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Articles |
1
Institute of Clinical Neuroscience, Department of Psychiatry and Neurochemistry, and
2
Department of Neurology, Göteborg University, Sahlgrenska University Hospital, SE-43/#80 Mölndal, Sweden.
3
Institute of Chemistry, Department of Analytical
Chemistry, Uppsala University, PO Box 531, SE-751 21 Uppsala, Sweden.
a Author for correspondence. Fax 46-18-471-3692; e-mail jonas.bergquist{at}kemi.uu.se
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Met) in the protein transthyretin. Mass
spectrometric analysis can identify modification of proteins, such as
point mutations, acetylation, phosphorylation, sulfation, oxidation,
and glycosylation.
Methods: Matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF MS) spectra from
cerebrospinal fluid (CSF) drawn from a patient with FAP were compared
with CSF from controls. We also isolated transthyretin with a
Centrisart molecular size cutoff filter and performed high-accuracy
peptide mass mapping to localize the site of the amino acid
substitution (Val30Met).
Results: Mass spectra of transthyretin were produced directly from human CSF as well as from CSF after a simple prepurification method without immunoprecipitation. On-target tryptic digestion and MALDI-MS verified mass spectrometric peak identification. The point mutation was still detectable in CSF after hepatic transplantation.
Conclusions: It is possible to diagnose FAP by a rapid MALDI-TOF MS analysis using only 100 µL of CSF, with only 250 nL actually consumed on target. The approach may also be useful to monitor production of mutated transthyretin by choroid plexus, especially after liver transplantation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Met) (1)(2)(3)(4). The
amyloid-generating mechanism has not yet been totally clarified, but it
is hypothesized that the single amino acid substitution produces a
confirmation change into an unstable amyloidogenic variant of the TTR
tetramer. The unstable form of tetramer undergoes a partial acid
denaturation that leads to the formation of monomeric intermediates
that can aggregate to form amyloid fibrils. The dominant symptom
resulting from the fibril formation is a progressive peripheral
neuropathy, although the age at onset and symptomatology are dependent
on the genetic background (5). Amyloidosis in the cardiac
conduction system, the gastrointestinal tract, and the vitreous body
often is symptomatic, whereas amyloid deposits in the kidney and
adrenals are usually asymptomatic. Until recently, the disease
was untreatable, leading to death, but liver transplantations have been
carried out worldwide with encouraging results. The progression of the
FAP polyneuropathy seems to be completely halted by liver
transplantation, but the observation time since the first liver
transplantation has been <10 years. A rapid screening system of
patients with suspicious TTR-related amyloidosis is therefore of great
importance. Mass spectrometric analysis often can identify modification of proteins, such as point mutations, acetylation, phosphorylation, sulfation, oxidation, and glycosylation (6)(7)(8)(9). However, mass spectrometric methods in general have relatively low tolerance for salts, buffers, and other sample impurities. One feature of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that makes it especially promising for mass spectrometric analysis of biological samples is its ability to detect biomolecules in complex mixtures in the presence of large molar excesses of salts, buffers, and other species (10)(11). Because of these qualities, MALDI MS has been utilized to study proteins and peptides in serum, blood, urine, tissue extracts, and whole cells (12)(13)(14)(15)(16). MALDI-TOF MS, introduced 1987 by Karas et al. (17), also has a very high mass range, up to at least 1 MDa, and the mass resolution is sufficient to separate protonated from sodium-containing molecular ions up to at least 10 kDa (18)(19)(20). Sequence information is especially important for the identification of proteins and may be obtained by MALDI MS analysis together with the use of enzymatic digestion followed by postsource decay of the resulting peptides or database-based peptide mapping (21)(22)(23).
We present results from MS analysis of TTR in human cerebrospinal fluid
(CSF) directly as well as from CSF separated after a simple
prepurification method. This method does not include any need for
immunoprecipitation. On-target tryptic digestion and MALDI MS verified
mass spectrometric peak identification. The total analysis can be
performed from only 100 µL of CSF (the MALDI analysis consumes only
250 nL of CSF, allowing multiple re-analysis). The method was
successfully demonstrated using CSF from a patient with a known TTR
mutation (FAP type I; Val30Met).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Case history for the FAP patient.
The female patient, born in
1964, is of Portuguese extraction (from the Oporto area, the focus of
Portuguese FAP, or Andrade neuropathy) and now lives in Göteborg,
Sweden. Several members of her family suffer from FAP. Her brother, who
suffered from a more advanced FAP caused by the TTR variant
Val30Met mutation, which is the most common
mutation that causes FAP, was earlier treated with liver
transplantation (24)(25). Beginning in March
1993, the FAP patient experienced para- and subsequently
tetrahypesthesia, which progressed slowly. Her gait was normal, and she
was able to walk a long distance although she was fatigable. She
suffered from some deep pain in her legs, particularly at rest. Repeat
electromyography showed a progressive reduction of motor response
amplitudes in her feet and probably in her hands. A routine CSF
examination was normal. The glucose load test results and urine amino
acid excretion were normal, and porphyrins were negative. An amyloid
123I-SAP scan (performed by Prof. M. Pepys at Hammersmith
Hospital, London, UK) showed evidence of amyloid deposits in the
kidneys. DNA analysis and Southern blot (performed by Prof. G.
