Evaluation of Protein Depletion Methods for the Analysis of Total-, Phospho- and Glycoproteins in Lumbar Cerebrospinal Fluid

Yuko Ogata,† M. Cristine Charlesworth,† and David C. Muddiman*,†,‡
Mayo Proteomics Research Center and Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905
Received December 25, 2004

A proper sample preparation, in particular, abundant protein removal is crucial in the characterization of low-abundance proteins including those harboring post-translational modifications. In human cerebrospinal fluid (CSF), approximately 80% of proteins originate from serum, and removal of major proteins is necessary to study brain-derived proteins that are present at low concentrations for successful biomarker and therapeutic target discoveries for neurological disorders. In this study, phospho- and glycoprotein specific fluorescent stains and mass spectrometry were used to map proteins from CSF on two-dimensional gel electropherograms after immunoaffinity based protein removal. Two protein removal methods were evaluated: batch mode with avian IgY antibody microbeads using spin filters and HPLC multiple affinity removal column. Six abundant proteins were removed from CSF: human serum albumin (HSA), transferrin, IgG, IgA, IgM, and fibrinogen with batch mode, and HSA, transferrin, IgG, IgA, antitrypsin, and haptoglobin with column chromatography. 2D gels were compared after staining for phospho-, glyco- and total proteins. The column format removed the major proteins more effectively and approximately 50% more spots were visualized when compared to the 2D gel of CSF without protein depletion. After protein depletion, selected phospho- and glycoprotein spots were identified using mass spectrometry in addition to some of the spots that were not visualized previously in nondepleted CSF. Fifty proteins were identified from 66 spots, and among them, 12 proteins (24%) have not been annotated in previously published 2D gels.
Keywords: cerebrospinal fluid • human • two-dimensional gel electrophoresis • mass spectrometry • glycoprotein • phosphoprotein • protein depletion • low-abundance protein

1. Introduction
Cerebrospinal fluid (CSF) is a physiological fluid largely produced by the choroid plexus that surrounds the brain and is in continuum with the extra-cellular fluid of the central nervous system. The total volume of human CSF is approximately 140 mL, and it is produced at the rate of 0.3-0.4 mL/min, carrying metabolites from various tissues within the brain.1 CSF is also in contact with the blood plasma through the blood-brain barrier, thus resembling an ultrafiltrate of plasma in its protein constituents. Due to its proximity to the brain and the central nervous system, CSF is an excellent medium for the physiological study of neurological disorders. For example, measurement of β-amyloid and hyperphosphorylated τ protein in CSF has become a valuable diagnostic tool for Alzheimer’s disease in recent years.2-4 In attempts to find biomarkers for neurological disorders, the CSF proteome has been characterized in many studies,5-11 and thus far, nearly
* To whom correspondence should be addressed. David C. Muddiman, Ph.D., Medical Sciences Building 3-115, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905. Phone: (507) 284-1997. Fax: (507) 284-9261. E-mail: muddiman.david@mayo.edu. † Mayo Proteomics Research Center. ‡ Department of Biochemistry and Molecular Biology. 10.1021/pr049750o CCC: $30.25 © 2005 American Chemical Society

70 proteins have been identified using 2-D gels followed by mass spectrometric analysis.6 For successful biomarker and therapeutic target discoveries, it is advantageous to be able to identify differentially expressed proteins globally and to assess the level of post-translational modifications for low-abundance proteins. The concentration of proteins in CSF ranges from 0.2 to 0.8 mg/mL (0.3-1% of serum protein concentration). Many serum proteins are present, including albumin, which constitutes 35-80% of total protein.12 Thus, removal of such abundant proteins can dramatically improve the number of proteins to be identified by reducing the dynamic range of protein levels in the biological fluid to better match that of the downstream analytical platform.13 For example, in 2D SDS-polyacrylamide gel (SDS-PAGE) analyses, ∼5 times more CSF can be loaded onto the gels and spots that are typically masked by abundant proteins can be visualized. Many studies have been reported using CSF as a biological specimen;5-11 however, few have taken advantage of protein depletion methods.13 Several methods are available for the removal of proteins that are present in high concentrations in physiological fluid. Currently, the most widely used albumin removal method is Cibracon Blue chromatography; however, this method is highly nonspecific, and it is known to bind
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numerous other proteins.14 Fractionation with organic solvents can also reduce the dynamic range of CSF by separating proteins by their molecular weights.15 Though the separation is not complete with this method, it is sufficient for 2Dchromatography and mass spectrometry or “shotgun” proteomics approach, and more than 300 proteins were identified in CSF including proteins that were identified using only a single peptide.15 The fractionation method has been also used for 2D PAGE analysis of CSF using reversed-phase solid-phase extraction to separate proteins according to their hydrophobic properties.11 Though numerous new proteins were identified in this study, multiple gels were needed to be developed from a single sample. Recently, immunoaffinity based multiple protein removal methods were developed by GenWay Biotech Inc. (up to 12 proteins) and Agilent Technologies (up to 6 proteins), which are effective and specific in removing abundant proteins.13,16 The first immunoaffinity protein depletion for CSF was demonstrated by Maccarrone et al.13 using the Agilent multiple affinity removal system followed by 2Dchromatography and mass spectrometry,17 and they identified 100 proteins. However, such immunoaffinity protein depletion methods have not been fully evaluated using 2D PAGE with post-translational modification specific stains, and have not been compared to other multiple immunoaffinity protein removal systems. In this study, avian IgY antibody microbeads in spin filters and a prepacked HPLC multiple affinity removal column were evaluated for the removal of six abundant proteins (HSA, transferrin, IgG, IgA, IgM, and fibrinogen with microbeads, and HSA, transferrin, IgG, IgA, antitrypsin, and haptoglobin with HPLC column) from a pooled CSF sample. A pooled sample from 25 healthy individuals was used to obtain an averaged biological specimen. Following the protein removal, 2D gels were compared after staining for phospho-, glyco-, and total proteins using post-translational modification specific fluorescent stains. These stains offer broad dynamic ranges (5001000-fold) for quantification, and do not interfere with mass spectrometric analyses for protein identification. The proteomic study method we describe here, major protein depletion f 2D PAGE f post-translational modification mapping and quantitative analysis f mass spectrometric protein identification, is effective and will be useful in future biomarker discoveries using CSF.

