Prefractionation of Cerebrospinal Fluid to Enhance Glycoprotein

Cerebrospinal fluid is one source of biomarkers for Alzheimer's disease. Proteomic analysis has been used for screening of disease-influenced proteins...
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Prefractionation of Cerebrospinal Fluid to Enhance Glycoprotein Concentration Prior to Structural Determination with FT-ICR Mass Spectrometry Carina Sihlbom,*,† Pia Davidsson,‡ and Carol L Nilsson†,§ Institute of Medical Biochemistry, Go¨teborg University, Box 440, SE-40530 Go¨teborg, Sweden, Discovery Medicine/Molecular Pharmacology, AstraZeneca R&D Mo¨lndal, SE-43183 Mo¨lndal, Sweden, and National High Magnetic Field Laboratory, Tallahassee, Florida 32310 and Department of Chemistry and Biochemistry, Florida State University, Florida 32306 Received July 12, 2005

Glycoproteins in cerebrospinal fluid are found to be altered in Alzheimer patients compared to healthy control individuals. We have utilized micro-solution isoelectric focusing and affinity chromatography, prior to gel electrophoresis to enable site-specific structural determination of the N-linked glycans in apolipoprotein J with the use of FT-ICR MS. The albumin depletion method is the most suitable as prefractionation method of CSF prior to 2-DE for structural determination of glycoproteins in the study of neurodegenerative disorders. Keywords: glycoprotein • prefractionation • CSF • 2-DE • albumin removal • FT-ICR MS

Introduction Cerebrospinal fluid (CSF) circulates within the ventricles of the brain and surrounds the spinal chord. CSF offers a unique window to diagnose central nervous system (CNS) disorders and to clarify the basic molecular mechanism of CNS pathologies. For the identification of the proteins and in the search of biomarkers, CSF has been studied previously by proteomic methods.1-6 Alterations in protein glycosylation have been related to human disease states, such as congenital disorders of glycosylation (CDG)7 and the spongiform encephalopathies.8 The structural elucidation of glycoproteins is a challenge because of their inherent complexity and heterogeneity in biological systems. CDG results in severe mental and physical disabilities. 2-DE and MS analysis of plasma from CDG patients have revealed increased fucosylation and branching of glycans attached to transferrin and R-1-antitrypsin, relative to normal controls.7 Previous reports indicates that glycosylation of various proteins is also altered in Alzheimer’s disease (AD). Glycosylation of acetylcholinesterase and butyrylcholinesterase increases in the CSF as a function of the duration of AD but these proteins cannot be used as early biomarkers of the disease.9 Glycosylation of tau is an early abnormality that may facilitate hyperphosphorylation of tau in AD brain.10 The activity of asp-2 is dependent on the extent of N-glycosylation and thereby may affect the formation of amyloid 39-42 β-peptides, which is the main component of amyloid plaques * To whom correspondence should be addressed. Phone: +46 31 773 3049. Fax: +46-31-41 61 08. E-mail: [email protected]. † Go¨teborg University. ‡ AstraZeneca R&D Mo¨lndal. § National High Magnetic Field Laboratory and Florida State University.

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Journal of Proteome Research 2005, 4, 2294-2301

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found in AD brain.11 Proteome analysis of AD and control brain were recently applied to investigate changes in glycosylation using a carbohydrate-specific stain.12 The glycosylation of collapsin response mediator protein 2 (CRMP-2) was reduced in AD, while the glycosylation of glial fibrillary acidic protein was increased. CRMP-2 regulates the assembly and polymerization of microtubules and is associated with neurofibrillary tangles in AD. We have previously combined 2-D gel electrophoresis and MS to analyze glycoproteins in CSF from patients with AD.13 Several of the altered proteins in the comparison between AD patients and controls were isoforms of glycoproteins such as R-1-antitrypsin, β-trace protein, R-1β-glycoprotein, apolipoprotein J, and apolipoprotein E. Apolipoprotein J (apoJ) is a glycoprotein with the capability to interact with a broad spectrum of molecules, among them the amyloid beta peptide connected with AD. Due to its co-localization with fibrillar deposits in systemic and cerebral amyloid disorders, apoJ is also considered to be an amyloid-associated protein. Although its function is uncertain apoJ has been implicated in a wide variety of physiological and pathological processes, a role that may vary according to the protein maturation, sub-cellular localization, and the presence of certain tissue- or cell-specific factors.14 Recently, it was shown that apoJ promotes amyloid plaque formation as well as its associated neuritic toxicity and is likely to play an important role in AD pathology.15 The low protein concentration in CSF (about 0.3 mg/mL), the highly abundant protein, albumin, and the high salt concentration that would interfere an electrophoretic separation makes the sample preparation a crucial step. If high protein loads of unfractionated CSF are used in attempts to detect low abundance proteins, it will induce precipitation near the electrodes of the gel strip and cause extensive smearing in 10.1021/pr050210g CCC: $30.25

