In contrast, Carrette and coworkers15 showed an
N-terminal truncated cystatin C at m/zcalc ⫽ 12.5kDa
in human CSF as a result of long-term storage at
⫺20°C in healthy subjects and patients with dementia
of various origin. These two reports raise many controversies about the actual role of truncated cystatin C
and also about the site of truncation. We further investigated this finding using linear MALDI-TOF-MS
technology to study the CSF from MS patients compared with healthy control subjects and patients with
other neurological diseases.
Subjects and Methods
Patients and Cerebrospinal Fluid Collection
From the 1Centro Studi sull’Invecchiamento, Fondazione “G.
D’Annunzio,” Chieti; 2Dipartimento di Scienze Biomediche, Università “G. D’Annunzio” di Chieti e Pescara; 3Centro Europeo
Ricerca sul Cervello, IRCCS-Fondazione S. Lucia, Roma; 4Dipartimento di Oncologia e Neuroscienze, Università “G. D’Annunzio”
di Chieti e Pescara; 5Dipartimento di Medicina di Laboratorio, Policlinico di Tor Vergata; and 6Dipartimento di Medicina Interna,
Università di Roma Tor Vergata, Roma, Italy.
Received Jun 12, 2006, and in revised form Jul 25. Accepted for
publication Aug 4.
This article includes supplementary materials available via the internet at
Published online Sep 27, 2006, in Wiley InterScience
( DOI: 10.1002/ana.20968
Address correspondence to Dr Urbani, Centro Studi
sull’invecchiamento (Ce.S.I.), Centre of Excellence on Aging, Università degli Studi di Chieti e Pescara, Via Colle dell’ Ara, Chieti
Scalo, 66100, Chieti, Italy. E-mail:
Thirty-seven patients with definite MS16,17 were included in
this study (complete clinical data are available online in the
supplementary material). Expanded Disability Status score
was obtained at the time of CSF acquisition. Clinical diagnosis was confirmed by cerebral and spinal magnetic resonance imaging studies and the presence of oligoclonal bands
in CSF. Patients had not been treated with steroids in the
month before study entry.
Thirty CSF samples were used as control group: 5 healthy
control subjects (ie, subjects undergoing spinal anesthesia for
orthopaedic noninflammatory conditions; 1 female and 4
male subjects); 11 patients with dementia, including 3 patients with suspected Alzheimer’s disease; 6 patients with
Guillain–Barré syndrome; 5 stroke patients; 1 patient with
amyotrophic lateral sclerosis; 1 case of encephalomyelitis; and
1 case of Leber’s disease. The diagnosis in each of these patients was defined according to individual disease diagnostic
criteria. The study was conducted in accordance with the
© 2006 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
principles of the Helsinki Declaration (World Medical Association, 1997).
After lumbar puncture, each sample was centrifuged at
10,000g for 10 minutes, transferred in polypropylene tubes,
and stored at ⫺80°C or ⫺20°C depending on the specific
Linear Matrix-Assisted Laser Desorption Ionization
Time-of-Flight Mass Spectrometric Analysis
Sample (10␮l CSF with 10␮l TFA 0.1%) preparation was
performed by ZipTip (Millipore tip size P10; Millipore,
Bedford, MA) C4 tips, as reported previously.11 All analyses
were performed with a Reflex IV MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany).
Cerebrospinal Fluid Protein Purification
and Identification
CSF samples stored at ⫺20°C for 3 months were fractionated by ultrafiltration using Microcon (Millipore) to enrich
proteins with a molecular mass in the 10- to 30kDa range.
This liquid portion was separated and fractionated by
high-performance liquid chromatography mass spectrometry.
Forty liquid fractions were collected from 24 to 50 minutes
of the chromatographic run. Fractions containing the 12,536
Thompson (Th) protein signal were digested with trypsin
and used for protein identification by mass spectrometric
analysis, as reported previously.11 The routine FindPept Tool
software ( was used to
calculate the accurate mass differences in the protein sequence.
Cystatin C Signals in Cerebrospinal Fluid MatrixAssisted Laser Desorption Ionization Mass Spectra
In Figure 1, the mass spectra is highlighted in the region of the cystatin C signal obtained from the CSF
samples of a MS patient and healthy control subjects,
analyzed after 3 months of storage at ⫺80°C (see Figs
1A, B) and after 3 months at ⫺20°C (see Figs 1C, D).
In Figures 1C and D, an unidentified peak at
m/zcalc ⫽ 12,536 Th appears that is absent in the mass
spectra acquired after storage at ⫺80°C. This signal
was associated with a truncated isoform of cystatin C,
as Carrette and coworkers15 and Irani and colleagues14
reported. In these two works, conflicting data are presented about the site of truncation and the functional
role of this modified isoform.
Our experiments univocally assign the 12,536 Th
present in ⫺20°C stored samples as cystatin C (National Center for Biotechnology Information accession
number gi兩14278690). Further experiments demonstrate that the site of truncation is in the N-terminal
region of the protein and result from the loss of the
1-8 peptide (SSPGKPPR) (complete mass spectrometry
characterization available online in the supplementary
Annals of Neurology
Vol 62
No 2
August 2007
Fig 1. Mass spectra region of the cystatin C signals obtained
from the same cerebrospinal fluid (CSF) samples of a multiple
sclerosis (MS) patient and a healthy control subject analyzed
after 3 months of storage at ⫺80°C (A: MS patient; B:
healthy control subject) and after 3 months at ⫺20°C (C:
MS patient; D: healthy control subject). (C, D) The peak at
m/zcalc ⫽ 12,536 Th is highlighted, associated with a truncated isoform of cystatin C, as reported previously.
