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Identification of N-Glycosylation Changes in the CSF and Serum in Patients with Schizophrenia Johannes L. Stanta,†,‡ Radka Saldova,†,§ Weston B. Struwe,§ Jennifer C. Byrne,§ F. Markus Leweke,| Matthius Rothermund,⊥ Hassan Rahmoune,‡ Yishai Levin,‡ Paul C. Guest,‡ Sabine Bahn,‡ and Pauline M. Rudd*,§ Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom, NIBRT Dublin Oxford Glycobiology Laboratory, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland, Central Institute of Mental Health, University of Heidelberg, Mannheim, Germany, and Department of Psychiatry, University Medical Faculty, Muenster, Germany Received March 15, 2010

Schizophrenia is a major neuropsychiatric disorder that affects 2% of the population worldwide. No biochemical diagnostic tests are available, and patients must undergo lengthy clinical evaluation periods before an accurate diagnosis can be given. Blood and cerebrospinal fluid are candidates for the identification of potential biomarkers for this disease. We have identified several N-glycans that distinguish first onset, unmedicated schizophrenia patients from healthy individuals. This is the first report of the N-glycome from low abundance serum proteins and cerebrospinal fluid. The tetraantennary tetrasialylated glycan with a polylactosamine extension, A4G4LacS4, from low abundance serum proteins showed a 2-fold increase in serum from male schizophrenia patients. Gender specificity was also demonstrated as the triantennary trisialylated glycan containing the SLex epitope was increased significantly in male schizophrenia patients on both high and low abundance serum proteins. Levels of bisecting and sialylated glycans in the cerebrospinal fluid showed a general down-regulation in schizophrenia patients and a 95% positive predictive power for distinguishing patients from controls. These changes are consistent with the reported down-regulation of β-1,4-mannosyl-glycoprotein 4-βN-acetylglucosaminyltransferase III and β-galactoside R-2,3/6-sialyltransferases in the prefrontal cortex from schizophrenia patients. These alterations in the N-glycosylation signature could be used potentially for early diagnosis and monitoring of patients after treatment. Keywords: human plasma glycome • human CSF glycome • glycosylation in schizophrenia • biomarker • cerebrospinal fluid • CSF • depleted serum • N-linked glycans • diagnostic

Introduction Schizophrenia is a devastating mental illness with 2% prevalence in the general population, with no available empirical diagnostic test. This makes diagnosis a subjective and lengthy process, often taking several months or even years before an accurate assessment can be established. This lack of a diagnostic test causes a substantial delay in providing appropriate treatment for patients and highlights the need to identify biomarkers that can advance the development of diagnostic tools. The onset of the illness occurs usually in late adolescence but this differs substantially between men and women, with that in men averaging in their early 20s and in women averaging in their late 20s to early 30s.1 * To whom correspondence should be addressed. Pauline M. Rudd, NIBRT Dublin Oxford Glycobiology Laboratory, Conway Institute, University College Dublin, Dublin 4, Ireland. Tel: +353-17166728. Fax: +353-17166950. E-mail: [email protected]. † These authors contributed to this paper equally. ‡ University of Cambridge. § University College Dublin. | University of Heidelberg. ⊥ University Medical Faculty.

4476 Journal of Proteome Research 2010, 9, 4476–4489 Published on Web 06/28/2010

The brain and central nervous system (CNS) are assumed to be the seat of the pathology of neuropsychiatric disorders, such as schizophrenia. Cerebrospinal fluid (CSF) is seen as a prime source for schizophrenia biomarker discovery since it is likely that molecular imbalances and differential protein expression manifested in this body fluid are due to its proximity to brain. Analysis of CSF may provide more specific biomarkers, treatment strategies and drug targets,2 as well as shed more light on the etiology of the disease. Despite the importance of CSF, a body-fluid that is obtained by less invasive means is desirable for development of a diagnostic test and would possess greater practical value. Serum and plasma are used regularly for diagnostic purposes. These fluids are used to transport molecular information between the brain and the periphery and can therefore reflect the physiological status of an individual. Though easily accessible, these body fluids have several disadvantages. First, they are comprised of a proteome that spans several orders of magnitude in concentration.3 Second, the comprising proteins have a naturally high dynamic range, which means that changes in protein abundance must exceed the normal range to be considered biologically relevant.4 10.1021/pr1002356

 2010 American Chemical Society

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N-Glycosylation Changes in Patients with Schizophrenia To overcome these challenges, serum samples have to undergo fractionation. In this study, we have analyzed high and low abundance serum proteins separately. The 20 most abundant proteins, which make up 95% of the protein concentration, consist mostly of albumin, immunoglobulins and acute phase proteins. While this fraction contains important information on the abundant serum proteins, it masks the thousands of proteins that are present in lower concentrations.3 The low abundance proteins hold promise as disease biomarkers because they may be derived from physiological systems that are altered by disease. Given the inheritable nature of schizophrenia, many research groups have attempted to identify schizophrenia susceptibility genes. These studies have confirmed the importance of genes in the etiology but have failed to relate the genetic risk to variants in gene products, suggesting that the underlying genetics are an extremely complex feature of schizophrenia, which is also reflected in the extreme heterogeneity of the disease. Riley et al.5 suggest that schizophrenia is genetically mediated rather than determined and argue the importance of environmental factors in combination with a genetic predisposition. Unsurprisingly, genetic studies have not resulted in identification of robust targets for use in diagnosis. Biomarker sources that hold more promise are the proteins, protein modifications and metabolites, which can display the influence of the key factors and their interactions, as well as reflect the environmental determinants. Glycosylation is one of the most common and most complex post-translational modifications (PTMs) of secreted and cell surface proteins6 and plays a critical role in protein-protein, protein-cell, and cell-cell interactions including antibody binding, protein degradation, cellular endocytosis, and protease protection.7-9 The interaction of these factors will affect the circulation time of proteins in body fluids. Glycosylation is cell-type specific10,11 and reproducible for a given physiological state. Glycoproteins occur usually as a collection of glycoforms that differ in the structure of the component oligosaccharides. Glycoform abundance and glycan structures can be altered significantly in disease, resulting in a characteristic signature as a means of investigating complex pathophysiological processes and revealing novel biomarkers and drug targets.2,12 In the field of biomarker discovery, serum glycan profiles of whole, undepleted serum have already yielded valuable information about various diseases such as congenital disorders of glycosylation,13,14 along with cancer,11,15 fibrosis as it occurs in liver cirrhosis,16and inflammation17,18 as associated with rheumatoid arthritis.19 For example, a decrease in galactosylation and sialylation on IgG occurs in rheumatoid arthritis19 and administration of sialylated IgG has a protective anti-inflammatory effect on the disease.20 There is also evidence suggesting an involvement of glycosylation in the development of schizophrenia.21 Barbeau et al.22 looked at the polysialylated neural cell adhesion molecule (PSA-NCAM) in postmortem hippocampus, using immunohistochemical analysis, and found a 20-90% reduction in the number of PSANCAM reactive cells compared to that found in control brains. Overall expression levels of NCAM were unchanged, suggesting that PTMs rather than protein expression or alternative splicing have an influence in the underlying mechanisms leading to the development of schizophrenia. Normal-phase high-performance liquid chromatography (NPHPLC) separation techniques provide a means of facilitating