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Figure 1. Experimental design. A pooled CSF sample was reduced in volume using 10 kDa MW cutoff filters, placed in appropriate buffered solutions, and applied to two immunoaffinity protein removal methods (Batch mode with GenWay microbeads using spin cups and Agilent HPLC column). 2D gels were developed using the flow-through from the two columns, and compared to a 2D gel of undepleted CSF after staining for total, phospho-, and glycoproteins. 2D gels were also developed using the eluate from the two columns for comparison after staining for total proteins. P in Figure 1 indicates a protein concentration assay.

2. Experimental Section
The schematic representation of the general experimental design is shown in Figure 1. A pooled CSF sample was reduced in volume using 10 kDa MW cutoff filters, placed in appropriate buffered solutions, and applied to two different immunoaffinity protein removal systems (GenWay spin cups and Agilent HPLC column). 2D gels were developed using the flow-through from the two matrixes, and compared to a 2D gel of undepleted CSF after staining for total, phospho-, and glycoproteins. 2D gels were also developed using the eluate from the two matrixes for comparison after staining for total proteins. The “P” in Figure 1 indicates a protein concentration assay to determine the amount of total protein present after each step. 2.1. CSF Samples. The CSF samples were pooled from 25 healthy individuals (15 females and 10 males), with ages ranging from 24 to 73. Each CSF sample was collected prospectively from patients who are undergoing diagnostic outpatient lumbar puncture at Mayo Clinic Rochester as part
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of their routine care. (IRB numbers: 443-04 and 1319-03.) The specimens were coded and frozen immediately at -80 °C. The concentration of the pooled sample was 328 µg/mL, determined with the Bradford method using bovine serum albumin (BSA) as a standard. 2.2. Batch Mode Microbeads Protein Removal. A 2.0-mL portion of pooled CSF was thawed, and immediately transferred to a Centricon, YM-10 (10 kDa MW cut off; Millipore, Billerca, MA) to reduce the volume by centrifugation at 5000 × g. After the volume reduction to 230 µL, the filter was reversed to collect the sample at 200 × g for 1 min. 370 µL of Tris buffered saline (TBS), pH 7.4 was added to the filter, sonicated for one minute, vortexed, and collected at 200 × g for one minute (590 µL total). 10 µL of this solution was set aside for Bradford protein assay. Meanwhile, 750 µL of avian IgY antibody microbeads (1.2 mg serum protein purification capacity, Seppro Mixed 6; GenWay, San Diego, CA) in TBS were placed in an eppendorf tube, and after the beads settled to the bottom, the supernatant (TBS) was removed by pipetting. To 750 µL settled beads, 580 µL of CSF in TBS was applied and incubated for 20 min on a rocking platform. After incubation, the bead slurry was transferred to two Economy mini-spin filter cups (800 µL capacity; Pierce, Rockford, IL), and centrifuged (2000 × g for 10 s) to collect the flow through. The beads were washed with 375 µL TBS per filter, and the flow-through was collected by centrifugation (2000 × g for 10 s), and combined with the initial flow-through. 10 µL was set aside for protein assay from this combined flowthrough. The beads were washed 3 times with 375 µL washing buffer (TBS with 0.05% Tween-20), and the retained major proteins were eluted with a stripping buffer (0.1 M glycine, pH 2.5). The flow-through and the eluate were stored at -80 °C separately until 2D PAGE analysis. 2.3. HPLC Column Protein Removal. 2.5 mL of pooled CSF was thawed, and immediately transferred to a Centricon, YM10 (10 kDa MW cut off) to reduce the volume by centrifugation