N-Truncated Cystatin C as a Marker of Ex Vivo
Degradation Processes
The relative intensity ratio of the m/zcalc ⫽ 12,536 Th
versus m/zcalc ⫽ 13,360 Th forms of cystatin C in the
CSF of MS patients has been investigated on a cohort
of 21 subjects. These data have been compared with a
control group.
The t test was applied to discriminate the two
groups investigated showing a nonincreased relative ratio of this truncated form ( p ⫽ 0.28) in MS patients
(Fig 2). However, the N-truncated cystatin C is always
absent in spectra obtained from CSF samples stored at
⫺80°C, and the relative intensity ratio was measured
considering the background level at the specific m/z
Subsequently, we analyzed eight selected CSF samples from MS patients after a 3-month storage at
⫺80°C versus aliquots of the same samples kept 3
months at ⫺20°C. The relative intensity ratio of the
12,536 signal versus 13,360 signal reported in Figure
3A clearly shows the selective ex vivo generation of this
isoform when CSF samples are stored at ⫺20°C ( p ⬍
The in vitro N-truncation of cystatin C occurred in
truncated form of cystatin C at m/zcalc ⫽ 12,536 Th,
which was always absent in the fresh samples or in
samples kept at ⫺80°C, in all CSF samples investigated when left for more than 10 days at ⫺20°C. This
truncation occurred in all the CSF samples analyzed
regardless of the underlying neurological status, indicating a storage-related phenomenon rather than physiological or pathological processing of the protein. Our
data are in agreement with Carrette and coworkers’
findings15 in dementia, and demonstrate that such a
degradation occurs at ⫺20°C also in MS patients,
causing an erroneous interpretation of this truncated
form of cystatin C as a biomarker for MS. Subsequent
investigations allowed the identification of the site of
Fig 2. Relative intensity ratio distribution of cystatin C truncated form/cystatin C full length (12,536/13,360) in the investigated groups (multiple sclerosis [MS] vs control group
[CG]). The CG includes 5 healthy control subjects, 10 patients with dementia, 5 patients with Guillain–Barré syndrome, 2 stroke patients, 1 patient with amyotrophic lateral
sclerosis, and 1 case of encephalomyelitis. Samples stored at
⫺80°C were analyzed immediately after thawing to avoid
oxidation or other artifact modifications.
all CSF samples stored at ⫺20°C independently from
the diagnosis. In fact, the truncated form of the protein
appeared (see Fig 3B) in samples kept ⫺20°C already
after 10 days, either belonging from MS patients (n ⫽
16) or control group (n ⫽ 6).
Irani and colleagues14 describe a C-terminal cleaved
cystatin C in CSF as a marker of MS by measuring the
relative intensity ratio of the truncated form 12.5kDa
versus the intact molecule at 13.6kDa by surfaceenhanced laser desorption ionization TOF-MS. In a
previous study, Carrette and coworkers15 reported an
N-truncated isoform of cystatin C with mass of
12.5kDa in CSF samples from patients with various
dementia diseases when the CSF specimens were stored
3 months at ⫺20°C. However, addressing the same
cleaved form at 12.5kDa cystatin C, these data are in
contrast either for the site of the truncation or for the
clinical functional insight. Here, we have pursued a
thorough characterization of this phenomenon, exploring both the distribution and preanalytical requirement
of the truncated cystatin C in CSF of MS patients and
patients with other neurological diseases. The linear
MALDI-TOF-MS data region of the cystatin C (see
Fig 1) was not characterized by any statistical difference in the protein profiling in all patients investigated
regardless of the diagnosis, not reproducing the data
showed in the previous work of Irani and colleagues.14
Our results show an N-terminal eight-amino acid–
Fig 3. (A) Box and whisker plot of the relative intensity ratio
of the 12,536 versus 13,360 signal on 8 selected cerebrospinal
fluid (CSF) samples from multiple sclerosis (MS) patients after
3 months of storage at ⫺80°C (A) versus aliquots of the same
samples kept 3 months at ⫺20°C (B). (B) Comparison of the
relative intensity ratio of CSF samples kept more than 3
months at ⫺20°C versus samples stored more than 3 months
at ⫺80°C. Control group (CG) included patient affected by
dementia (n ⫽ 1), patients with cerebral vascular diseases
(n ⫽ 3), patients with Guillain–Barré syndrome (n ⫽1), and
Leber’s hereditary optic neuropathy case (n ⫽ 1).
Del Boccio et al: Cystatin C Degradation in CSF
truncation in the N-terminal region, resulting in the
loss of the 1-8 peptide (SSPGKPPR).
In conclusion, direct MALDI-TOF-MS approach
for proteomics investigations may lead to the development of novel laboratory tests for clinical molecular
markers, but preanalytical processing standardization is
a mandatory step for clinical proteomics studies, to
avoid erroneous identification of biomarkers.
This study was supported by the Italian National Grant Program
(“Progetto Ricerca Finalizzata; IRCCS-S. Lucia #186,” G.F.,
C.D.I.) and by Italian Minister of Research (PRIN
2004055300_003, G.F., A.U.).
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