23,24

comparative glycomics analyses. This approach has four main advantages over other platforms such as mass spectrometry (MS), tandem MS, liquid chromatography-MS, capillary electrophoresis (CE), high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD)24,25 and lectin arrays.26 First, with the NP-HPLC approach, glycans are labeled stoichiometrically in a structurally unbiased manner with 2-aminobenzamide (2-AB), allowing accurate quantitative measurements and relative comparison between samples.25 Second, this method enables the analysis of glycan isoforms based on sequence and linkage (for example, core R1-6 fucosylation can be distinguished from R1-3 outer arm fucosylation). Third, neutral and charged glycans can be analyzed at the same time, without the removal of sialic acids required for many other glycomics platforms. Finally, glycan size and linkage result in a specific elution position that can be converted to glucose units (GUs) using a dextran hydrolysate standard ladder. GUs are comparable between HPLC platforms and make interpretation easier as they can be used in conjunction with GlycoBase, a web-based GU database collection http://glycobase.nibrt.ie:8080/database/show_nibrt.action. Here, we describe the first profiling study of CSF glycans. Only a few studies have looked at the structural properties of N-linked glycans in CSF11,27 targeting individual proteins rather than the total glycan pool. Hakansson and colleagues27 analyzed isoforms of the two most abundant glycoproteins in CSF which are β-trace (present in at least five isoforms), and R1antitrypsin (present in at least three isoforms).27,28 The use of quantitative chromatographic methods to analyze the total glycan pool of unmedicated schizophrenia patients and comparison of these with healthy controls, can reveal significant changes between disease groups and explain disease characteristics.29 Changes in the N-glycan signature, related to disease status, could lead to the development of more specific diagnostics compared to current methods and give additional insights into the glycosylation machinery, identifying which glycosidases or glycotransferases may be involved in the etiology of schizophrenia.30,31

Materials and Methods Samples Selection and Collection. The ethical committees of the Medical Faculties of the Universities of Cologne and Muenster approved the protocols of this study including procedures for sample collection and analysis. Informed consent was given in writing by all participants and clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The subjects were antipsychotic-naive patients diagnosed with first episode paranoid schizophrenia and matched healthy controls. Diagnosis was assessed according to DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, fourth edition) and ICD 10 (International Statistical Classification of Diseases and Related Health Problems 10th Revision) by experienced psychiatrists using the Structured Clinical Interview for DSM-IV (SCID). The patients were diagnosed with schizophrenia (F20) or paranoid schizophrenia (F20.0) and had a DSM-IV score of 295.30. Blood samples were collected via venous puncture of subjects into S-Monovette 7.5 mL serum tubes (Sarstedt; Numbrecht, Germany). Serum was prepared by placing samples at room temperature for 2 h for blood coagulation, followed by centrifugation at 4000× g for 5 min. The resulting supernatants were stored at -80 °C in Low Binding Eppendorf tubes (Hamburg, Germany) prior to analysis. CSF was collected via Journal of Proteome Research • Vol. 9, No. 9, 2010 4477

research articles lumbar puncture and stored at -80 °C in Low Binding Eppendorf tubes until use. All samples were collected midmorning. Healthy controls were comprised of hospital staff and colleagues and these were screened for severe combined immunodeficiency. Patients and controls were excluded from the study if they had any known comorbidity such as diabetes, heart disease, thyroid disease, liver cirrhosis, autoimmune disease, any recent infections, or a history of substance abuse. A total of 54 individuals (23 schizophrenia patients and 31 controls) who had not been treated previously with antipsychotic medication were analyzed. In many cases, serum and CSF from the same subject were available. The age distribution of CSF samples was 22 ( 3 and 23 ( 3 for patients and controls, respectively. For serum, the age distribution was 30 ( 7 and 29 ( 8 years for patients and controls, respectively. Samples were chosen to have a narrow age window to eliminate agedependent influences on glycosylation which were observed in previous studies.32-34 Serum Samples Preparation. Thirty eight serum samples (19 schizophrenia and 19 controls) were processed with 9 quality control samples. The quality controls were 9 aliquots of the same sample from a healthy control, processed at the same time as the other samples in this study. Serum samples were taken from the sample bank and thawed on ice. Eight microliter aliquots of each sample were used for high abundance serum protein depletion, using the PROTEOPREP 20 Plasma Immunodepletion Kit according to the manufacturer’s instructions (Sigma, Dorsett, UK). Before loading onto the equilibrated column, 8 µL sample (95 mg/mL protein) was diluted to 100 µL with EQ buffer (10 mM sodium phosphate, pH 7.4 and 150 mM NaCl) and filtered. The sample was then added to the resin and incubated for 20 min. Low Abundance Proteins. The flow-through was collected after centrifugation at 1000× g and washed twice with 100 µL EQ buffer. The three flow-through fractions were then combined, the sample was concentrated, and the buffer was exchanged into ammonium bicarbonate (50 mM) using a 5 kDa molecular weight cutoff spin column. Proteins more than 5 kDa were retained in ammonium bicarbonate buffer and used for the analysis. High Abundance Proteins. The proteins that bound to the resin were eluted with 2 mL ES buffer (0.1 M Glycine-HCl, pH 2.5 containing 0.1% of Octyl β-D-glucopyranoside) per column using a vacuum manifold. The elution step was repeated three times and the four elution fractions of the same sample were combined. After adding 400 µL Trizma base (1 M) to neutralize the solution, an aliquot of 2 mL from this pool was used for the analysis. CSF Samples Preparation. Thirty-two CSF samples (14 schizophrenia and 18 controls) were processed with 5 quality control samples. CSF samples were thawed on ice, and the 50 µL aliquots were transferred directly into a 96-well plate, evaporated to dryness, and resuspended in 7 µL of H2O. Release and Fluorescent Labeling of Glycans. Samples were reduced and alkylated. Each sample was added to a 96-well plate well in a volume of 7 µL. Four microliters of DTT (0.5 M) was added followed by incubation at 65 °C for 15 min. One microliter of iodoacetamide (0.1 M) was added and incubated at room temperature for 30 min. The sample was then cast in a gel (22 µL Protogel, 11.25 µL Tris-HCl (1.5 M, pH 8.8), 1 µL sodium dodecyl sulfate (10%), 1 µL Ammonium peroxodisulphate (10%), 1 µL N,N,N,N′-tetramethyl-ethylenediamine) in the well of the plate. After setting, gels were transferred to a 4478