 2005 American Chemical Society

Prefractionation of CSF to Enhance Glycoprotein Concentration

the second dimension. Albumin can be removed by either dyeligands such as the widely recognized Cibacron Blue16 or specific antibodies.17 Low-abundance CSF proteins are difficult to characterize and affinity chromatography for removal of albumin18 has been used. The limitation of affinity chromatography using Cibacron Blue is that some other proteins that bind to albumin would also be retained on the affinity column. At present, many commercial albumin-removal kits have been designed with minimum nonspecific binding and for use with both serum and CSF.19,20 Another, more general method for prefractionation is microsolution isoelectric focusing (sol-IEF).21,22 In this strategy, samples are prefractionated into a few well-defined pools by small volume chambers separated by thin polyacrylamide based membranes containing immobilines at specific pH’s. Earlier conventional preparative IEF apparatus such as the Rotofor23 is divided into twenty chambers with no separation barriers and about 0.5 mL in resulting volume of each chamber. This apparatus yields more dilute fractions that are incompatible with direct subsequent analysis by 2-D gels. The development of the sol-IEF technique by Zuo and Speicher22 was commercialized by Invitrogen (ZOOM fractionator) and no sample processing is necessary between the prefractionation and the first dimension in gel electrophoresis. Our previous studies indicated the need for sample prefractionation in order to characterize protein glycoforms.13 The aim of the present study was to investigate the efficiency of prefractionation by albumin affinity columns or sol-IEF prior to 2-D gels and the potential improvement in the MS data for characterization of glycoproteins in CSF of AD patients. The study of large biomolecules such as glycopeptides demands high performance of the mass spectrometer in terms of resolution and mass accuracy. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides the high mass accuracy24 needed to improve the specificity for protein database search results and prediction of glycoforms. In addition, FT-ICR MS/MS has shown broad application in structural biochemical studies of post-translational modifications of proteins.25

Experimental Section Two different types of prefractionation of CSF were used, micro-solution isoelectrofocusing (sol-IEF) with a ZOOM IEF Fractionator (Invitrogen, CA, USA) and albumin affinity chromatography using the Montage Albumin Deplete kit (Millipore Corp., MA, USA). After the prefractionation step both samples were further separated with 2-D gel electrophoresis and the sol-IEF samples were also separated with 1-D SDS-PAGE. CSF Samples. CSF samples were obtained from the Clinical Neurochemical Laboratory, Sahlgrenska University Hospital/ Mo¨lndal, Sweden. Lumbar puncture was performed in the L4L5 vertebral interspace. The CSF samples were centrifuged at 2000 × g (4 °C) for 10 min to eliminate cells and other insoluble material, and stored at -80 °C. The CSF used in this study was taken from a pool of CSF samples from healthy individuals. Solution Isoelectrofocusing. The maximum protein load recommended for the ZOOM IEF Fractionator (Invitrogen, Carlsbad, CA, USA) is 2 mg and therefore 7 mL of CSF was used. The CSF proteins were precipitated at least 2 h at -20 °C with acetone at the ratio 1:4 v/v. The mixture was centrifuged at 10 000 × g for 15 min and the pellet was air-dried. The CSF protein pellets were dissolved in a buffer containing 9 M urea, 4% 3-[(cholamidopropyl) dimethylammonio] propanosulfonate