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w/v agarose in SDS-PAGE running buffer with a trace of bromophenol blue dye). Two protein standard mixtures were used: CandyCane glycoprotein molecular weight standards, and PeppermintStick phosphoprotein molecular weight standards (Molecular Probes), which were used to assess the specificity of the fluorescent stains. Paper wicks were wetted with 2 µL of each standard and inserted in the agarose on either side of the IPG strips. The gels were electrophoresed for 12 min at 140 V and 6 h at 200 V in running buffer containing 25 mM Tris, 0.1% SDS, and 190 mM glycine. 2.4.3. Gel Staining. One gel from nondepleted and the two depleted CSF gels were stained using three different dyes (Figure 1): Pro-Q Diamond for phosphoproteins, Pro-Q Emerald 488 for glycoproteins, and Sypro Ruby for total proteins (Molecular Probes). The two gels of proteins eluted from the affinity matrixes were stained only with Sypro Ruby for total proteins. For differential staining, gels were fixed in 500 mL fixing solution (50% v/v MeOH and 10% v/v acetic acid) overnight. Additional fixation was done for 1 h with 500 mL of the above fixing solution to ensure that all the SDS was washed out of the gels. After washing 4 times with 500 mL MilliQ water for 15 min each, the gels were incubated in 500 mL of Pro-Q Diamond for 3 h and 15 min, and destained with 500 mL of 20% v/v acetonitrile and 50 mM sodium acetate (pH 4.0) for 1 h. The destain step was repeated 4 times over 4 h. To remove the destain solution, the gels were washed with 500 mL of MilliQ water 2 times before gel images were scanned. After image scanning, gels were washed in 3% v/v acetic acid overnight, preceding the ProQ Emerald stain process. The gels were oxidized prior to ProQ Emerald staining in 500 mL of 0.5% w/v periodic acid and 3% acetic acid for 1 h. After oxidization, the gels were washed with 500 mL of 3% acetic acid 3 times (20 min/wash), and incubated with 250 mL of ProQ Emerald stain for 2.5 h. The gels were washed again with 500 mL 3% acetic acid for 30 mim once, for 45 min 3 times, and with 500 mL MilliQ water for 15 min twice. After gel image scanning, the three gels were incubated with 400 mL Sypro Ruby stain overnight. After destaining with 500 mL solution of 10% v/v methanol and 7% v/v acetic acid for 30 min, the three gels were washed with 500 mL MilliQ water for 10 min twice before image scanning. The remaining two gels (eluates from GenWay microbeads and Agilent HPLC column) were fixed in a 500 mL solution of 50% v/v methanol and 10% v/v acetic acid for 30 min, washed with 500 mL 5% v/v methanol for 15 min, and incubated in Sypro Ruby stain overnight. The gels were destained using a 500 mL solution of 10% v/v methanol and 7% v/v acetic acid for 30 min and washed with 500 mL MilliQ water for 10 min twice before they were scanned. Gel incubations in solutions were done with gentle agitation on an orbital shaker, and all gel staining and destaining processes were done in the dark by covering the gel containers tightly with aluminum foil. 2.4.4. Image Analysis. The gel images were scanned using a Bio-Rad Molecular Imager FX (Pro Plus) using excitation at 532 and 555 nm long pass filter for ProQ Diamond and Sypro Ruby, and excitation at 488 and 530 nm band-pass filter for ProQ Emerald. The images were then processed using PDQuest (v 7.2) gel analysis software (Bio-Rad). After cropping, re-sizing, and filtering using the filtering wizard with a filter size of 9 × 9 pixels to eliminate speckles, the spots were detected using the spot-detection wizard, by setting the same parameters for all gels. The spots were also inspected manually to remove
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at 5000 × g. After the volume reduction to ∼80 µL, the filter was reversed to collect the sample at 200 × g for 1 min. 60 µL of buffer A (proprietary phosphate-salt solution, pH 7.4, containing 0.02% NaN3; Agilent, Wilmington, DE) was added to the filter, sonicated for one minute, vortexed and collected at 200 × g for one minute. This step was repeated again, and a total of 200 µL was collected. After setting aside 25 µL for the Bradford protein assay, the CSF protein in buffer A was applied to multiple affinity removal HPLC column (4.6 × 100 mm, 2.4 mg serum protein purification capacity; Agilent), at a flow rate of 0.5 mL/min during the flow-through collection, and at 1 mL/min during elution using a BioLogic Pathfinder chromatography system (Bio-Rad, Hercules, CA). The chromatography was monitored with a UV detector at 280 nm and a conductivity meter. Antibody-bound proteins were eluted with buffer B (proprietary urea buffer, pH 2.5; Agilent) at 1 mL/min flow rate. To neutralize the pH, 1 M Tris-HCl, pH 8.0, was added at 1/10 of the total volume. Both the flow-through and the neutralized eluate were stored at -80 °C separately for further 2D gel analyses. 2.4. 2D Electrophoresis. 2.4.1. IEF. A total of five gels were developed (Figure 1): flow-through and eluate fractions from both the HPLC column and microbeads in spin cups, and CSF without depletion. For the first dimension isoelectric focusing, ReadyStrip IPG strips, 18 cm, nonlinear (pH 3-10; Bio-Rad) were used. The five protein solutions were desalted and concentrated using 10 kDa MW cutoff filters. When the volumes were reduced down to ∼60 µL, 100 µL of 20 mM Tris-HCl (pH 8.0) was added to the filters, and further reduced down to ∼60 µL again. This washing step was repeated for a total of 3 times. After this desalting step, the filters were reversed, and the samples were collected by centrifugation for 1 min. Then to each filter, 20 µL of dissociation solution (7 M Urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 1% 3-10 Pharmalyte, Amersham Biosciences, Piscataway, NJ.) was added, sonicated for 1 min, vortexed lightly, and collected as before. CSF protein in each sample was further diluted with rehydration buffer (7 M Urea, 2 M thiourea, 4% CHAPS, 60 mM DTT, 0.5% Pharmlalyte, 0.25% 3-10 NL IPG buffer, Bio-Rad) to obtain 100 µg protein in 380 µL of dilution solution. The concentration of protein was determined using the Bradford method. The protein concentration values were also verified using a RediPlate EZQ protein quantification kit (Molecular Probes, Eugene, OR) following their standard protocols. Both methods gave the same results within experimental error. CSF protein samples in rehydration buffer were then applied to the IPG strips (100 µg/380 µL/strip) and rehydrated overnight under mineral oil to prevent drying. The strips were focused using PROTEAN IEF unit (Bio-Rad) with a one step program. A limiting current of 30 µA per strip was maintained, and the strips were focused for total of 80 000 Vh. After IEF, the strips were stored at -80 °C until SDS-PAGE analysis. 2.4.2. SDS-PAGE. Five 20 × 20 × 0.1 cm 12% acrylamide gels were cast using a solution of 12% acrylamide/bis (29/1), 0.375 M Tris/HCl (pH 8.8), 0.05% w/v ammonium persulfate, and 0.025% v/v N,N,N′,N′-tetramethylethylenediamine (TEMED) in MilliQ water. The IEF strips were equilibrated for 10 min with ∼5 mL each of 1% w/v DTT in equilibration buffer (20% v/v glycerol, 0.38 M Tris base, pH 8.8, 6 mM urea, and 2% w/v SDS), and for 15 min with 2% w/v iodoacetamide in equilibration buffer with gentle agitation. The IPG strips were loaded onto the 12% acrylamide gels with agarose sealing solution (1%