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Stanta et al. Whatman Protein Precipitation FF plate for three washings with acetonitrile and NaHCO3 (50 mM). N-Glycans were released by digestion with 5 mUnits of N-glycosidase F (PNGase F, Roche Applied Science) per well and incubation at 37 °C overnight. Glycans were eluted from the gel with 3 washes of H2O and acetonitrile. The elution solution was evaporated to dryness, resuspended in fresh formic acid (1%, v/v), and dried before fluorescent labeling with 2-AB using the 2-AB labeling kit according to the manufacturer’s instructions (Ludger Ltd.). The samples were transferred onto pieces of 3MM Whatman chromatography paper to remove excess 2-AB. The chromatography paper was washed three times with acetonitrile before the labeled glycans were eluted with H2O. The eluents were dried down and resuspended in 25-50 µL of H2O for the HPLC analysis. Exoglycosidase Digest. Exoglycosidase arrays were used in combination with a 3 h HPLC run. The following exoglycosidases were used: Arthrobacter ureafaciens sialidase (ABS, EC 3.2.1.18), Streptococcus pneumoniae sialidase (NAN1, EC 3.2.1.18), bovine kidney R-fucosidase (BKF, EC 3.2.1.51), bovine testes β-galactosidase (BTG, EC 3.2.1.23), and β-N-acetylglucosaminidase cloned from S. pneumonia, expressed in Escherichia. coli (GUH, EC 3.2.1.30), all purchased from ProZyme. A glycan aliquot with a good spectrum was digested 18 h at 37 °C in 50 mM sodium acetate buffer, pH 5.5. Before HPLC analysis, the enzymes were removed using Micropure-EZ protein-binding filters from Millipore. The water-soluble glycans were collected in the flow-through which was evaporated to dryness and resuspended in 100 µL water-acetonitrile (20: 80) for HPLC analysis. 2-AB-labeled glycans from serum were available in sufficient quantities for the digest array but 2-AB CSF glycans were pooled to yield sufficient material. For this, patient and control samples were pooled separately. Analysis of Glycans with NP-HPLC. NP-HPLC was performed as described in ref 25. A TSK-Gel Amide-80 4.6 × 250 mm column (Anachem, Luton, U.K.) was used on a 2695 Alliance separations module (Waters, Milford, MA) equipped with a Waters temperature control module and a Waters 2475 fluorescence detector. Solvent A was 50 mM ammonium formate buffer at pH 4.4. Solvent B was acetonitrile. The column temperature was set to 30 °C. For the exoglycosidase digest analysis, a 3 h HPLC gradient was used. For comparative screening of disease and control samples, a 1 h HPLC gradient was used. The 1 h gradient started with 35% A and increased continuously over 48 min to 47% A at a flow rate of 0.8 mL/ min, followed by an increase of 47% to 100% A over 1 min, washing with 100% A for 4 min, returning to 35% A over 1 min and re-equilibration of the column with 35% A for 6 min. The 3 h gradient started with a linear gradient of 20% A and increased over 152 min to 58% A at a flow rate of 0.4 mL/min. Samples were injected in 80% acetonitrile. Fluorescence was measured at 420 nm with excitation at 330 nm. The system was calibrated using an external standard of hydrolyzed and 2-AB-labeled glucose oligomers to create a dextran-standard ladder. Every tenth injection was a dextran-standard ladder, which was used to calibrate the HPLC for normalization of peaks across runs. Glycan Analysis and Structure Allocation. GU values were calculated by the Empower GPC software from Waters which fits a fifth order polynomial distribution curve to the dextran ladder. The structures were assigned through GlycoBase search, an online GU value and glycan structure database

N-Glycosylation Changes in Patients with Schizophrenia

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Figure 2. Box plot of the significant changes in schizophrenia in LAS (a) A3F1G3S3 (U19), (b) A4G4LacS4 (U23) and HAS (c) A3F1G3S3 (predominant, H6). Both LAS N-glycan peaks showed elevated levels in male schizophrenia patients.

Figure 1. Typical glycan profile from healthy volunteers of (a) HAS, highlighting the glycan peaks that were significantly changed (H1-H6); (b) LAS and (c) CSF proteins. The most abundant A2G2S2 glycan structure is pictured.

http://glycobase.nibrt.ie:8080/database/show_nibrt.action, and retention shifts after exoglycosidase treatment. The NP-HPLC glycan chromatogram was used to quantitatively compare glycan peaks. Individual peaks were normalized to the total peak area and the percentage (%) area of each peak was compared.