research articles hydrate (CHAPS), 35 mM Tris, 65 mM dithiothreitol (DTT) and bromphenol blue with a total volume of 3.5 mL. After agitation for 1 h, 35 µL of ZOOM Carrier Ampholytes, pH 3-10, were added to the sample for a final concentration of 1% ampholytes. The ZOOM IEF Fractionator apparatus consists of a cylindrical pipe with five sample chambers divided by disposable gelbased disks. Here, the apparatus was used to fractionate CSF into the intervals between pH 3.0, 4.6, 5.4, 6.2, 7.0, and 10.0. Assembling and protein loading of the ZOOM IEF Fractionator was performed according to the manufacturer’s instruction except for the preparation of the running buffers. Anode buffer were prepared by mixing 10.8 g urea, 3.3 mL Novex IEF Anode Buffer (50×) and deionized water to a total volume of 20 mL. Cathode buffer were prepared by mixing 10.8 g urea, 2.0 mL Novex IEF Cathode Buffer pH 3-10 (10×) and deionized water to a total volume of 20 mL. The CSF proteins were isoelectrically focused in the ZOOM fractionator for 30 min at 100 V, 80 min at 200 V, followed by 600 V until equilibrium as indicated by the decrease in current until a stable value of 0.6 mA (approximately 4 h) was reached. The fractionated sample from each chamber (670 µL) were removed with a pipet and ZOOM Carrier Ampholytes (pH 3-10) were added to a final concentration of 1% before the separation on narrow pH IPG strips. 170 µL (25%) of each fraction was loaded on the 7 cm IPG strips. Albumin Affinity Chromatography. Most of the available albumin depletion kits are manufactured for low volumes resulting in a need for concentration of the CSF. We used 5 kDa membrane cutoff centrifuge tubes (Amicon Ultra-4 Centrifugal Filters, Millipore Corp., MA, USA) for the concentration and desalting of 4 mL CSF. The spin conditions were 6800 × g (7500 rpm with Beckman, Avanti J-25, Corona, CA, USA) and 4 °C for approximately 30 min, until a retentate volume of 300 µL was left. Albumin depletion was performed by affinity chromatography (Montage Albumin Deplete kits, Millipore Corp., MA, USA). The composition of the column is proprietary, but the affinity resin is claimed to be specially formulated to bind albumin without binding significant amounts of other serum or plasma proteins. The concentrated CSF, 150 µL out of 300 µL, was mixed with an equal volume of the equilibrium buffer (supplied in the kit). The affinity column was equilibrated with 400 µL buffer and centrifuged for 2 min at 2000 rpm (approximately 500 × g). The eluate was discarded and the equilibration procedure repeated once. The column was loaded with half of the volume (150 µL) of the sample mixture (concentrated CSF and equilibrium buffer) and centrifuged for 2 min at 2000 rpm. The eluate was recovered from the collection tube, added back into the affinity column insert and centrifuged again for a second albumin depletion processing. After the second processing of the sample, the eluate was transferred to a separate collection tube and the same procedure was applied to the remaining 150 µL of sample mixture. The sample eluates were pooled into one tube. The column was loaded with 200 µL wash buffer provided in the kit, centrifuged 2 min at 2000 rpm, and the eluate was added to the sample. The wash procedure was repeated once. The sample eluate, 700 µL, was divided in two 1.5 mL tubes and 1.3 mL ice-cold acetone was added to 350 µL sample eluate. Proteins were precipitated in -20 °C for 2 h prior to 2-D gel electrophoresis. The mixture was centrifuged at 10 000 × g for 15 min and the pellet was air-dried. Journal of Proteome Research • Vol. 4, No. 6, 2005 2295

research articles To test the performance of the affinity column and to ensure that minimum of nonspecific binding has occurred, the bound proteins were eluted according to the manufacturer, and the proteins precipitated with the same procedure as the albumindepleted CSF sample. 2-D Gel Electrophoresis. Sample Preparation for the Albumin-Depleted CSF. The protein pellet was dissolved in 7 µL of a buffer containing 0.35 M sodium dodecyl sulfate (SDS) and 150 mM dithiothreitol (DTT) and boiled for 3 min. The sample was diluted with 40 µL of isobuffer (9 M urea, 4% 3-[(cholamidopropyl) dimethylammonio] propanosulfonate hydrate (CHAPS), 35 mM Tris, 65 mM DTT, bromphenol blue) and 40 µL of rehydration buffer (9M urea, 4% immobilized pH gradient (IPG) buffer, 18 mM DTT, bromphenol blue) and agitated for 1 h. After this step the two sample tubes divided prior to the precipitation were pooled together again. IEF. Isoelectric focusing was performed with IPG strips, pH 4.7-5.9, 7 cm (BioRad, Hercules, CA, USA), in the Ettan IPGphor IEF System (Amersham Biosciences/GE Healthcare, England). Active rehydration was performed at 30 V. The focusing step was completed at 20 000 Vh with a program of 250 V for 15 min and a gradient up to 4000 V during 2 h at 20 °C. 2-D SDS-PAGE. After equilibration of the IPG strips in buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, bromphenol blue) containing 1% DTT for 15 min and 2.5% iodoacetamide for a further 15 min, the second dimension separation was carried out by use of the Nu-PAGE gel system (NOVEX, San Diego, CA, USA) combined with MOPS running buffer [1 M 3-(N-morpholino) propane sulfonic acid (MOPS), 1 M Tris, 69 mM SDS, 20 mM EDTA] for about 45 min. Gel Staining. Gels were stained with SYPRO Ruby Protein Stain (Molecular Probes, Leiden, The Netherlands) according to the supplier’s protocols and scanned in 2-D 2920 Master Imager (Amersham Biosciences/GE Healthcare, England). The scan parameters were: excitation at 540 nm, emission at 630 nm during 3 s with 16-bit pixel density and image resolution of 150 µm. The optical density of protein spots is proportional to protein concentration. Glycoprotein spots were excised for further analysis by mass spectrometry. To visualize the enrichment of glycoproteins the gels were stained with Pro-Q Emerald 300 Glycoprotein stain (Molecular Probes, Leiden, The Netherlands) scanned in a Fluor-S MultiImager (BioRad, Hercules, CA, USA), excitation with UV-light 290-365 nm, emission at 530 nm during 30 s, 16-bit pixel density, image resolution of 200 µm and subsequently stained with SYPRO Ruby Protein Stain (Molecular Probes, Leiden, The Netherlands) according to the supplier’s protocols. The gels were scanned again in order to obtain an image of the protein pattern. The scan parameters for Sypro Ruby stained gels were as stated above for the 2-D 2920 Master Imager (Amersham Biosciences/GE Healthcare, England). 1-D SDS-PAGE. In-solution IEF fraction pH 4.6-5.4 was also analyzed by SDS-PAGE. A 100-µL portion of the sample fraction was mixed with 25 µL of SDS-sample buffer (4×) and divided into three lanes on a 12% Bis-Tris gel (Novex, San Diego, CA, USA). After separation (200V) and staining of the gel with Sypro Ruby the whole lanes below 60 kDa were cut into four pieces and digested with trypsin. In-Gel Trypsin Digestion. The method described by Shevchenko et al. was applied with minor modifications. Briefly, the gel pieces were washed twice in 100 µL of H2O/CH3CN (1:1 v/v) for 15 min. The gel pieces were dried in a vacuum 2296