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obvious artifacts, such as speckles and lines along the edge of the gels before comparative spot analysis. 2.5. Silver Staining and in-Gel Trypsin Digestion. The SDSPAGE for HPLC column flow-through was used for protein identification. Before the spots were excised, the gel was silver stained for visualization of the spots using the Blum method18 with minor modifications. The gels were developed with a solution of 3% w/v sodium carbonate, 0.04% formaldehyde, and 0.0004% v/w sodium thiosulfate until the desired spot abundance was reached. Staining was stopped with 5% ethylenediaminetetraacetic acid (EDTA) for 10 min. The gels were stored in MilliQ water at 4 °C until spot cutting. All spots were excised manually and stored at -20 °C until trypsin digestion. After rehydration with 200 mM Tris-HCl (pH 8.2), protein spots were destained with a solution of 15 mM potassium ferrocyanide and 50 mM sodium thiosulfate for ∼2 min followed by MilliQ water wash 4 times or until yellow color of potassium ferrocyanide is no longer visible. In addition, the proteins were reduced with 20 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 50 mM Tris-HCl (pH 8.2) at 55 °C for 20 min followed by dehydration with acetonitrile twice. The proteins were also alkylated with a buffer containing 40 mM iodoacetamide in 50 mM Tris-HCl (pH 8.2) for 20 min at room temperature followed by dehydration with acetonitrile. After rehydrating the gel pieces with 20 mM Tris-HCl (pH 8.2) and dehydrating again with acetonitrile, the protein was digested for 5 h with 10 µL (2 ng/µL) trypsin (Promega Corporation, Madison WI) in 20 mM Tris-HCl (pH 8.2) at 37 °C. The peptides were extracted using 20 µL 2% v/v trifluoroacetic acid (TFA) after 30 min incubation, then with 20 µL acetonitrile after 5 min incubation. The pooled extracts were concentrated to less than 5 µL in vacuo, and brought up in 0.1% TFA for protein identification by mass spectrometry. 2.6. Mass Spectrometry and Database Analysis. Mass spectra were obtained by nano-flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The peptide mixture from each gel piece was first trapped onto a 75 µm × 5 cm ProteoPep C18 PicoFrit nanoflow column and eluted with a 0.1% formic acid/acetonitrile gradient using a Michrom Paradigm MS4 (Michrome BioResoruces inc, Auburn, CA) coupled to a ThermoFinnigan LTQ linear ion trap mass spectrometer (ThermoElectron, San Jose, CA). The mass spectrometer was set to continuously scan for ions in the m/z ) 375-1600, automatically switching to MS/MS mode on the ions with abundances exceeding 2000 ion counts. The MS/MS data were searched against tryptic peptide sequences from the SWISS-PROT database using Mascot (Matrix Sciences London, UK) search algorithms19 running on a 10 node cluster. All searches were conducted with variable or differential modifications allowing +16 for methionine sulfoxide, and +57 for carboxamidomethyl-cysteines. The search was restricted to trypsin generated peptides allowing for up to 3 missed cleavages. The taxonomy was set to humans. Peptide mass tolerance was ( 1.5 Da, and fragment mass tolerance was set to ( 0.8 Da. At the above settings, probability based MOWSE scores20 > 44 indicated identity or extensive homology (p < 0.05, -10*log(P)), where P is the probability that the observed match is a random event. Protein scores were derived from ion scores as a nonprobabilistic basis for ranking protein hits.19 In other words, protein scores > 44 indicated protein identifications with an error rate of 5% or less. In this study, proteins were identified using the following additional criteria: (1) sequence
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tags for two or more peptides; (2) at least one of the peptide’s ion score was > 44.

3. Results and Discussion
To evaluate the relative efficiency of batch versus column mode for removal of abundant proteins, and compare two heterogeneous immunoaffinity depletion methods, we have evaluated GenWay IgY beads using spin filter cups and Agilent multiple affinity removal HPLC column by comparing 2D gels before and after the protein depletion. The general experimental scheme described above is shown in Figure 1. 3.1. High-Abundant Protein Depletion. Since the protein concentration of CSF is relatively low, the volume of the pooled sample was first reduced as suggested by the affinity matrix manufacturers using 10 kDa filters and placed in appropriate buffers for protein depletion. Two aliquots (2.0 and 2.5 mL) of the pooled CSF (328 µg/mL) were reduced in volume, and placed in either TBS (pH 7.4) for microbeads or Buffer A (phosphate-salt solution, pH 7.4) for the HPLC column at final sample volumes of 580 µL and 175 µL, respectively. The column purification capacities were approximately 1.2 mg and 2.4 mg of serum protein for the microbeads and the HPLC column, respectively, which translates to 3.7 and 7.3 mL of the pooled CSF sample in this study. Thus, the purification was conducted under the recommended capacities for both methods. During the volume reduction and buffer exchange using 10 kDa cut off filter, approximately 10% of the protein was lost, which was in concordance with expected protein loss noted in Millipore user’s manual for the filters. During the affinity purification, on the average, 24.6% and 18.0% of proteins were obtained in the flow-through from the microbeads and the HPLC column, respectively. The difference in yields may be also due to the fact that the two columns removed different proteins. Four out of six major proteins were common to both, but two were different. Among the four common proteins removed, IgA did not yield notable spots on the eluate gels as the concentration of IgA in CSF is relatively low (∼1 µg/mL compared to IgG at ∼25µg/mL).12 The Agilent column removes antitrypsin, which was present at relatively high concentration as indicated by the high spot abundance shown in the 2D gel of the eluate from the HPLC column in Figure 2 (E). Haptoglobin was also effectively removed by this matrix. On the contrary, two proteins removed by the GenWay microbeads, IgM and fibrinogen were not present at high concentrations as suggested by the lack of major spots in the 2D gel of the eluate from the beads in Fugure 2D. In addition, the relatively low concentration of IgM in CSF has also been reported previously.12 According to the study done by Reiber in 2001, the IgM/IgG concentration ratio is 1/125 in CSF, while it is ∼ 1/10 in serum.12 We attribute the difference in the yield to both different column formats and proteins that were removed. 3.2. Comparative 2D SDS-PAGE Analysis of CSF with and without Protein Depletion. 2D gels of the flow-through from the two columns were compared to the one without protein depletion after staining for total proteins, and shown in Figure 2B,C. Both GenWay microbeads and Agilent column flowthrough gels were depleted of 4 common major protein spots (HSA, transferrin, IgG, and IgA) which were seen in the 2D gels of eluate from the two protein removal methods (D and E). However, albumin depletion with the beads was not complete. In Figure 2, a faint streak was present in the gel of flow-through from the microbeads (B), where albumin spots were present in the nondepleted CSF gel. In addition, 1D gel analysis of the