Results N-Glycosylation of Human Serum and CSF. Thirty-eight serum samples (19 schizophrenia and 19 controls) were depleted of the 20 most abundant proteins. This yielded high abundance serum proteins (HAS) and low abundance serum proteins (LAS). These samples, along with 32 CSF (14 schizophrenia and 18 controls), were processed for N-glycan analysis using quantitative NP-HPLC and exoglycosidase digestions, with structural assignments made using database matching (GlycoBase; http://glycobase.nibrt.ie:8080/database/show_nibrt. action). This is the first time the total N-glycan analysis on LAS and CSF has been reported, including full structural assignments. All samples were processed simultaneously and blindly in the N-glycan analysis. Figure 1 shows typical NP-HPLC profiles of the released N-glycans in: (a) HAS, (b) LAS, and (c) CSF proteins. The N-glycan pools were different. The most abundant peak in each profile, containing predominantly glycan A2G2S2, is indicated in Figure 1. A2G2S2 comprised 34% of the glycan structures in HAS, consistent with previous studies,25 48% in LAS and 20% in CSF. N-Glycosylation Changes in First Onset Schizophrenia Patients. Glycan structures in the LAS, HAS, and CSF samples were assigned by exoglycosidase digestion followed by high resolution HPLC analysis. This was followed by analysis using the high-throughput protocol established in the Dublin-Oxford Glycobiology Laboratory at the University College Dublin24,35 to investigate differences between schizophrenia patients and controls. A two-sided ANOVA analysis was employed to calculate a p-value for the medical status, gender, and any interaction between genders and disease. High Abundance Serum Proteins. The HAS fraction contained the following glycoproteins: IgG, IgA, IgM, IgD, R1-Acid Glycoprotein, Complement C1q, Complement C3, Transferrin,

Complement C4, Ceruloplasmin, Fibrinogen, and R1-Antitrypsin. The following proteins were also included: Albumin, Prealbumin, Apolipoprotein B, R2-Macroglobulin, Apolipoprotein A-I, Plasminogen, Haptoglobin, and Apolipoprotein A-II. Whole serum N-glycan profiles are well characterized,25,33 and the structures have been identified with exoglycosidase digests and MS. As expected, the HAS chromatogram is almost identical to the chromatogram from whole serum33 with respect to peak number and abundance. Figure 1a shows a typical chromatogram, and the glycans were assigned on the basis of Royle et al.25 using Glycobase. Peak H6 was increased significantly in male schizophrenia patients (by 30%) and decreased significantly in female patients (by 25%) compared to controls (Figure 2). This peak contains five underlying N-glycans: A3F1G3S3 (predominant), A4G4S2, A4G4S3, FA3G3S3, and FA3BG3S3 (Table 1). Four peaks with 14 underlying N-glycans were significantly altered with gender with no disease interaction: H1+H2 (FA2, A2B, A1G1), H3 (A2G2, M6, A2BG2, A2G1S1), H4 (FA2BG2S1, A2F1G2S1) and H5 (A3G3S3 (predominant), A3BG3S3, Man9Glc1, A4G4S1 and A3F1G3S2) (Table 2). Low Abundance Serum Proteins. Figure 3 shows a typical chromatogram of the total N-glycan pool from the LAS fraction and digestion with exoglycosidases. The glycans in each peak are described in detail including % areas in undigested and digested profiles in Supplementary Table S1 (Supporting Information). A4G1GlcNAc (X11), derived from A4G4LacS4 (U23), was present in decreased levels in the ABS, BTG digest compared to the undigested or ABS digested N-glycan pool. Furthermore, this glycan could not be quantified in the ABS, BTG, BKF digest because its abundance was too low for a high signal-to-noise ratio. This effect was also seen by Royle et al.25 and might be due to unspecific activity of the BTG exoglycosidase which results in digestion to A4 of this structure. Glycan peaks that were significantly changed by disease or gender are indicated by arrows in the presented exoglycosidase digestions. Two N-glycans were changed significantly between disease and control: A4G4LacS4 (U23) and A3F1G3S3 (U19) (Figure 2 and Table 3). These glycans were significantly higher in male schizophrenia patients. The A4G4LacS4 peak was doubled in male schizophrenia patients compared to the effect seen in other groups (Figure 2b). The false discovery rate36 was 0.058 for A4G4LacS4 which indicates that the observed change was unlikely to be a false positive. Values from female schizophrenia patients and healthy controls were at the same level as controls and showed little variability. A3F1G3S3 was also significantly higher in male schizophrenia patients when compared to male Journal of Proteome Research • Vol. 9, No. 9, 2010 4479

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Table 1. Summary of the Significant N-Glycan Changes between Schizophrenia Samples and Controls on HAS Proteinsa

a

SD, Standard deviation; avg., average; SZ, schizophrenia.

b

Two-way ANOVA interaction by gender and medical status.

Table 2. Summary of the Significant N-Glycan Gender Changes on HAS Proteins from First Onset Schizophrenia Patients and Controlsa

b

a No changes between disease samples and controls were observed on these N-glycans. SD, Standard deviation; avg., average; SZ, schizophrenia. Two-way ANOVA interaction by gender and medical status.

controls, but the change was less than 16% and therefore may be too low to be considered biologically relevant. Five peaks with 10 underlying N-glycans were significantly changed with gender with no disease interaction: U1 (FA2, A2B, A1[3]G1), U11 (FA2G2S(6)1, A2BG2S(6)1), U15 (FA3G3S(6)1, A3BG3S(6)1), U17 (A3G3S3, A3F1G3S2) and U22 (A4F1G4S4) (Table 4). CSF. Figure 4 shows a typical chromatogram of the N-glycan pool from CSF and the digestion products achieved using arrays of exoglycosidases. The glycans in each peak are described in detail including % areas in undigested and digested profiles in 4480

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Supplementary Table S2 (Supporting Information). Glycans that were significantly different in schizophrenia patients and that show a gender difference are illustrated by the arrows following the exoglycosidases digestions. Four peaks with 7 underlying N-glycans were changed between disease and control (Table 5). Peaks C17 (FA2BG2S2, FA2G2S2, A3F1G3), C18 (A3G3S(6)1, FA4G3) and C20 (A4BG4, A3G3S2) were significantly lower in disease without significant gender interaction (Figure 5). Peak C3 (FA2B, M5, A2[6]G1, A3B) showed significant gender interactions (Table 5) and was significantly higher in female schizophrenia patients and lower

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N-Glycosylation Changes in Patients with Schizophrenia