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centrifuge, rehydrated in 10-20 µL of digestion buffer (25 mM NH4HCO3) containing 12.5 ng/µL sequencing grade trypsin (Promega, Madison, WI, USA) and incubated at 37 °C overnight. Any remaining digestion was stopped with 10 µL 2% HCOOH (Merck, Darmstadt, Germany) in water. The supernatant was collected, and the peptides were extracted twice with 30 µL of 2% HCOOH in water/CH3CN (1:1). The combined supernatants were evaporated to dryness in a vacuum centrifuge. Tryptic digests were reconstituted in 15 µL 0.1% HCOOH. Mass Spectrometry and Database Analysis. Nano-LC-MS/ MS of the tryptic peptides was performed in a 20 cm × 50 mm i.d. fused silica column packed in-house with ReproSil-Pur C18AQ porous (120Å) C18-bonded particles (Dr. Maisch GmbH, Ammerbuch, Germany). Two-microliter sample injections were made with a HTC-PAL auto sampler (CTC Analytics AG, Zwingen, Switzerland), connected to an Agilent 1100 binary pump (Agilent Technologies, Palo Alto, CA, USA). The peptides were trapped on a precolumn (4.5 cm × 100 or 130 mm i.d.) packed with 3 mm C18-bonded particles (Hydrosphere (120 Å), YMC Co. Ltd., Kyoto, Japan) in a valve switching configuration. The nanoLC-ESI-MS interface was constructed in-house, a tapered emitter tip made by fused-silica capillary (20 mm i.d., 150 mm o.d., Polymicro) was connected toward the steel screen. The voltage applied to the union was +1.4 kV and the eluent was electrosprayed from the emitter tip. The gradient was 0-50% CH3CN, starting with HCOOH 0.2% in water (100 nL/min) for 40 min. LC-MS data were obtained in a hybrid linear ion trap FT-ICR MS (LTQ-FT, Thermo Electron, Bremen, Germany), equipped with a 7 T magnet. The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Survey MS spectra (from m/z 400-1600) were acquired in the FT-ICR and the four most abundant doubly, triply or quadruply protonated ions in each FT-scan were selected for MS/MS in the linear ion trap followed by detection in the FT cell. Mass values for peptides that could not be matched to the identified protein sequence, by Mascot (Matrix Science, London, United Kingdom) were examined for the presence of glycosylation by use of the GlycoMod tool (http://us.expasy.org/tools/glycomod). The Swiss-Prot accession number corresponding to the protein identity and unmatched monoisotopic masses was entered, and a mass deviation of 10 ppm was tolerated. The number of possible hexoses (Hex) was set to 3-10, N-acetylhexosamines (HexNAc) 2-10, deoxyhexoses 0-3, and N-acetylneuraminic acids (NeuAc) 0-6.