Holmgren, Umeå University Hospital, Umeå, Sweden) showed that the
patient carries the TTR variant Val30Met (FAP)
mutation.
The patient received a liver transplant on July 7, 1995. Her neurological condition stabilized, and repeat quantitative neurological examination at 4-month intervals showed a slight stationary neurological deficit. Neurophysiological examination on March 16, 1998 disclosed that the amplitude reduction had essentially stopped after the liver transplantation. Nerve conduction velocity was normal, and all other values were unchanged. The liver transplantation and subsequent immunosuppression were uncomplicated apart from a meningitis in November 1997. Virological analysis showed a probable Epstein-Barr infection. The CSF inflammatory markers had normalized in the sample for the present study, which was obtained on November 11, 1998.
Control CSF.
Control samples were obtained from patients
without symptoms or signs of major neurological or psychiatric
disorders, who were undergoing lumbar puncture for diagnostic purposes.
Routine CSF analyses gave values within the reference
intervals, without any signs of inflammation or damage to the
blood-brain barrier function.
centrisart molecular size cutoff filter
To perform a simple prepurification of the TTR, we used a
Centrisart (Sartorius AG) centrifuge membrane with a molecular
cutoff at 100 kDa. The membrane was prerinsed and wetted with water
before 100 µL of CSF and 100 µL of water were put into the
centrifuge tube. The floater with the membrane was then re-inserted.
After centrifugation for 10 min at 2500g (4 °C), the
supernatant was discarded and 100 µL of water was added to the CSF
below the membrane. After centrifugation for 10 min at 2500g
(4 °C), the supernatant was discarded and the desalted concentrate
was used for MALDI-MS analysis.
maldi ms
Materials.
Angiotensin II (1045.5 amu), corticotropin
fragment 18–39 (2464.2 amu), equine cytochrome c (12 360.1 amu),
equine myoglobin (16 951.5 amu), and porcine trypsin (23 463.5 amu)
were purchased from Sigma Chemical Company and used as calibrators.
Cytochrome c (1.7 µmol/L) and myoglobin (3.3 µmol/L)
dissolved in 1 g/L trifluoroacetic acid (TFA) in ultrapure
water (MilliQ Plus; Millipore) were used as an internal protein
calibration solution. For internal calibration of proteins in filtered
CSF, 10 µL of filtered CSF was mixed with 5 µL of protein
calibration solution. The MALDI matrices used were
3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid), purchased from
Fluka Chemie AG, and 
-cyano-4-hydroxy-cinnamic acid (CHCA),
purchased from Aldrich Chemie GmbH. Sinapinic acid (30 g/L, saturated)
and CHCA (15 g/L, saturated) were dissolved in 1 g/L TFA in
acetonitrile-water (1:1, by volume).
Sample preparation.
Samples were prepared by the so-called
seed layer method (26): A matrix seed layer first was
created by depositing a droplet (0.5 µL) of a 1 g/L solution of
matrix dissolved in acetonitrile on the highly polished, stainless
steel sample probe. Thereafter, equal volumes (5 µL) of the matrix
and analyte solutions were mixed in a test tube, and a droplet (0.5
µL; i.e., 250 nL of CSF) of matrix-analyte mixture was deposited on
the matrix seed layer. The sample was then left to dry completely in
air.
Enzymatic digestion.
After initial MALDI analysis of
undigested TTR in CSF, selected fractions on the target were digested
using on-target digestion: To the selected sample spot, 2 µL of
trypsin was added (Promega; 0.1 g/L in 0.1 g/L HCl was diluted
1:100 in 100 mmol/L fresh
NH4HCO3). The protein
content in each spot was calculated to be 
15–20 ng, so the amount
of trypsin was 100 pg/ng. The target was placed in a moist
chamber at 37 °C for 1 h, and then dried at room temperature;
the sample spot was then dissolved in 1 µL of 1 g/L TFA in
acetonitrile-water (1:1, by volume) and dried before MALDI-MS analysis.
Apparatus.