Five peaks with 10 underlying N-glycans were significantly changed between the genders with no link to schizophrenia: C1 (A2B), C2 (FA2, A3), C5+C6 (FA2[6]G1, A3G1, FA3B), C7 (FA2[3]G1, FA4) and C8 (FA2[3]BG1, M6) (Table 6). The multivariate analysis of all glycans in the pool was done with a partial least-squares-discriminant analysis (PLS-DA). The biggest changes, with the greatest influence on the PLS-DA separation, were 4 chromatogram peaks with 11 underlying N-glycans: C3 (FA2B, M5, A2[6]G1, A3B), C17 (FA2BG2S2, FA2G2S2, A3F1G3), C18 (A3G3S(6)1, FA4G3) and C20 (A4BG4, A3G3S2) (Figure 5 and Table 5). The PLS-DA model was used to determine the predictive power of the CSF glycan profile using the “leave-1/3-out” cross-validation method. Two-thirds of the samples were randomly selected and used as a learning set to build a PLS-DA model. The other third was considered as unknowns and the PLS-DA model used to predict their disease classification. The correct or erroneous classification of these was used to determine the true predictive power of the developed PLS-DA model. This procedure was repeated eight times with different random selections of samples. The averaged results showed a positive predictive value of 95% (1 in 18 positive values was false) and a negative predictive value of 88% (1 in 8 negative results was false) (Figure 6). This represents a good predictive power and holds potential for the development of a diagnostic test.

Discussion

Figure 3. Typical NP-HPLC chromatograms of the glycans from LAS proteins after different exoglycosidase digestions. (A) Chromatograms of (a) the undigested sample; (b) the sample digested with ABS; (c) with ABS and BKF; (d) ABS and BTG; (e) ABS, BKF, BTG. (B) NP-HPLC profiles are zoomed in to highlight small peaks. A summary of the structures underlying the peaks can be found in Supplementary Table S1 (Supporting Information). Glycans significantly changed in schizophrenia patients and between genders are pictured with the arrows following exoglycosidases digestions.

in male schizophrenia patients when compared to the average of the healthy controls (Figure 5).

There are several reports demonstrating that the glycosylation pattern of serum glycoproteins can depend on pathophysiology. To study the pathology of schizophrenia and discover diagnostic biomarkers, we analyzed serum and CSF glycans of patients with schizophrenia and compared these to profiles of healthy controls. The use of glycans as a robust and reproducible source of specific and sensitive biomarkers has been illustrated with the breast cancer biomarker, CA15-3, which showed an increase in sensitivity from 61.5 to 78.5% when a glycan biomarker (sLex) was included in the test.37 In this study, we are the first to report N-glycan analysis on the CSF and low abundance proteins. We have determined the structures and their abundance by NP-HPLC. In addition, this work has extended the current knowledge on serum glycomics by analyzing N-glycans derived from low abundance serum proteins. We have used these novel N-glycan pools to determine differences in glycosylation in schizophrenia that can form the basis of a diagnostic test. Differences were identified in the levels of several N-glycans in serum and CSF samples from first onset, unmedicated schizophrenia patients. Furthermore, the subgroup analysis of gender and disease revealed gender specific differences in glycosylation in schizophrenia as well as unisex markers for the disease. N-Glycosylation Changes in Serum from Schizophrenia Patients. The strategy for identifying a serum glycan biomarker for schizophrenia was to investigate N-glycans on HAS proteins separately from N-glycans of LAS proteins. Three peaks from serum were promising in separating disease from controls: U19 (A3F1G3S3) and U23 (A4G4LacS4) from LAS protein fraction and H6 (A3F1G3S3 (predominant), A4G4S2, A4G4S3, FA3G3S3 and FA3BG3S3) from HAS protein faction (Figure 2). All of the changed serum glycans were complex type N-glycans containing sialylated N-acetyllactosamines. The elevation of these structures was observed only in males with schizophrenia. The length and hydrophilicity of these N-glycan structures means that they extend away from the protein and are therefore ideal Journal of Proteome Research • Vol. 9, No. 9, 2010 4481

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Table 3. Summary of the Significant N-Glycan Changes between Schizophrenia and Control on LAS Proteins

a

SD, Standard deviation; avg., average; SZ, schizophrenia.

b

Two-way ANOVA interaction by gender and medical status.

Table 4. Summary of the Significant N-Glycan Gender Changes on LAS Proteins from First Onset Schizophrenia Patients and Controlsa

b

a No changes between disease samples and controls were observed on these N-glycans. SD, Standard deviation; avg., average; SZ, schizophreni. Two-way ANOVA interaction by gender and medical status. c R(2-6) sialic acid linkage confirmed with NAN1 exoglycosidase digest (data not shown).

candidates to interact with lectin-like receptors, for example selectins, expressed by other cells.38 A4G4LacS4 has a GU value of 11.78 and the unsialylated form has a GU of 10.69. No unsialylated A4G4Lac was found in the N-glycan pools of either serum fraction suggesting that sialyltransferases are not affected in schizophrenia, which is supported by the fact that there was no decrease in sialylated glycans in serum. The increase in polylactosamines in male schizophrenia patients could be a result of increased glyco-transferase activity during the late stage of N-glycan processing in the Golgi. Possible candidates for this up-regulation could be a β-1,4galactosyltransferase and a β-1,3-N-acetylglucosaminyltransferase (β3GnT) which are responsible for the synthesis of polylactosamines.38 Their increased activity would explain the 4482

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elevation of the polylactosamine N-glycans. So far, none of the encoding genes for these transferases have been associated with schizophrenia or neurological diseases. Alternatively, the increased lactosamine glycosylation could be a result of an increased residence time in the Golgi apparatus which correlates with slower transit times of this organelle system.39 A3F1G3S3 was significantly elevated in male schizophrenia patients on LAS (peak U19) and HAS (peak H6) (Figure 2). This structure contains sialyl Lewisx (sLex) epitope. Elevated levels of sLex N-glycans have previously been linked to cancer malignancy40,41 and chronic inflammation.42,43 Chronic inflammation has been linked to schizophrenia44 and therefore, the increase in sLex in male schizophrenia patients may be indicative of inflammatory status. The sLex epitope was also found in peaks U17 (A3F1G3S2, together with A3G3S3, which is

N-Glycosylation Changes in Patients with Schizophrenia

Figure 4. Typical NP-HPLC chromatograms of the glycans from CSF proteins after different exoglycosidase digestions. (A) Chromatograms of (a) the undigested sample, the sample digested with (b) ABS; (c) ABS, BTG; (d) ABS, BTG, BKF; (e) ABS, BTG, BKF, GUH. (B) NP-HPLC profiles are zoomed in to highlight small peaks. A summary of the structures underlying the peaks can be found in Supplementary Table S2 (Supporting Information). Glycans that significantly changed in schizophrenia patients and between genders are pictured with the arrows following exoglycosidases digestions.