Results and Discussion Two approaches, in-solution IEF and albumin affinity chromatography, to enrich low-abundance proteins in CSF were tested with the aim of characterization of glycoprotein isoforms in apoJ. We have previously used micro-narrow IPG strips (pH 4.7-5.9) in the first dimension for separation of protein isoforms in CSF. Evaluation of the prefractionation method was based on the amount of processed CSF that could be loaded on the IPG strips, which was also reflected in the 2-D gel image, as well as the resulting mass spectrometry data. Solution Isoelectrofocusing with a ZOOM IEF Fractionator. Focusing of proteins in the ZOOM IEF Fractionator apparatus was finished when the reading of the current has stopped decreasing and come to a steady-state. No more than 2 mA were allowed during the run and the focusing was finished at 0.6 mA after approximately 4 hours. We evaluated the efficiency of protein focusing by separation of 7 mL CSF into four

Prefractionation of CSF to Enhance Glycoprotein Concentration

Figure 1. 2-D gel electrophoresis of 7 mL in-solution isoelectric focused CSF in fraction 1 (pH 3.0-4.6), fraction 2 (pH 4.6-5.4), fraction 3 (pH 5.4-6.2) and fraction 4 (pH 6.2-7.0) with broad and narrow IPG strips. a) IPG strips pH 3-10 showing a complete protein focusing and b) IPG strips pH 4.7-5.9 detecting the six isoforms of apoJ, as circled on the gel for fraction 2 and missing in fraction 1 and 3.

fractions on the full range (pH 3-10) 2-D gels, as shown in Figure 1a. It was clear, that in the pH range of interest (pH 4.5-5.4), corresponding to fraction 2, there was no overlap from the nearby fractions 1 and 3. We could also confirm that six protein isoforms of apoJ were detected in the analysis of fraction 2 with narrow IPG strips (pH 4.7-5.9) 2-D gels, Figure 1b, which could not be seen in the corresponding analysis of fractions 1 and 3. Albumin is found in fraction 3 in the pH range 5.4-6.2. We increased the CSF volume to 9 mL, but the focusing time increased to about 6 h. Although a stable current was obtained, the focusing was not complete and the results were hard to reproduce. The IEF fractions from 7 mL CSF were separated with 2-D gel electrophoresis and 1-D SDS-PAGE, respectively, prior to mass spectrometry analysis. Albumin Depletion with Affinity Chromatography. The maximum volume applied to the affinity chromatography columns was 400 µL and the recommended use with serum/ plasma was 100 µL sample mixed with an equal volume of buffer. The protein concentration in CSF is about 100 times lower than in serum/plasma, because CSF has a total protein amount of about 0.3-0.4 mg/mL. Centrifuge tubes with 5 kDa membrane cutoff were used to reduce the volume and to remove salt from the crude CSF, resulting in a concentration factor of fifteen to twenty. The possible loss of proteins by binding to the membrane was tested by narrow pH range 2-D

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Figure 2. 2-D gel electrophoresis, pH 4.7-5.9, of (a) 2 mL albumin-depleted CSF stained with glycoprotein-specific Pro-Q Emerald dye, (b) total protein Sypro Ruby fluorescent dye, and (c) the affinity chromatography column strip, total protein stained. The circle shows the position of the six isoforms of apoJ. The enrichment of glycoproteins was verified with glycoprotein specific stain and only a small amount of the glycoprotein apoJ had bound to the affinity column.

gel analysis of 25 µL concentrated CSF compared to 500 µL of crude CSF. A slight total protein loss could be observed by comparing the gel images (data not shown). Concentrated CSF corresponding to 1, 2, and 4 mL of crude CSF was prepared for the affinity chromatography columns with the proportions as described in the Experimental Section. The resulting albumin-depleted and protein precipitated CSF was loaded on narrow IPG strips (pH 4.7-5.9) 2-D gels. The aim was to load as high amounts of CSF as possible. The most reproducible 2-D gels were obtained when using concentrated CSF corresponding to 2 mL of crude CSF, as in Figure 2b. We verified the enrichment of glycoproteins with glycoprotein specific stain, as shown in Figure 2a. It was also concluded that a small amount of the glycoprotein apoJ had bound to the affinity column when comparing Figure 2, parts b and c. Gel Image Comparison of the Two Prefractionation Methods. In previous proteomic analysis, we could load 500 µL of crude (nonprefractionated) CSF on 7 cm pH 4.7-5.9 IPG strips. In Figure 3, the crude CSF is compared to the albumin-depleted and in-solution IEF-prefractionated CSF. Since the same CSF pool has been used in all experiments, the data obtained from the albumin-depletion experiments and IEF fractionator can be compared. The six spots of apoJ showed great improvement Journal of Proteome Research • Vol. 4, No. 6, 2005 2297