All MALDI analyses were performed with a Reflex
MALDI-TOF mass spectrometer (Bruker-Franzen Analytik GmbH). Samples
were irradiated with a nitrogen laser (VSL-337; Laser Science). A
circular gradient neutral density filter (cat. no. 28650;
Oriel) permitted continuous attenuation of the laser beam down to 1%
of the laser’s output energy. The ion source and flight tube were
evacuated by turbo pumps to a pressure of <4.0 x
10-6 Pa. The instrument was equipped
with a two-stage electrostatic reflectron and a delayed extraction
(time-lag-focusing) ion source. Spectra were acquired in linear mode at
an accelerating voltage of 25 kV, and in the reflected mode at an
accelerating voltage of 20 kV. Mass spectra were analyzed using Bruker
software on a Sun Sparcstation. The sample potential/first-electrode
potential ratio was optimized to achieve optimal resolution for the
studied proteins/peptides. The sample probe was made of highly polished
stainless steel. Because the spectrometer was equipped with a video
camera, visual inspection of the sample inside the Bruker Reflex II
MALDI-TOF mass spectrometer was possible.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Several peaks were detected in the range where TTR and its
conjugated forms should appear (m/z 13 700–14 100). In
the mass spectra from the controls (Fig. 1
, A–C), a peak at
approximately m/z 13 881 dominated. In previous
studies, TTR conjugated with cysteine
(m/zcalculated = 13 881.4 for
[M+H]+) was the most prominent
TTR-related peak in mass spectra from healthy subjects
(27)(28). In one of the controls (Fig. 1A
), a
peak at m/z 13 938 was discernable. This peak could be TTR
conjugated with cysteinylglycine
(m/zcalculated = 13 938.4 for
[M+H]+), a conjugated form of TTR that also had
been detected previously with mass spectrometric analysis
(29). The same m/z range in the MALDI spectrum
from CSF drawn from a patient with FAP contained two peaks at
m/z 13 762 and m/z 13 794 (Fig. 1D
). The
observed peaks could tentatively be identified as the free forms of the
wild-type TTR (m/zcalculated =
13 762.4 for [M+H]+) and the mutated
(Val30Met) TTR
(m/zcalculated = 13 794.4 for
[M+H]+).
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In addition to TTR, we observed both forms of cystatin C known to be
present in CSF, the nonhydroxylated form
(m/zcalculated = 13 344.3 for
[M+H]+) and the hydroxylated form
(m/zcalculated = 13 361.3 for
[M+H]+), together with
ß2-microglobulin,
(m/zcalculated = 11 730.3 for
[M+H]+; Fig. 1
). With external calibration, the
discrepancy between the observed and calculated masses was <200 ppm.
However, to get an indisputable identity of the TTR, we chose to
perform a rapid purification step.
maldi-ms of isolated ttr
Mass spectrometric detection of specific proteins present in low
concentrations in CSF is complicated by the high salt concentration
(140 mmol/L sodium and 120 mmol/L chloride), as well as other
compounds, e.g., lipids (15 mg/L), glucose (600 mg/L), and proteins
(350 mg/L) (30). To obtain less complex protein samples and
decrease the salt content, the CSF was fractionated using a Centrisart
molecular size cutoff filter before analysis by MALDI MS. In the mass
spectra for the filtered CSF fractions, the signal-to-noise
ratio of TTR was substantially improved compared with the mass spectra
from untreated CSF (Fig. 2
). Furthermore, two internal calibrants (cytochrome c and
myoglobin) were added to the filtered CSF to utilize internal
calibration and thereby optimize the mass accuracy. With the improved
signal-to-noise ratio and better mass accuracy, we were able to
conclude that the most likely matches for the observed peaks in the
mass spectra from the controls (Fig. 2
, A–C) were the free form of
wild-type TTR (m/zcalculated = 13 762.4
for [M+H]+), sulfated TTR
(m/zcalculated = 13 842.3 for
[M+H]+), cysteinylated TTR
(m/zcalculated = 13 881.4 for
[M+H]+), cysteinylglycine-conjugated TTR
(m/zcalculated = 13 938.4 for
[M+H]+), and glutathione-conjugated TTR
(m/zcalculated = 14 067.4 for
[M+H]+). The discrepancy between the observed
and calculated masses for the dominating peaks was typically
<100 ppm (Table 1
).
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In the MALDI spectrum for filtered CSF drawn from a patient with FAP,
the tentative matches for the observed peaks were as follows: free form
of wild-type TTR (m/zcalculated =
13 762.4 for [M+H]+), and free form of mutated
(Val30Met) TTR
(m/zcalculated = 13 794.4 for
[M+H]+). The spectrum also contained two
smaller peaks at m/z 13 882.1 and m/z 13 938.0
for [M+H]+. These peaks could possibly be
identified as TTR conjugated with cysteine
(m/zcalculated = 13 881.4 for
[M+H]+), and cysteinylglycine
(m/zcalculated = 13 938.4 for
[M+H]+).