research articles predominant) and U22 (A4F1G4S4) in LAS (Supplementary Table S1, Supporting Information, and Table 4) and in H4 (A2F1G2S1 together with FA2BG2S1) and H5 (A3F1G3S2 together with A3G3S3-predominant, A3BG3S3, Man9Glc1, A4G4S1) in HAS (Table 2). These peaks showed a significant gender difference, although these differences were of the same magnitude as those between disease and control. A3G3F1S3 is present on many high abundance proteins such as fetuin, antitrypsin, haptoglobin and transferrin45 and A4G4S2 and A4G4S3 are on R1-acid glycoprotein46 and haptoglobin.45 Therefore, these changes in glycosylation could be also related to the expression levels of these proteins in schizophrenia. Furthermore, the lifespan of a protein in the bloodstream can be changed due to a change in glycosylation. Sugars protect proteins from protease digestion and the presence of sialic acid serves to reduce clearance by the asialoglycoprotein receptor.38 A defect in glycosylation can thus directly influence the protein concentration in the circulation. N-Glycosylation Changes in CSF from Schizophrenia Patients. The findings of this study suggest that abnormalities in N-glycosylation are present in first onset schizophrenia patients and may explain important pathogenic mechanisms in schizophrenia. The results from the PLS-DA analysis showed that the CSF N-glycosylation pattern is effective in distinguishing between disease and control (Figure 6). Given the predictive values of 95% (positive) and 85% (negative), this may lead to the development of a useful diagnostic test for schizophrenia. The features with the greatest influence on the PLS-DA separation were 4 chromatogram peaks with 11 underlying N-glycans: C3 (FA2B, M5, A2[6]G1, A3B), C17 (FA2BG2S2, FA2G2S2, A3F1G3), C18 (A3G3S(6)1, FA4G3), and C20 (A4BG4, A3G3S2) (Figure 5 and Table 5). All structures were found to be significantly lower in CSF from all schizophrenia patients compared to controls, except for peak C3, which was found to be lower only in male schizophrenia patients and higher in the female schizophrenia subjects. The general trend of significantly lower relative abundance of the above-mentioned glycans may reflect the down-regulation of the glycosyltransferases. This supports recent transcriptomics findings from Narayan et al.21 These researchers investigated expression differences in glyco-relevant genes in the prefrontal cortex of predominantly male schizophrenia patients. They used a glycobiology focused chip to investigate genes encoding glycosyltransferases, carbohydrate-binding proteins, proteoglycans and adhesion molecules. They reported decreased expression of genes encoding for glycan transferases in the N- and O-linked glycan biosynthetic pathways and glycosphingolipid metabolism in brains from schizophrenia patients with short-term illness. Genes relevant for the N-glycan biosynthesis pathway, MAN2A2, MGAT3, ST6GAL1, and ST3GAL2, were down-regulated by approximately 0.8-fold.21 The changes from the predominant peaks observed in the current study related to the changes of the glycosyltransferases in the N-glycan biosynthesis pathway found by Narayan et al.21 are summarized in Figure 7. Many of our observed glycan changes are consistent with the glycosyl-transferase downregulations that were found by Narayan et al.21 The percentile contribution of each structure to the HPLC peak is indicated in Table 5 and Figure 7, which gives an indication of the effect of the glycan structure on the glycosyltransferase up or down regulation. The glycans that could be linked to the biosynthesis pathway are the most abundant structures of the peak and Journal of Proteome Research • Vol. 9, No. 9, 2010 4483

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Table 5. Summary of the N-Glycans That Were Significantly Different in CSF Samples from Schizophrenia Patients When Compared to Controlsa

a Significant p-values are highlighted in bold. The proposed structures underlying these peaks are also included. SD, Standard deviation; avg., average; SZ, schizophrenia. b Two-way ANOVA interaction by gender and medical status.

Figure 5. Box plots of the changes between CSF samples from schizophrenia patients when compared to CSF control samples. Peaks C17, C18, and C20 were significantly lower in schizophrenia patients. Peak C3 showed gender specific differences in schizophrenia.

hence contribute greatly to its abundance. However, these findings will have to be confirmed with a more detailed analysis of the individual glycan structures. 4484

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MGAT3 encodes for β-1,4-mannosyl-glycoprotein 4-β-Nacetylglucosaminyltransferase III (GlcNAc-T III) which is responsible for the synthesis of bisecting glycans in the Golgi apparatus. This would explain our observation of decreased FA2B, FA2BG2S2, and A4BG4 in schizophrenia subjects. FA2B is the predominant glycan in peak C3 (Table 5), which showed lower levels in males and higher levels in females (Figure 5). FA2BG2S2 is the predominant glycan in peak C17 and A4BG4 contributes 50% to peak C20 (Table 5) and they both showed a decrease in male and female schizophrenia patients. Narayan et al.21 also investigated gender influences on their results and did not find any significant correlations, which may be because there were only 3 female subjects in their sample set of 19. This could explain why this study only found a down-regulation of MGAT3. GlcNAc-T III is also an important regulatory transferase because it competes with GlcNAc-Ts IV and V, which synthesize tri and tetraantennary glycans, for a biantennary glycan. In addition, once a bisecting GlcNAc is attached, the glycan can no longer be processed with GlcNAc-T IV or V to yield further branches. MGAT4 and MGAT5 encode GlcNAc-Ts IV and V, respectively and these were expressed at

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N-Glycosylation Changes in Patients with Schizophrenia a

Table 6. Summary of Significantly Changed N-Glycans between the Genders in CSF Samples

a Significant p-values are highlighted in bold. SD, Standard deviation; avg., average; SZ, schizophrenia. medical status.