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Figure 3. Typical gel images with 2-D gel electrophoresis, pH 4.7-5.9, stained with Sypro Ruby, showing the six isoforms of apoJ for (a) 500 µL nonprefractionated CSF, (b) 2 mL albumin-depleted CSF by affinity chromatography and (c) isoelectric focused CSF in fraction pH 4.6-5.4 corresponding to 1.8 mL of crude CSF. The numbered spots were excised and trypsin digested. The resulting MS data for spot 1-6 is shown in Tables 2 and 3. Table 1. FT-ICR-MS Data of a Selection of Glycopeptides, in the sol-IEF 1-D Gel Band Corresponding to the Mass Range of apoJ glycopeptide mass (Da)

delta ppm

3392.375

7.1

3837.629a 3887.602a 4033.666a

6.3 0.9 2.5

4049.595 4050.689a

5.4 4.0

4399.780 4621.975 4783.944

1.3 2.4 5.0

glycostructure

(HexNAc)2 (Deoxyhexose)1 (NeuAc)3 + (Man)3 (GlcNAc)2b (Hex)1 (HexNAc)1 + (Man)3 (GlcNAc)2 (Hex)2 (HexNAc)2 (NeuAc)2 + (Man)3 (GlcNAc)2b (Hex)2 (HexNAc)2 (Deoxyhexose)1 (NeuAc)2 + (Man)3 (GlcNAc)2 (Hex)4 (HexNAc)2 (NeuAc)2 + (Man)3 (GlcNAc)2 (Hex)3 (HexNAc)2 (Deoxyhexose)2 (NeuAc)1 + (Man)3 (GlcNAc)2 (Hex)1 (HexNAc)7 (NeuAc)2 + (Man)3 (GlcNAc)2b (Hex)4 (HexNAc)1 + (Man)3 (GlcNAc)2 (Hex)1 (HexNAc)1 (Deoxyhexose)1 (NeuAc)3 + (Man)3 (GlcNAc)2b

a Also detected in the albumin depleted CSF 2-D gel spots. b Dissociation spectrum with the peak for the glycoform antennae HexHexNAcNeuAc collected.

as demonstrated by better isomer detection and spot intensity. The spot intensity was estimated to 4-8-fold for the albumindepleted CSF (corresponding to 2 mL crude CSF) by comparing the numbers from the gel image scan. For the in-solution IEF method, 25 vol % of the fractionated sample could be loaded on the strips, which corresponds to 1.8 mL of crude CSF. The 2298

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reason for the lower enrichment by using in-solution IEF method is not clear but binding to plastic material in the apparatus and to the chamber disks could be possible. The final sample volume in each chamber in the ZOOM fractionator could not be decreased. The total amount of spots did not change drastically with approximately 120 spots detected for crude CSF and 140 spots for albumin-depleted CSF, 70 spots for in-solution isoelectric focused CSF in the narrow pH range 2-D gel images shown in Figure 3. It is not possible to make any comparison between the prefractionation methods using the number of spots detected because some spots coalesced due to overloading of some of the proteins. Also, in the insolution isoelectric focused CSF gel image, some proteins migrating above the pH range (5.4) for fraction 2 are excluded. Mass Spectrometry Analysis. Because the 2-D gel of the insolution pre-fractionated CSF was not as intense as the gel from albumin-depleted CSF and the mass spectrometry data revealed only very few glycopeptides in some of the isomeric spots, we decided to also enrich glycoproteins with 1-D SDSPAGE followed by nano-LC-MS/MS. Fifteen percent of fraction 2 (pH 4.6-5.4), corresponding to 1 mL of crude CSF, was separated on a 12% NuPAGE Bis-Tris gel. ApoJ was found in the second of the four bands cut below 60 kDa in the nanoLC-FT-ICR-MS/MS analysis. Accurate mass assignments of

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Prefractionation of CSF to Enhance Glycoprotein Concentration Table 2. FT-ICR-MS Data for the Glycopeptides, Found in the Six Isoforms of apoJ, Figure 3b, for the Albumin Affinity Chromatography Processed CSF mass (Da) spot

1

2

3

4

5

6

3368.33 3782.54

3368.32 3742.62 3887.60 4033.67 4050.69 4086.56

3019.18 3222.25 3368.31 3742.57 4033.67 4050.69 4086.57

3002.20 3206.26 3222.28 3368.32 3384.32 3620.50 3742.56 3782.54 4033.67 4050.68 4086.58

2857.15 3019.21 3206.31 3222.29 3238.30 3368.35 3384.35 3620.52 3742.58 3782.57 4033.67 4050.69 4086.57

3192.37 3206.27 3368.33 3742.58 3782.56 3837.52 4033.67 4050.69 4057.56 4086.57

tryptic peptides were submitted in database queries to provide protein identification. Unmatched peptide masses were submitted to the Glyco-Mod software tool in order to obtain suggested assignments of glycan composition. Table 1 shows the glycopeptides confirmed with dissociation spectra of a glycoform antennae and/or also found in the albumin-depleted CSF 2-D gel spots selected from the more than 40 theoretical glycopeptides of apoJ found in the sol-IEF 1-D gel band corresponding to the mass range of apoJ. Some of the glycopeptides (not reported here) did also match to other proteins detected in this 1-D gel band such as apolipoprotein E, haptoglobin and zinc-R-2-glycoprotein. We suspect retention of the in-gel digested glycopeptides in the gel to be an underlying factor for poor MS data yielded from the intensively