In all MALDI spectra from filtered CSF, both from the FAP patient and from the controls, both forms of cystatin C are observed (not shown). However, the TTR peaks are the dominating peaks in the mass spectra.
maldi-ms of tryptic peptides from ttr
To verify the identification of TTR and find the site of the amino
acid substitution (Val30Met), on-target
tryptic digestion and MALDI mass spectrometric analysis were performed.
By comparing the mass spectra of tryptic peptides from the CSF of a
healthy control and a patient with FAP, we could identify the tryptic
peptides with the site of the amino acid substitution (Fig. 3
and Table 2
). The amino acid sequence coverage for TTR was 85% (108 of 127
amino acids), and the mutation was localized to amino acids 22–34.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fractionation of the CSF with a Centrisart molecular size cutoff filter before analysis substantially improved the sensitivity, and several more forms of TTR were detected. Although the molecular mass of TTR (13 762 Da) is much lower than 100 kDa, the TTR molecules are preferably trapped by this membrane (probably because of the tertiary structure of the protein or hydrophobic-hydrophilic interactions). The use of a filter with smaller cutoff (10 kDa) did not provide the same efficient purification (data not shown). In the mass spectra from the control CSF, the free form of TTR as well as dihydroxylated, sulfated, cysteine-conjugated, cysteinylglycine-conjugated, and glutathione-conjugated forms were identified. The wild-type and the mutated form of free TTR dominated the mass spectrum from FAP CSF. The mass spectrum also contained two smaller peaks that could possibly be identified as TTR conjugated with cysteine and cysteinylglycine.
We verified the identification of TTR and located the site of the amino
acid substitution (Val30Met) to amino acids
22–34 by performing on-target tryptic digestion and MALDI mass
spectrometric analysis.
The TTR conjugates observed in the mass spectra from the fractionated CSF from the controls are all linked near the NH2 terminus at Cys10 (28). No tryptic peptides from amino acid sequence 1–15 were observed in the mass spectra from the on-target tryptic digestion. Prior experiments have shown that it is difficult to observe these tryptic fragments with mass spectrometric analysis (28)(31)(32). Théberge et al. (28) suggested that because the TTR 1–9 and TTR 10–15 tryptic peptides are very hydrophilic, they elute with the solvent front when the mixture of tryptic fragments is fractionated with HPLC before MALDI-TOF MS analysis. However, this explanation is not valid for the experiment performed in this study and the experiments described in Refs. (31)(32) because no fractionation was performed between the tryptic digestion and the mass analysis. A possible explanation is that the efficiency of the enzymatic digestion is lowered by the conjugate or that the mass is altered in a nontrivial way.
Westman et al. (32) showed previously that some of the most abundant proteins in human CSF can be identified directly with MALDI-TOF MS. However, only a fraction of the many thousands of proteins present in CSF can be detected directly without fractionation or concentration. Compared with previously described methods, the method of isolating and sequencing TTR with a Centrisart molecular size cutoff filter is fast and simple, and the required CSF volume is very small (31)(32)(33). On the other hand, the method is probably less general and is not suitable for proteins present in CSF in very low concentrations (31)(32)(33).
TTR is predominantly synthesized by the liver and in the choroid plexus
(34). Little serum TTR transverses the blood-brain barrier,
and thus only a minor part of CSF TTR (<10%) is derived from the
liver (35). This fact explains why we detected
Val30Met TTR in the CSF from the
liver-transplanted FAP patient. The present method provides a means of
monitoring for possible central nervous system pathology in FAP
patients after liver transplantation, as a result of the continuous
synthesis of mutated TTR from the choroid plexus. Data have been
presented on symptoms from the meninges and the pituitary area in FAP
patients, including superficial siderosis after bleedings caused by
amyloid deposits (36)(37)(38). The possibility of monitoring CSF
TTR can thus provide a diagnostic tool in patients who have undergone
liver transplantation earlier or to follow the progress of the disorder
within the central nervous system.
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Acknowledgments |
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Footnotes |
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-cyano-4-hydroxy-cinnamic acid. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  D. de Boer, M. M. Erps, W. K.W.H. Wodzig, and M. P. van Dieijen-Visser Inadequate Attempts to Measure the Microheterogeneity of Transthyretin by Low-Resolution Mass Spectrometry Clin. Chem., July 1, 2005; 51(7): 1299 - 1300. [Full Text] [PDF]
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H. R. Bergen III, S. R. Zeldenrust, M. L. Butz, D. S. Snow, P. J. Dyck, P. J. B. Dyck, C. J. Klein, J. F. O'Brien, S. N. Thibodeau, and D. C. Muddiman Identification of Transthyretin Variants by Sequential Proteomic and Genomic Analysis Clin. Chem., September 1, 2004; 50(9): 1544 - 1552. [Abstract] [Full Text] [PDF]
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