Definitions of acute encephalopathy and febrile seizures
Because acute encephalopathy is a generic term for acute brain dysfunction usually preceded by infection, encephalopathy related to infection as acute encephalopathy was not excluded [1, 2].
Acute encephalopathy: The criteria for diagnosis of encephalopathy were 1) impaired consciousness, 2) signs of increased intracranial pressure due to brain edema, 3) convulsions and seizures, 4) slow activity on electroencephalography lasting more than 24 h after acute onset, and 5) no bacteria or fungi on CSF culture [1, 2, 9].
Febrile seizures: Febrile seizures were defined as 1) seizures with fever and impaired consciousness lasting less than 24 h without neurological sequelae, 2) usually occurring between 3 months and 5 years of age, and 3) without evidence of intracranial infection or defined cause [4–7, 9].
Two studies were performed on cerebrospinal fluid (CSF) from patients at the Department of Pediatrics, Nippon Medical School Chiba Hokusoh Hospital. In an initial study (experiment 1) for discovering the new, novel biomarkers, CSF from febrile seizure subjects (n = 28) and patients with encephalopathy (n = 8), including influenza encephalopathy (n = 3), encephalopathy due to rotavirus (n = 1), human herpes virus 6 (n = 1) were used for the SELDI analysis. In a second study (experiment 2) for validating the markers, SELDI analysis was performed on CSF from a second set of febrile seizure subjects (n = 14) and encephalopathy patients (n = 5). Diagnosis was made by at least two pediatric neurologists based on clinical findings, routine examinations described above, and lumber puncture performed at the onset of illness.
All CSF specimens were collected for diagnostic tests and the remaining portions of the specimens were used for SELDI-TOF MS investigation. Following collection, samples were centrifuged at 1500 rpm for 5 min to remove cells, divided into aliquots and immediately frozen on dry ice and stored at -80°C. The Institutional Review Board at Nippon Medical School, Chiba Hokusoh Hospital approved the collection and investigation of samples and written informed consent was obtained from all subjects.
SELDI-TOF MS is a system that enables rapid protein profiling, identification, and characterization from crude biological samples by selective capture of subclasses of proteins with specific physical or biochemical characteristics. The molecular weight as well as the quantity of individual proteins absorbed on each ProteinChip array is then directly assessed by a time-of-flight mass spectrometer, generating quantitative protein mass profiles for individual CSF specimens. Comparative protein expression profile analysis highlights any CSF protein species which are aberrantly expressed in the CSF of encephalopathy patients compared to febrile seizure subjects.
Before analysis of the CSF, experiments were designed to optimize the ProteinChip array conditions for the SELDI-TOF MS proteomics tests. Three chip types, Q10 (strong anion exchange surface), CM10 (weak cation exchange surface), and IMAC30 (metal binding surface) (Bio-Rad), were tested along with a variety of binding/washing conditions, including pH 4.5, 6.5, and 8.0 binding/washing buffers. The ProteinChip array condition that produced optimal results was the Q10 ProteinChip array with 50 mM Tris-HCl, pH 8.0 binding and wash steps, and was therefore used in this study.
For all experiments, a 96-well ProteinChip cassette-compatible bioprocessor was used. The ProteinChip array spot surface was equilibrated with wash buffer (50 mM Tris-HCl, pH 8.0) for 10 min prior to use. CSF specimens were centrifuged and fresh aliquots of the CSF specimens were stored at -80°C. For analysis, these samples were slowly thawed on ice, centrifuged (10000 × g, 4°C, 15 min). Equal amounts of CSF protein (1.5 μg) diluted with 9 volumes of washing buffer were applied to each ProteinChip array spot. ProteinChip arrays were then incubated in a humidity chamber at room temperature for 60 min with shaking at 200 rpm. Unbound proteins were removed by washing three times for 5 min with binding buffer and then briefly washed with HPLC grade water. An energy absorbing molecule (EAM), saturated sinapinic acid (SPA), in a solution containing 50% acetonitrile/5% trifluoroacetic acid, was applied to the ProteinChip array spot surface. The arrays were air dried and proteins captured on individual spots were evaluated using the PBSIIc ProteinChip reader (Bio-Rad). The ProteinChip reader was externally calibrated with protein and peptide standards (Bio-Rad) containing human angiotensin I (1296.5 Da), Fibrinopeptide B (1570.6 Da), porcine Dynorphin A [209-225] (2147.5 Da), human ACTH [1-24] (2933.5 Da), human β-endorphin [61-91] (3465.0 Da), bovine insulin (5733.6 Da), bovine Ubiquitin (8564.8 Da), bovine cytochrome-C (12230.9 Da), bovine superoxide dismutase (15591.4 Da), equine myoglobin (16951.5 Da), bovine β-lactogloburin A (18363.3 Da), horseradish peroxidase (43240.0 Da), bovine albumin (66410 Da), and chicken conalbumin (77490.0 Da) bovine IgG (147300.0 Da). External calibration provided a 0.1% mass accuracy. The ionized proteins were detected and their molecular mass/charge (m/z) ratios determined using time-of-flight mass spectrometry (TOF-MS) analysis with a detection range m/z of 1,570-12,230 for low molecular range, 5,733 to 43,240 for mid range and 16,951-147,300 for high molecular range. The setting for cluster formation at the first pass was set as S/N of 20% in all spectra, and for the second pass S/N was set to 2%. The cluster mass window was set to 0.3%. In one experiment, 713 peaks were identified. The protein concentration in CSF was determined using Bradford methods (Bio-Rad) in a procedure consistent with the manufacturer's instructions.