fluorescent stained 2-D gel spots, compared to the analysis of the 1-D gel. But there is also an extensive improvement in sensitivity by the use of nano-LC electrospray as compared to the analysis of the sol-IEF 2-DE spots (circled in Figure 3c) with direct electrospray FT-ICR MS (results not shown). The six isoforms of apoJ (Figure 3) were excised and trypsin digested from the 2-D gel of the albumin-depleted CSF. Table 2 shows the accurate masses of the multiply protonated ions that matched to theoretical glycopeptides of apoJ found in the nano-LC-FT-ICR-MS analysis. Table 3 shows the corresponding glycopeptide structures identified by GlycoMod for the MS data in Table 2. In the 2-D gel spots from the albumindepleted CSF, eighteen glycopeptides in the six isoforms of apoJ were found compared to only six glycopeptides from the unfractionated CSF.13 Some of the theoretical glycopeptides were fragmented and showed the typical patterns associated with loss of lactosamine (HexHexNAc) and of the antennae, including sialic acid (HexHexNAcNeuAc). An example of fragmentation is the triply protonated glycopeptide at m/z 1296.875 [M ) 3887.602 Da], that was automatically selected for ion trap dissociation. This glycopeptide was found both in the 1-D gel band from the sol-IEF prefractionation and in the 2-D gel spot 2 from the albumin-depleted CSF. Glyco-Mod predicted the glycoform (Hex)2(HexNAc)2(NeuAc)2 + (Man)3(GlcNAc)2 attached to the apoJ peptide 372-385 (LANLTQGEDQYYLR) (Figure 4a), with mass accuracy of 1 ppm. The structure was confirmed by fragments produced in the ion trap (Figure 4b). High mass accuracy is very helpful in distinguishing different isoforms of glycopeptides, because of the existence of oligosac-

Table 3. Glycopeptide Structures Identified by GlycoMod of apoJ, Spots 1 to 6 from the Desalted and Albumin Depleted CSF 2-D gel (Figure 3b) glycopeptide mass (Da)

error ppm

glycan composition

peptide sequence

2857.152 3002.204 ” 3019.176 3192.372 ” 3206.260 ” ” 3222.249 3238.304 ” ” ” 3368.310 ” ” 3384.318 ” ” 3620.498 3742.565 3782.540 3837.518 ” 3887.603a 4033.670 4050.688 4057.560 ” ” ” ” 4086.562 ”

-3.3 1.5 1.5 5.6 6.8 7.0 -4.3 4.9 5.1 3.2 4.6 4.7 4.7 8.4 3.9 4.0 -5.0 -4.7 8.0 -8.3 8.9 0.9 5.5 -1.9 6.1 1.2 3.4 3.9 -0.9 -4.4 6.3 6.4 -7.3 4.6 4.6

(Hex)2 (HexNAc)1 (Deoxyhexose)2 + (Man)3(GlcNAc)2 (Hex)2 (HexNAc)1 (Deoxyhexose)1 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)1 (HexNAc)1 (Deoxyhexose)2 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)3 (HexNAc)2 (NeuAc)1 + (Man)3(GlcNAc)2 (HexNAc)4 (Deoxyhexose)1 + (Man)3(GlcNAc)2 (HexNAc)5 + (Man)3(GlcNAc)2 (Hex)3 (HexNAc)3 + (Man)3(GlcNAc)2 (Hex)2 (HexNAc)4 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)2 (HexNAc)3 (Deoxyhexose)1 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)3 (HexNAc)3 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)4 (HexNAc)1 (Deoxyhexose)2 + (Man)3(GlcNAc)2 (Hex)3 (HexNAc)2 (Deoxyhexose)2 + (Man)3(GlcNAc)2 (Hex)4 (HexNAc)2 (Deoxyhexose)1 + (Man)3(GlcNAc)2 (Hex)3 (HexNAc)1 (NeuAc)2 + (Man)3(GlcNAc)2 (Hex)3 (HexNAc)4 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)3 (HexNAc)3 (Deoxyhexose)1 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)4 (HexNAc)3 + (Man)3(GlcNAc)2 (Hex)3 (HexNAc)1 (Deoxyhexose)1 (NeuAc)2 + (Man)3(GlcNAc)2 (Hex)4 (HexNAc)3 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)4 (HexNAc)2 (Deoxyhexose)2 + (Man)3(GlcNAc)2 (Hex)3 (HexNAc)3 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)2 (HexNAc)2 (Deoxyhexose)1 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)4 (HexNAc)3 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)2 (HexNAc)2 (NeuAc)3 + (Man)3(GlcNAc)2 (Hex)5 (HexNAc)2 (Deoxyhexose)2 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)2 (HexNAc)2 (NeuAc)2 + (Man)3(GlcNAc)2 (Hex)2 (HexNAc)2 (Deoxyhexose)1 (NeuAc)2 + (Man)3(GlcNAc)2 (Hex)3 (HexNAc)2 (Deoxyhexose)2 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)7 (HexNAc)4 + (Man)3(GlcNAc)2 (Hex)5 (HexNAc)2 (Deoxyhexose)2 (NeuAc)2 + (Man)3(GlcNAc)2 (Hex)6 (HexNAc)4 (Deoxyhexose)1 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)6 (HexNAc)5 (Deoxyhexose)1 (NeuAc)1 + (Man)3(GlcNAc)2 (Hex)1 (HexNAc)3 (NeuAc)3 + (Man)3(GlcNAc)2 (Hex)6 (HexNAc)1 (NeuAc)3 + (Man)3(GlcNAc)2 (Hex)5 (HexNAc)1 (Deoxyhexose)1 (NeuAc)3 + (Man)3(GlcNAc)2