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Figure 2. 2D gel of CSF before and after major protein depletion. All gels were stained for total proteins. The 2D gels for the flowthrough (B and C) show the CSF protein spots after removing 6 major proteins: HSA, transferrin, IgG, IgA, IgM, and fibrinogen with GenWay microbeads in batch mode, and HSA, transferrin, IgG, IgA, antitrypsin, and haptoglobin with Agilent HPLC column. The 2D gels of eluate (D and E) shows the major proteins that were captured on the columns during the purification.

same flow-through also revealed a faint HSA protein band for microbead treated CSF in batch mode, whereas the HSA band was not observed for the column depleted CSF (data not shown). This incomplete HSA removal may be due to the differences in the column formats (incubation of beads with approximately equal volume of sample solution vs HPLC column) leading to different chromatographic efficiencies. Protein spots were detected using PDQuest (v 7.2) gel analysis software (Bio-Rad) by setting the same parameters for all gels. Various parameter settings were tested; however, all results reported approximately the same numbers of spots for nondepleted CSF and microbead depleted CSF, whereas 1.5 times as many spots were detected for column depleted CSF. With a parameter setting that yielded > 95% spot detection, the numbers of spots detected were 441, 445, and 707 for nondepleted CSF, batch mode and column depleted CSF, respectively. A comparative analysis of the spots were also done using PDQuest software between the gel images of CSF with and without major protein depletion by creating a match set among the three gels. To create the match set, the reference gel was set to the column depleted CSF gel as it had the largest number of spots, and 28 land-marks (spots that are common to all gels) were set in all quadrants of the gels by manual inspection. 59% of spots in the 2D gel of HPLC column flow-through (Figure 2C) were unique (spot quantity ratio > 20/1) when compared to the 2D gel of nondepleted CSF, whereas in the 2D gel of microbead flow-through (Figure 2B), 51% of spots were unique. In other words, large percentages of unique spots were visualized in both gels; however, more unique spots were visualized in the gel of column depleted CSF as expected from the higher number of spots observed using column protein depletion. 45% of spots in the CSF gel were unique when compared to both batch mode and column treated gels. This reaffirms the fact that a large number of spots were removed from the nonde-

pleted CSF gel (Fiture 2A) by removing the abundant proteins, including fragments. 3.3. 2D Gels of CSF Stained for Glyco- and Phosphoproteins. Both glycosylation and phosphorylation of proteins are not only common in biological systems, but also play important roles in a protein’s function. Both modifications are involved in various cellular processes such as signal transduction,21 metabolism, receptor activity, reproduction, and growth.22-25 In CSF, a pronounced increase in the phosphorylation of protein has been specifically associated with Alzheimer’s disease, whereas an increase in tau protein alone can be observed in other neurological disorders.3 Therefore, it is of great importance to study the differentially expressed levels of post-translational modifications in biomarker discovery. Figure 3 shows the 2D gels of nondepleted CSF (left), batch mode microbead (center) and column (right) protein-depleted CSF after staining for glyco- (upper row) and phosphoproteins (lower row). In the 2D gel of microbead depleted CSF, it is apparent that antitrypsin is glycosylated, and that other glycoprotein spots are obscured by the large spots due to antitrypsin. For this reason also, it is more beneficial to use an immunoaffinity medium that can remove antitrypsin. Though glycoproteins were observed in all quadrants of the gels, phosphoproteins were mainly observed in the higher molecular weight range with acidic pI’s, as phosphate groups add negative charges to the proteins. A comparison of glycoproteins and phosphoproteins also revealed proteins that are both glycosylated and phosphorylated as in R-2-HS-glycoprotein, a protein know to change its function upon phosphorylation in fetal plasma.26 As the gel of column depleted CSF yielded the largest number of spots, the differentially stained gel images for this gel were colorized, and the overlaid image is shown in Figure 4, with red, green and blue colors indicating total, glyco- and phosphoproteins, respectively. As red and green colors generate
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Figure 3. 2D gels stained for glyco- and phosphoproteins. The 2D gels of nondepleted CSF (A and D), microbead (B and E) and HPLC column (C and F) depleted CSF were compared. Some prominent glycoproteins (A, B, and C) such as antitrypsin and prostaglandin D2 synthase are annotated. A comparison of glycolproteins and phosphoproteins (D, E, and F) revealed proteins that are both glycosylated and phosphorylated as in R-2-HS-glycoprotein.

Figure 4. Overlaid image of total (red), glyco- (green), and phosphoproteins (blue) for the 2D gel of HPLC column depleted CSF. Mixing of green and red colors generate yellow, which also indicates moderate level of glycosylation. CandyCane protein standards placed at the acidic end of the gel contained the following glycoproteins: glycoproteins: R2-macroglobulin (MW ) 180 000 Da), Glucose oxidase (MW ) 82 000), R1-acid glycoprotein (MW ) 42 000 Da). PeppermintStick protein standards placed at the basic end of the gel contained the following phosphoproteins: Ovalubumin (MW ) 45 000), β-casein (23 600).

yellow when mixed, the yellow spots in the Figure 4 also indicate moderate levels of glycosylation. Most notable posttranslational modification is the glycosylation of prostaglandin D2 synthase (PDGS) noted in Figure 3. PDGS originates exclusively from the brain; however, the biological function of this protein remains unknown.12 It has been reported that the concentration of PGDS does not vary significantly in different pathological conditions including dementia, hydrocephalus, neuropathy optic neuritis, multiple sclerosis and demyelinating syndrome, but it was reduced by 2-fold in the CSF from brain tumor patients.7,27 Current diagnostic relevance of PDGS comes
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from its use as a marker to detect CSF in nasal secretion from patients with rhinorrhea.28 Since PDGS is a brain derived protein, studies that assess the levels of post-translational modification should prove useful in providing more insight to PGDS functions in CSF. 3.4. Mass Spectrometric Identification of Protein Spots. A total of 66 protein spots from the gel of the Agilent column flow-through were used for identification by mass spectrometry after trypsin digestion. The locations of the spots are indicated in Figure 5 and numbered as follows: glycoprotein spots (g1g10), phosphoprotein spots (p1-p14), and selected number of spots from all quadrants of the gel, including spots that were not visualized in the CSF gel without protein depletion (142). In addition, 2 spots were excised from the undepleted CSF gel that matched the column depleted gel spots (c1-c2) for comparison though they were not well visualized in the undepleted CSF gel. From the above protein spots, 50 proteins were identified using Mascot search algorithms,19 and listed in Table 1, including their theoretical molecular weights of the protein (Mr), isoelectric points (pI), accession numbers, % peptide coverage used for protein identification (% coverage), and probably based MOWSE scores20 (protein score). With the parameters used for database search that were described in the Experimental Section, probability based MOWSE scores20 >44 indicated identity or extensive homology with an error rate of 5% or less. In this study, the proteins that were identified all had protein scores >90, and multiple peptides were used for identification with at least one of the peptide’s ion scores >44. Many of the proteins identified confirmed the previously published results; however, among the proteins identified, 12 proteins have not been annotated in previously published 2D gels (Table 1). One of the proteins identified, procollagen C-proteinase enhancer protein, was also identified from the location of the nondepleted CSF gel that matched to the locations in the Agilent flow-through gel (Figure 5). The protein spots for this protein in nondepleted CSF gel were not well visualized and would not have been excised for identification without abundant protein depletion to help locate the spots.