Figure 6. PLS-DA plot calculated using data obtained by screening 32 CSF samples (14 disease samples versus 18 control samples). Each object in the graph represents one individual. For each individual, the position in the plot is a compact representation of all glycan peaks. Objects that appear close to each other are generally similar across all variables. Peaks C3, C17, C18, and C20 had the greatest influence on calculating the components 1 and 2 of the PLS-DA plot.

normal levels in schizophrenia,21 which would amplify the outcome of the decreased MGAT3, resulting in lower levels of biantennary bisected glycans. ST6GAL1 and ST3GAL2 encode β-galactoside R-2,3/6sialyltransferases in the Golgi which attach sialic acids to the glycan structures in the final step of the N-glycan pathway. We found decreased abundance of peaks contain-

b

Two-way ANOVA interaction by gender and

ing sialylated structures (C17 (FA2BG2S2, FA2G2S2); C18 (A3G3S(6)1) and C20 (A3G3S2)) in schizophrenia samples when compared to healthy individuals (Table 5). Glycans contain R2,3- and/or R2,6-sialic acids but the two enzymes responsible have a different prevalence. R2,6-Linked sialic acid is more often found on biantennary glycans, whereas R2,3-linked sialic acid is more often found on tri and tetraantennary glycans.47 In future work, the sialic acid linkage will be determined for all glycan structures in CSF to reveal whether there is a schizophrenia-specific sialic acid linkage bias. Down-regulation of sialyltransferases implies that overall processing of sialylated glycans may be reduced in schizophrenia. Sialic acids alter conformation of glycoproteins and gangliosides and their physical properties. Ca2+ ions bind to the sialic acid residues of gangliosides, which are located in clusters on neuronal and synaptic membranes.38 Sialic acid may function in supplying Ca2+ ions to the neuronal cells.38 Polysialic acid is involved in neural cell migration, axonal guidance, synapse formation and functional plasticity of the nervous system.38 Decrease of sialyltransferase activity in neuronal tissue is associated with neurodegenerative disease and impaired mental function.38 Narayan et al.21 also found that MAN2A2 was decreased in schizophrenia patients. This gene encodes for an R-mannosidase that catalyzes the first step in the biosynthesis of complex N-glycans in the Golgi. It controls the conversion Journal of Proteome Research • Vol. 9, No. 9, 2010 4485

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Figure 7. Glycan processing and changes in schizophrenia. Pictured are the changes in the relevant glycans from this study that were also found by Narayan et al.21 along the N-glycan processing pathway in the Golgi apparatus. The glycotransferase expression that was found decreased by Narayan et al. is highlighted in red with a gray background. Glycans that were found decreased in CSF from schizophrenia patients in our study are framed in red. The glycan changes from our results with gender interaction are framed in green. Glycan structures were determined as peaks which can consist of more than one structure. The percentile contribution of a structure to a peak is also indicated.

of hybrid to complex N-glycans by hydrolyzing N-glycans in the final step of the N-glycan maturation pathway. A decrease in the activity of this enzyme would result in an overall decrease in complex N-glycan levels and an increase in high mannose glycan levels. Such an effect could not be determined from the present results because the contribution of high mannose glycans in most peaks was too low to detect such small changes. Given the coherence between the transcriptional findings and our results (Figure 7), CSF glycan pattern profiling could represent a useful method that may be translated to blood when possible, for diagnosing schizophrenia and monitoring disease progress and treatment. Furthermore, these findings suggest that transcriptional changes in the N-glycan pathway in schizophrenia may be reflected in the abundance of individual glycan types in the CSF proteome. The CSF proteome is not only derived from the brain but it is also produced in the CNS by two different mechanisms. First, choroid plexuses in the brain ventricles secrete CSF constituents originating from the blood, following active and passive transport through the blood brain barrier.48 Second, around 20% of the CSF proteome is derived from drainage of interstitial liquid of the nervous tissues. This is of particular importance as this can reflect the physiological and the pathological status of the CNS. Glycoproteins are involved in numerous immunological and regulatory pathways. A glycan imbalance on proteins would be expected to have a major impact on the whole organism. Such deficits could explain the diverse pathological effects that are associated with schizophrenia, such as those involving immunological reactions and cell signaling. Disruption of glycan biosynthesis at the onset of the illness could result in widespread downstream effects. 4486

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N-Glycosylation Gender Differences in All Samples. Gender differences in glycosylation are not well understood and have only recently been discovered in total serum proteins in a study from Knezevic et al.33 in which the whole serum N-glycan profiles of 1008 individuals were analyzed. Although the current study involved a smaller sample set, a number of the gender specific findings from Knezevic et al. in serum samples were confirmed. Our sample set was age-matched to rule out any age influence on the data. The only consistent gender specific changes were found in both high and low abundance serum N-glycome in peaks containing FA2, A2B, and A1G1 (H1+H2 and U1). The differences showed the same trend, with females having a lower level of these N-glycans. These findings were unrelated to the medical status. The present study shed a light on the gender specificity of the glycan biomarkers for schizophrenia. Gender differences were observed in all sample types in our study and schizophrenia N-glycan changes in serum showed significant changes in one sex. It is well-known that male schizophrenic patients have an earlier age of disease onset than female patients.49 Gender differences in N-glycans from CSF proteins may reflect gender brain differences. Brain development and maturation are slower in males. This makes them vulnerable for longer to detrimental brain insults during brain development and is thought to be an explanation for the earlier development of schizophrenia in males.50 Different sex hormone levels and regulation could be an explanation for N-glycan gender differences. Serum estrogen levels regulate secretion of pituitary gland released hormones, many of which are glycosylated, such as luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone and chorionic gonadotrophin. These pituitary gland hormones have been linked to schizophrenia. However, in our study, we did

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N-Glycosylation Changes in Patients with Schizophrenia not find any of the characteristic glycohormone glycans changed between disease and control. Glyco-Structural Findings and Comparison between Central and Peripheral Candidates Markers. The complete structural determination of CSF and LAS glycosylation was presented in this study. The data show that NP-HPLC N-glycan analysis gives detailed information on glycosylation, suitable for the determination of characteristic changes that can potentially be used as biomarkers. The structures were determined using exoglycosidase array digestions and selected structures were verified by MS. HPLC and MS structural analyses are sensitive methods but require significant sample analysis times in the discovery phase. The results of this study will speed-up future N-glycan pool analyses from CSF and serum in future discovery projects. The high sensitivity of the NP-HPLC platform allowed detailed structural information of LAS and CSF N-glycan pools to be obtained. The HAS chromatogram is almost identical to the chromatogram from whole serum33 in terms of number of peaks and their abundance. This suggests that whole serum glycomics only covers HAS proteins and loses significant information, coded by the LAS proteins. We have detected 28.7% bisecting N-glycans present in CSF (Supplementary Table S2, Supporting Information, ABS+BTG+BKF digest). On the basis of the findings from Hakkansson et al.27 many of the bisecting structures in CSF may be derived from β-trace, which is the most abundant glycoprotein in CSF51 and present in more than six glycoforms. The profiles were characteristic for the derived specimen and the protein pool may be used as a reflection of the physiological status of the individual. Glycan pattern changes can reveal aberrant glycosylation machinery and contribute to the understanding of the disease. Due to the high sensitivity of the platform, which allows detection of N-glycans in femtomole quantities, analysis requires only minimal amounts of sample. A single sample requires as little as 8 µL serum or 50 µL CSF for well resolved N-glycan patterns. This can be easily obtained from patients or existing biobanks to enable further validation studies of various diseases with a link to altered glycosylation. The current findings may form the basis for the discovery of more specific biomarkers and disease monitoring methods.