290-299 HNSTGCLRMK 290-299 HNSTGCLRMK 290-299 HNSTGCLRMK 290-297 HNSTGCLR 287-297 EIRHNSTGCLR 287-297 EIRHNSTGCLR 290-299 HNSTGCLRMK 290-297 HNSTGCLR 290-297 HNSTGCLR 290-297 HNSTGCLR 290-299 HNSTGCLRMK 290-299 HNSTGCLRMK 290-299 HNSTGCLRMK 81-89 KEDALNETR 290-297 HNSTGCLR 290-297 HNSTGCLR 290-299 HNSTGCLRMK 81-89 KEDALNETR 290-297 HNSTGCLR 290-299 HNSTGCLRMK 287-297 EIRHNSTGCLR 372-385 LANLTQGEDQYYLR 287-297 EIRHNSTGCLR 287-298 EIRHNSTGCLR 290-297 HNSTGCLR 372-385 LANLTQGEDQYYLR 372-385 LANLTQGEDQYYLR 372-385 LANLTQGEDQYYLR 290-299 HNSTGCLRMK 81-89 KEDALNETR 290-297 HNSTGCLR 290-297 HNSTGCLR 82-94 EDALNETRESETK 290-299 HNSTGCLRMK 290-299 HNSTGCLRMK

a

modification

Met-ox CysCAM CysCAM Met-ox CysCAM CysCAM CysCAM Met-ox

CysCAM CysCAM + Met-ox CysCAM CysCAM CysCAM CysCAM

CysCAM + Met-ox CysCAM CysCAM Met-ox

Fragmentation of glycopeptide [3887.603], also found in the sol-IEF 1-D gel, see Figure 4b.

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research articles

Sihlbom et al.

Figure 4. Nano-LC-FT-ICR-MS of one band from a sol-IEF 1-D gel corresponding to the mass range of apoJ. (a) The triply protonated glycopeptide at m/z 1296.875 [M ) 3887.60 Da], was automatically selected for ion trap dissociation. This glycopeptide was also identified in spot 2 from the albumin-depleted 2-D gel. b) CAD spectrum of the ion peak m/z 1296.875 (as shown in (a) above). From the fragmentation data it can be determined that the glycopeptide bears a biantennary glycan of the complex type.

charide combinations with small mass differences. The number of possible combinations decreases with increasing mass accuracy.

Conclusion For future studies of clinical CSF in relation to neurological disease, the restricted availability of CSF material must be considered and requires as low volumes as possible for experimental use. Solution isoelectric focusing allows prefractionation of up to 7 mL of CSF, but the volume of solution IEF fractions that can be loaded on 1- or 2-D gels is limited and only a part of the final fraction. The albumin depletion method, using 2 mL crude CSF, is the best suited for structural 2300

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determination of less abundant proteins for use with clinical CSF samples in the discovery of disease-specific glycoproteins. The 2-D gel spot intensities were remarkable improved using albumin depletion compared to solution IEF prefractionation.

Acknowledgment. The authors thank Kaj Blennow for providing the CSF, Ann Brinkmalm, Linda Paulson and Rita Persson for advice on and the use of the ZOOM fractionator, Hasse Karlsson for the nano-LC column and setup, Thomas Larsson for database searches, and Susanne Teneberg for proofreading. Financial support from the ICR facility at the National High Magnetic Field Laboratory (NSF CHE-99502), Vetenskapsrådet-Medicin, Swedish Society of Medicine and the

research articles

Prefractionation of CSF to Enhance Glycoprotein Concentration

Swedish Foundation for International Cooperation in Research and Higher Education (STINT) is gratefully acknowledged. The purchase of the LTQ-FT-ICR mass spectrometer was made possible through a grant from Knut and Alice Wallenberg Foundation.

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PR050210G

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