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Table 1. Summary of Proteins Identified from Spots Excised from the 2D Gel of Abundant Protein Depleted CSF Using a HPLC Column
spot no. accession protein ID Mr pI protein score % coverage

p1

p2

p3 p4

p5 p6 p7

p8 p9 p10 g1 g2 g3 g4 g5 g6

g7 g8 g9

g10 g11 g12 g13 g14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Q14515 P05155 P01024 P13591 Q14515 P05155 P13591 P05155 P00450 P04004 P51693 P04004 P04196 P01011 P51693 P08253 P01042 P01042 Q96KN2 P01019 Q12805 P01042 P01019 P01019 Q96KN2 P01042 P02765 P00751 P43652 P04217 P07225 P02790 P02790 P04217 P02748 P08697 P01028 P02769 P01019 P08697 P02748 P01011 P10909 Q96KN2 P01019 P01008 P01011 P01008 P01019 P01019 P01008 P02774 P01019 P02763 P19652 P41222 Q12860 P02679 P02774 P01028 P27169 P06396 P02570 P06727 P02774 P07711 P10909 P05156 P10909 P01024 P06396 P01019 P01024 P36955 P36955 P02675 P02675 P41222 P06733 P01024 P01023 P01024

SPARC-like protein 1 precursor Plasma protease C1 inhibitor precursora Complement C3 precursor Neural cell adhesion molecule 1b SPARC-like protein 1 precursor Complement C3 precursor Neural cell adhesion molecule 1b Plasma protease C1 inhibitor precursora Ceruloplasmin precursor Vitronectin precursor Amyloid-like protein 1 precursorb Vitronectin precursor Histidine-rich glycoprotein precursorb Alpha-1-antichymotrypsin precursor Amyloid-like protein 1 precursor b 72 kDa type IV collagenase precursora Kininogen precursor Kininogen precursor Glutamate carboxypeptidase-like protein 2 precursorb Angiotensinogen precursor EGF-containing fibulin-like extracellular matrix protein 1 precursor Kininogen precursor Angiotensinogen precursor Angiotensinogen precursor Glutamate carboxypeptidase-like protein 2 precursorb Kininogen precursor Alpha-2-HS-glycoprotein precursor Complement factor B precursor Afamin precursor Alpha-1B-glycoprotein precursor Vitamin K-dependent protein S precursora Hemopexin precursor Hemopexin precursor Alpha-1B-glycoprotein precursor Complement component C9 precursora Alpha-2-antiplasmin precursor Complement C4 precursor Serum albumin precursor Angiotensinogen precursor Alpha-2-antiplasmin precursor Complement component C9 precursora Alpha-1-antichymotrypsin precursor Clusterin precursor Glutamate carboxypeptidase-like protein 2 precursorb Angiotensinogen precursor Antithrombin-III precursor Alpha-1-antichymotrypsin precursor Antithrombin-III precursor Angiotensinogen precursor Angiotensinogen precursor Antithrombin-III precursor Vitamin D-binding protein precursor Angiotensinogen precursor Alpha-1-acid glycoprotein 1 precursor Alpha-1-acid glycoprotein 2 precursor Prostaglandin-H2 D-isomerase precursor Contactin precursor Fibrinogen gamma chain precursor Vitamin D-binding protein precursor Complement C4 precursor Serum paraoxonase/arylesterase 1a Gelsolin precursor Actin, cytoplasmic 1 Apolipoprotein A-IV precursor Vitamin D-binding protein precursor Cathepsin L precursora Clusterin precursor Complement factor I precursor Clusterin precursor Complement C3 precursor Gelsolin precursor Angiotensinogen precursor Complement C3 precursor Pigment epithelium-derived factor precursor Pigment epithelium-derived factor precursor Fibrinogen beta chain precursor Fibrinogen beta chain precursor Prostaglandin-H2 D-isomerase precursor Alpha enolase Complement C3 precursor Alpha-2-macroglobulin precursor Complement C3 precursor

75169 55119 187046 93303 75169 187046 93303 55119 122128 54271 72131 54271 59541 47621 72131 73825 71900 71900 56743 53121 54604 71900 53121 53121 56743 71900 3930 85479 69024 54176 75074 51643 51643 54176 63133 54531 192650 69248 53121 54531 63133 47621 52461 56743 53121 52569 47621 52569 53121 53121 52569 52929 53121 23497 23588 21015 113249 51479 52929 192650 39593 85644 41710 45343 52929 37540 52461 65677 52461 187046 85644 53121 187046 46313 46313 55892 55892 21015 47008 187046 163175 187046