Protein peaks were analyzed with the ProteinChip Data Manager software version 3.0 (Bio-Rad). Each study was repeated at least twice, with baseline subtraction, spectrum normalization, and peak detection performed using the ProteinChip software.
Data collection and statistical methods
All spectra were externally mass calibrated and peak intensities were normalized using total ion current. The mean peak intensity of the duplicates was used for statistical analysis. Statistical analysis was also performed using the Kruskal-Wallis H test. When differences were significant, the Mann-Whitney U test was used to determine the significance of differences between each group. Uncorrected p values were corrected by multiplying them by the number of comparisons (Bonferroni-Dunn correction) to calculate corrected p values.
Purification of the 4.8-kDa protein
Methodology for purifying the 4.8-kDa protein was developed for on-chip analysis and also scaled up to column chromatography. First, conditions of capture were defined using a combination of various binding/washing buffers (100 mM sodium acetate, pH 4.0 and 5.0; 50 mM sodium phosphate, pH 6.0 and 7.0; and 50 mM Tris-HCl, pH 8.0 and 9.0) and anion exchange (Q10) and cation exchange (CM10) ProteinChip arrays. The 4.8-kDa protein was captured by Q10 ProteinChip arrays with the pH 4.0-8.0 conditions. Then, 500 μl of a CSF sample was mixed with an equal volume of 50 mM sodium acetate buffer (pH 4.0) containing 0.1% octyl glucoside (Wako Chemicals, Osaka, Japan). A 100 μl aliquot of the mixture was passed through an anion exchange column (Q Sepharose Fast Flow, GE Healthcare, Tokyo, Japan) and eluted with 50, 100, 200, 300, 500, and 1000 mM NaCl in 50 mM sodium acetate buffer, pH 4.0 containing 0.1% octyl glucoside. Each reverse phase fraction was analyzed for the presence of the 4.8-kDa peak using NP20 ProteinChip arrays (Bio-Rad). Protein species in reverse phase fractions were concentrated using micro-C18 ZipTip (Millipore, Billerica, MA, USA) following manufacturer's instructions, and reduced and alkylated using iodoacetamide.
Peptide Mass Fingerprint (PMF) analysis of the 4.8-kDa peak
The 4.8-kDa peptide was subjected to peptide mass fingerprinting (PMF) using ProteinChip arrays. Briefly, purified species were applied onto a normal phase ProteinChip array (NP20) and reduced with 5 mM DTT/10 mM NH4HCO3, (pH 8.0) at 70°C. Then, 2 μg/ml of trypsin (Promega, Tokyo, Japan) in 10 mM NH4HCO3 (pH 8.0) was applied and incubated for 2 h at 37°C. Digested peptides were detected using the ProteinChip reader and the data were analyzed using the Mascot search engine.
Identification of the 4.8-kDa peptide by MS/MS analysis
For MS/MS analysis, the purified protein was digested overnight with trypsin at 37°C, desalted with a ZipTip (Millipore) eluted with 2 μl of an aqueous acetonitrile solution (containing 50% acetonitrile (v/v) and 0.1% formic acid), and loaded onto a GlassTip in preparation for quadrupole time-of-flight (Q-TOF) MS analysis. Q-TOF analysis was performed using a MALDI-QSTAR mass spectrometer (Applied Biosystems, Tokyo, Japan) operated in positive mode. The ions corresponding to the tryptic digest peptides from the 4810 m/z species were selected for subsequent collision-induced dissociation, and the resultant fragment ions were analyzed. The resulting MS/MS spectral information was submitted to the Mascot search engine for identification. Among the analyzed peaks, the m/z 1907 peak generated a good fragmentation pattern for the Mascot search. Search parameters and settings for the Mascot identification were as follows: database (NCBInr), taxonomy (Homo sapiens), enzyme (SemiTrypsin), fixed modification (none selected), variable modification (Carbamidomethyl (C)), peptide mass tolerance (± 0.3 Da), peptide tolerance (± 0.3 Da), MS/MS tolerance (± 0.15 Da), peptide charge (+1), monoisotopic and max one missed cleavages. Sixty one matching the ion was identified as gi|17136078, a neurosecretory protein VGF precursor [Homo sapiens] with score of 61 (score > 49 indicates identity) and gi|2244659, a neuro-endocrine specific protein VGF [Homo sapiens] with score of 61 (score > 49 indicates identity).