Conclusion The glycomics study presented in this paper is the first to analyze the N-glycan pool of CSF and LAS proteins and to resolve their N-glycan structures. We have shown that CSF and LAS samples can be used for high throughput glycomics analyses and to quantify differences in N-glycan profiles between these body fluids. The comparison of N-glycan profiles from first-onset unmedicated schizophrenia cases revealed molecular differences which can distinguish schizophrenia patients from controls with a high predictive power, with some of the alterations being gender specific. Our findings suggest that changes in protein glycosylation are associated with disease physiopathology and could be hold potential as diagnostic tools for schizophrenia. These findings are consistent with reported glycosyltransferase changes in the prefrontal cortex,21 which further implies that new approaches might focus on targeting glycosyltransferases for the development of novel neuropsychiatric treatment. Abbreviations: 2-AB, 2-aminobenzamide; ABS, Arthrobacter ureafaciens sialidase; β3GnT, β1,3-N-acetylglucosaminyltrans-

Figure 8. Structural symbols for the glycans and their linkages.

ferase; BKF, bovine kidney R-fucosidase; BTG, bovine testes β-galactosidase; CE, capillary electrophoresis; CNS, central nervous system; CSF, cerebrospinal fluid; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, fourth edition; Fuc, Fucose; GU, glucose units; GUH, β-N-acetylglucosaminidase cloned from S. pneumonia, expressed in Escherichia. coli; HAS, high abundance serum; HPAEC-PAD, high-performance anion exchange chromatography with pulsed amperometric detection; ICD 10, International Statistical Classification of Diseases and Related Health Problems 10th Revision; LAS, low abundance serum; Man, Mannose; MS, mass spectrometry; NAN1, Streptococcus pneumoniae sialidase; NANA, Sialic Acid; NPHPLC, normal-phase high-performance liquid chromatography; PNGase F, N-glycosidase F; PTMs, post-translational modifications. Structural Abbreviations Used. Structures are abbreviated in accordance to previous publications25,29 and as used in GlycoBase. Briefly, all N-glycans have two core N-Acetylglucosamines (GlcNAc) and a trimannosyl core. F at the start of the abbreviation indicates a core fucose linked R1-6 to the core GlcNAc; A[y]a represents the number a of antenna (GlcNAc) on the trimannosyl core linked to the R1-y mannose arm; B, bisecting GlcNAc linked β1-4 to β1-4 core mannose; Fb after Aa represents the number b of fucose linked R1-3 to antenna GlcNAc; Gc represents the number c of galactose linked β1-4 on antenna; S(z)d represents number d of sialic acids linked R2-z to the galactose. The graphical symbols shown in Figure 8 are used to represent the different sugar residues and linkages between them. Symbols encode the following monosaccharide structures: GlcNAc, filled square; mannose, open circle; galactose, open diamond; fucose, diamond with a dot inside; beta linkage, solid line; alpha linkage, dotted line.

Acknowledgment. We thank Dr. Jayne Telford for careful reading of this manuscript. This work was supported by The Stanley Medical Research Institute. Johannes L. Stanta is recipient of a DOC-fellowship of the Austrian Academy of Science. Supporting Information Available: Supplementary Tables 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Castle, D. J.; Murray, R. M. The neurodevelopmental basis of sex differences in schizophrenia. Psychol. Med. 1991, 21 (3), 565–75. (2) Dwek, M. V.; Brooks, S. A. Harnessing changes in cellular glycosylation in new cancer treatment strategies. Curr. Cancer Drug Targets 2004, 4 (5), 425–42. (3) Anderson, N. L.; Anderson, N. G. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics 2002, 1 (11), 845–67. (4) Karp, N. A.; Lilley, K. S. Design and analysis issues in quantitative proteomics studies. Proteomics 2007, 7 (Suppl 1), 42–50. (5) Riley, B.; Kendler, K. S. Molecular genetic studies of schizophrenia. Eur. J. Hum. Genet. 2006, 14 (6), 669–80.

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research articles

N-Glycosylation Changes in Patients with Schizophrenia assisted laser desorption/ionization-mass spectrometry following enzymatic release within sodium dodecyl sulphate-polyacrylamide electrophoresis gels: application to species-specific glycosylation of alpha1-acid glycoprotein. Electrophoresis 1998, 19 (11), 1950–9. (47) Nemansky, M.; Schiphorst, W. E.; Van den Eijnden, D. H. Branching and elongation with lactosaminoglycan chains of N-linked oligosaccharides result in a shift toward termination with alpha 2->3-linked rather than with alpha 2->6-linked sialic acid residues. FEBS Lett. 1995, 363 (3), 280–4.

(48) Spector, R.; Johanson, C. E. The mammalian choroid plexus. Sci. Am. 1989, 261 (5), 68–74. (49) Faraone, S. V.; Chen, W. J.; Goldstein, J. M.; Tsuang, M. T. Gender differences in age at onset of schizophrenia. Br. J. Psychiatry 1994, 164 (5), 625–9. (50) Goldstein, J. M.; Kennedy, D. N.; Caviness, V. S.; Brain Development, X. I. Sexual dimorphism. Am. J. Psychiatry 1999, 156 (3), 352. (51) Thompson, E. G. The CSF Proteins: A Biochemical Approach; Elsevier: Amsterdam, 1988.

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Journal of Proteome Research • Vol. 9, No. 9, 2010 4489