4.68 6.09 6.02 4.78 4.68 6.02 4.78 6.09 5.44 5.55 5.54 5.55 7.09 5.33 5.54 5.26 6.34 6.34 5.19 5.87 4.95 6.34 5.87 5.87 5.19 6.34 5.43 6.67 5.64 5.45 5.48 6.55 6.55 5.45 5.43 5.87 6.65 5.82 5.87 5.87 5.43 5.33 5.89 5.19 5.87 6.32 5.33 6.32 5.87 5.87 6.32 5.4 5.87 4.93 5.03 7.66 5.62 5.37 5.4 6.65 5.08 5.9 5.29 5.28 5.4 5.31 5.89 7.72 5.89 6.02 5.9 5.87 6.02 5.97 5.97 8.54 8.54 7.66 6.99 6.02 6 6.02

930 617 378 184 971 651 376 269 213 213 126 250 233 180 159 120 236 402 319 282 213 143 127 570 427 277 433 1025 629 232 134 257 250 393 822 379 218 179 103 443 145 980 105 1093 749 242 136 1490 563 744 264 141 742 136 115 194 175 302 140 317 116 224 486 207 311 112 321 167 275 151 433 494 138 807 533 146 146 149 517 509 162 93

32 9 9 6 34 13 10 12 7 10 5 12 11 11 9 4 9 11 29 13 13 8 5 21 29 12 23 26 25 12 7 20 19 18 22 26 3 11 5 29 6 51 6 47 31 21 8 55 24 26 23 5 32 17 11 21 6 20 14 3 9 15 33 20 24 10 15 5 14 2 22 19 1 32 24 10 13 17 29 10 2 10

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research articles
Table 1. (Continued)
spot no. accession protein ID Mr pI protein score

Ogata et al.

% coverage

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 c1 c2

Q15782 P07358 P00751 P00751 P06396 P02675 Q15113 Q15113 P36955 P36955 P17174 P36222 P40295 Q92876 Q92876 P02647 P02647 P02647 P81605 P81605 P81605 Q15113 Q15113

Chitinase 3-like protein 2 precursor Complement component C8 beta chain precursorb Complement factor B precursor Complement factor B precursor Gelsolin precursor Fibrinogen beta chain precursor Procollagen C-proteinase enhancer protein precursor Procollagen C-proteinase enhancer protein precursor Pigment epithelium-derived factor precursor Pigment epithelium-derived factor precursor Aspartate aminotransferase Chitinase-3 like protein 1 precursor Malate dehydrogenase Kallikrein 6 precursor Kallikrein 6 precursor Apolipoprotein A-I precursor Apolipoprotein A-I precursor Apolipoprotein A-I precursor Dermcidin precursorb Dermcidin precursorb Dermcidin precursorb Procollagen C-proteinase enhancer protein precursor Procollagen C-proteinase enhance protein precursor

43473 67003 85479 85479 85644 55892 47942 47942 46313 46313 46087 42586 36272 26838 26838 30759 30759 30759 11277 11277 11277 47942 47942

7.11 8.5 6.67 6.67 5.9 8.54 7.41 7.41 5.97 5.97 6.57 8.69 6.89 7.15 7.15 5.56 5.56 5.56 6.08 6.08 6.08 7.41 7.41

186 143 316 162 123 413 92 233 408 328 561 268 495 119 91 491 1230 587 94 157 196 322 198

8 4 12 4 6 25 13 13 17 11 20 21 27 9 13 44 65 45 24 37 44 15 14

a Proteins that have not been annotated before. b Proteins that have been identified only with 2D-chromatography-MS method. Note: p1-p10: phosphoprotein spots, g1-g14: glycoprotein spots, c1-c2: undepleted CSF gel spots that matched to Agilent gel spot 29 & 30.

Figure 5. Spots used for protein identification from the 2D gel of HPLC column depleted CSF. The spots were numbered as follows: glycoprotein spots (g1-g10), phosphoprotein spots (p1-p14), selected number of spots from all quadrants of the gel, including spots that were not visualized in the CSF gel without protein depletion (1-42), and spots identified from the nondepleted CSF gel for comparison (c1-c2). The spot numbers also correspond to the protein identifications listed in Table 1. A section (IgG heavy chain region) of this gel was also compared to the same section from the 2D gel of nondepleted CSF, showing dramatically increased number of spots visualized in the HPLC column depleted CSF gel. 844 Journal of Proteome Research • Vol. 4, No. 3, 2005

Protein Depletion Methods for CSF

research articles
Major protein depletion followed by differential staining for post-translational modifications and mass spectrometric protein identification is effective in obtaining significantly more information from the traditional 2D SDS-PAGE analysis of CSF. This method allows identification and quantitative analysis of brain derived proteins that are present at low concentrations and their levels of post-translational modifications. The general experimental scheme described here will be used for our future efforts in biomarker and therapeutic target discoveries for neurological disorders.

The number of protein spots excised was limited as the focus of this study was to elucidate the utility of major protein depletion in 2D PAGE analysis of low-abundant proteins in CSF. However, the large percentages of new proteins identified (24%) from the limited number of excised spots clearly indicate the utility of major protein depletion in conjunction with mass spectrometric analysis. There were 6 proteins that have not been annotated in previously published 2D gels but have recently been annotated using 2D-chromatography-mass spectrometry or “shotgun” proteomics approach after Agilent column protein depletion13 or protein fractionation using organic solvents.15 They were: complement component C8 β chain precursor, dermcidin precursor, glutamate carboxypeptidase-like protein 2 precursor, amyloid-like protein 1 precursor, histidine-rich glycoprotein precursor, neural cell adhesion molecule 1. Both 2D gel and 2D chromatography are useful in global and differential proteomic studies, and it is important that both techniques compliment each other. Our protein identification results did exactly so, by validating the results from the shotgun proteomic approach and by providing additional new protein identifications.

Acknowledgment. We thank Dr. Mark B. Keegan for providing CSF samples, and Professor Dominic M. Desiderio for his technical guidance in 2D PAGE. Thanks are also due to Mr. Benjamin Madden and Ms. Linda Benson for assisting with protein identification. The authors are grateful for the financial support of the Ruan Family Charitable Trust and the Mayo Clinic College of Medicine. References
(1) Milhorat, T. H. In Neurobiology of Cerebrospinal Fluid; Wood, J. H., Ed.; Plenum: New York, 1983; Vol. 2. (2) Andreasen, N.; Minthon, L.; Davidsson, P.; Vanmechelen, E.; Vanderstichele, H.; Winblad, B.; Blennow, K. Arch. Neurol. 2001, 58, 373-379. (3) Hu, Y. Y.; He, S. S.; Wang, X.; Duan, Q. H.; Grundke-Iqbal, I.; Iqbal, K.; Wang, J. Am. J. Pathol. 2002, 160, 1269-1278. (4) Hu, Y. Y.; He, S. S.; Wang, X. C.; Duan, Q. H.; Khatoon, S.; Iqbal, K.; Grundke-Iqbal, I.; Wang, J. Z. Neurosci. Lett. 2002, 320, 156160. (5) Yuan, X.; Desiderio, D. M. J. Proteome Res. 2003, 2, 476-487. (6) Sickmann, A.; Dormeyer, W.; Wortelkamp, S.; Woitalla, D.; Kuhn, W.; Meyer, H. E. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2002, 771, 167-196. (7) Terry, D. E.; Desiderio, D. M. Proteomics 2003, 3, 1962-1979. (8) Hammack, B. N.; Fung, K. Y.; Hunsucker, S. W.; Duncan, M. W.; Burgoon, M. P.; Owens, G. P.; Gilden, D. H. Mult. Scler. 2004, 10, 245-260. (9) Raymackers, J.; Daniels, A.; De Brabandere, V.; Missiaen, C.; Dauwe, M.; Verhaert, P.; Vanmechelen, E.; Meheus, L. Electrophoresis 2000, 21, 2266-2283. (10) Puchades, M.; Hansson, S. F.; Nilsson, C. L.; Andreasen, N.; Blennow, K.; Davidsson, P. Mol. Brain Res. 2003, 118, 140-146. (11) Yuan, X.; Desiderio, D. M. Proteomics 2005, 5, 541-550. (12) Reiber, H. Clin. Chim. Acta 2001, 310, 173-186. (13) Maccarrone, G.; Milfay, D.; Birg, I.; Rosenhagen, M.; Holsboer, F.; Grimm, R.; Bailey, J.; Zolotarjova, N.; Turck, C. W. Electrophoresis 2004, 25, 2402-2412. (14) Gianazza, E.; Arnaud, P. Biochem. J. 1982, 201, 129-136. (15) Zhang, J.; Goodlett, D. R.; Peskind, E. R.; Quinn, J. F.; Zhou, Y.; Wang, Q.; Pan, C.; Yi, E.; Eng, J.; Aebersold, R. H.; Montine, T. J. Neurobiol. Aging 2005, 26, 207-227. (16) GenWay Biotech: http://www.genwaybio.com/. (17) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., 3rd Nat. Biotechnol. 1999, 17, 676-682. (18) Blum, H.; Beier, H.; Gross, H. J. Electrophoresis 1987, 8, 93-99. (19) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-3567. (20) Pappin, D. J.; Hojrup, P.; Bleasby, A. J. Curr. Biol. 1993, 3, 327332. (21) Wells, L.; Vosseller, K.; Hart, G. W. Science 2001, 291, 2376-2378. (22) Faux, M. C.; Scott, J. D. Trends Biochem. Sci. 1996, 21, 312-315. (23) Krebs, E. G. Trends Biochem. Sci. 1994, 19, 439. (24) Posada, J.; Cooper, J. A. Mol. Biol. Cell 1992, 3, 583-592. (25) Hunter, T. Cell 1995, 80, 225-236. (26) Jahnen-Dechent, W.; Trindl, A.; Godovac-Zimmermann, J.; MullerEsterl, W. Eur. J. Biochem. 1994, 226, 59-69. (27) Saso, L.; Leone, M. G.; Sorrentino, C.; Giacomelli, S.; Silvestrini, B.; Grima, J.; Li, J. C.; Samy, E.; Mruk, D.; Cheng, C. Y. Biochem. Mol. Biol. Int. 1998, 46, 643-656. (28) Arrer, E.; Meco, C.; Oberascher, G.; Piotrowski, W.; Albegger, K.; Patsch, W. Clin. Chem. 2002, 48, 939-941. (29) Anderson, N. L.; Anderson, N. G. Electrophoresis 1998, 19, 1853-1861.

4. Conclusions
In CSF, large percentages (∼80%) of proteins originate from serum. In particular, albumin constitutes 35-80% of total protein in CSF. This leads to the extremely large dynamic range of protein concentrations (up to twelve orders of magnitude).29 To reduce this dynamic range to better match the analytical platform, immunoaffinity was used to deplete the major proteins present in CSF. Two protein depletion methods were evaluated: GenWay avian IgY antibody microbeads (Seppro) in spin cups and Agilent multiple affinity removal HPLC column. Both GenWay and Agilent immunoaffinity matrixes effectively removed major proteins from CSF. Between the two systems, Agilent column yielded a larger number of protein spots visualized using Sypro Ruby gel stain. The reasons are severalfold. Agilent column removed antitrypsin, which occupied the areas of the gels where a large number of spots from other proteins were present. Haptoglobin was also notably depleted by this method. In addition, the albumin depletion was not complete with GenWay beads. This incomplete depletion may be due to the different column formats (Spin column for GenWay beads and HPLC column for Agilent beads), and the fact that the beads used for this study had different capacities. GenWay also offers the prepacked columns for the Mixed 6 beads, and has recently made available Seppro Mixed 12 HPLC columns, which can remove 12 major serum proteins. Use of this column may resolve the above problems, in addition to ability to remove 6 more abundant proteins from CSF. Staining of glyco- and phosphoproteins using ProQ Emerald and Diamond stains, respectively, combined with major protein depletion provided new insights into the post-translational modification of CSF proteins. As the above-mentioned stains offer broad dynamic ranges, these staining methods will be useful in determining the differentially expressed levels of posttranslational modifications in CSF. Protein depletion and visualization of larger number of spots also lead to identification of proteins that have not been annotated in previously published 2D gels. 12 new proteins were identified including 6 proteins that have been annotated only with 2D-chromatography-mass spectrometry method after immunoaffinity protein depletion,13 or fractionation using organic solvents.15

PR049750O
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