Characterization of Tau in Cerebrospinal Fluid Using Mass Spectrometry

CNS/Pain, Department of Disease Biology, Södertälje, SE-151 85, Sweden, and Max-Planck-Institute for. Molecular Genetics, D-14195, Berlin, Germany...
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Characterization of Tau in Cerebrospinal Fluid Using Mass Spectrometry Erik Portelius,*,†,⊥ Sara F. Hansson,†,⊥ Ai Jun Tran,† Henrik Zetterberg,† Pierre Grognet,‡ Eugeen Vanmechelen,‡ Kina Höglund,§ Gunnar Brinkmalm,† Ann Westman-Brinkmalm,† Eckhard Nordhoff,| Kaj Blennow,† and Johan Gobom† Clinical Neurochemistry Laboratory, Department of Neuroscience and Physiology, University of Göteborg, Sahlgrenska University Hospital, S-431 80 Mölndal, Sweden, Innogenetics, Ghent, Belgium, AstraZeneca CNS/Pain, Department of Disease Biology, Södertälje, SE-151 85, Sweden, and Max-Planck-Institute for Molecular Genetics, D-14195, Berlin, Germany Received December 20, 2007

The neurodegenerative disorder Alzheimer’s disease (AD) is the most common cause of dementia in the elderly. The presence of neurofibrillary tangles, consisting of hyperphosphorylated tau protein, is one of the major neuropathologic characteristics of the disease, making this protein an attractive biomarker for AD and a possible target for therapy. Here, we describe an optimized immunoprecipitation mass spectrometry method that enables, for the first time, detailed characterization of tau in human cerebrospinal fluid. The identities of putative tau fragments were confirmed using nanoflow liquid chromatography and tandem mass spectrometry. Nineteen tryptic fragments of tau were detected, of which 16 are found in all tau isoforms while 3 represented unique tau isoforms. These results pave the way for clinical CSF studies on the tauopathies. Keywords: Alzheimer’s disease • immunoprecipitation • MALDI-TOF MS • cerebrospinal fluid • Tau • LC-MS/MS

Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that causes dementia in approximately 10% of individuals over the age of 65.1 One of its typical brain lesions is neurofibrillary tangles that consist of a hyperphosphorylated form of the microtubule-stabilizing protein tau.2–5 Hyperphosphorylation of tau causes the protein to detach from the microtubules, destabilizing the axons. This process promotes axonal and synaptic plasticity in the developing brain but is pathological in the adult brain, and it is specifically related to a group of disorders referred to as tauopathies, which includes AD and some forms of frontotemporal dementia (FTD).6 Molecular characterization of cerebrospinal fluid (CSF) tau presents an analytical challenge for several reasons. One is the high heterogeneity of the protein: in the adult human brain there are six different tau isoforms produced from a single gene by alternative mRNA splicing (Figure 1). This heterogeneity is compounded by extensive post-translational modifications, including phosphorylation, glycosylation, and oxidation of the protein.7 Of a potential 80 serine and threonine phosphorylation sites in the longest isoform, 39 different sites have been verified.8 Mounting evidence indicates that these play an important role in regulating the propensity of the protein to * Corresponding author. E-mail address: [email protected]. † University of Göteborg. ‡ Innogenetics. § AstraZeneca CNS/Pain. | Max-Planck-Institute for Molecular Genetics. ⊥ These authors contributed equally to this work.

2114 The Journal of Proteome Research 2008, 7, 2114–2120 Published on Web 03/20/2008

Figure 1. Six different tau isoforms produced from a single gene by alternative mRNA splicing are presented. The blue, green, and yellow boxes correspond to exon 2, 3, and 10, respectively.

aggregate. However, it is still not known whether the hyperphosphorylation of tau (triggered by an increased rate of phosphorylation and/or decreased rate of dephosphorylation) and tangle formation are a cause or a consequence of AD. Another challenge is the low concentration of tau in CSF, ranging from approximately 300 ng/L in healthy individuals to 900 ng/L in AD patients.9,10 Thus, a typical CSF sample volume of 3 mL contains less than 3 ng of tau, or approximately 65 fmol (based on the isoform 4R/2N). Considering that this quantity is distributed over many different modified forms and six splice variants, the amount available for analysis of each 10.1021/pr7008669 CCC: $40.75

 2008 American Chemical Society

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Characterization of Tau in Cerebrospinal Fluid Using MS

Figure 2. MALDI MS analysis of protease digested tau. The immunoprecipitation was conducted using the antibody BT2. The antibodies were immobilized (a) to the magnetic beads, and three different peaks corresponding to tau were detected. When BT2 was not cross-linked before the precipitation (b) the signal decreased for the three peaks. The m/z values in the mass spectra correspond to the protonated monoisotopic molecular masses (MH+).

Figure 3. CSF was subjected to perchloric acid extraction, and the antibodies were cross-linked before the immunoprecipitation. Four different antibodies were tested: (a) BT2, (b) HT7, (c) AT120, and (d) AT270. Using BT2 and HT7, three peaks corresponding to tau were detected. The peaks labeled with * in (d) were not to be identified. The m/z values in the mass spectra correspond to the protonated monoisotopic molecular masses (MH+).

molecular species falls close to the detection limit of state-ofthe-art mass spectrometric instrumentation. An enzyme-linked immunosorbent assay (ELISA) has been used extensively to determine the CSF tau concentration in clinical samples. Initial studies utilized antibodies insensitive to the modification status of the protein, thereby measuring the total tau (T-tau) concentration. Of approximately 50 studies conducted on AD patients and controls, almost all have shown an increase in T-tau in AD patients by approximately 300% with a sensitivity and specificity of 80–90%.9 However, when comparing the CSF T-tau concentration of AD patients with that

Figure 4. MALDI TOF mass spectrum showing peaks detected when the CSF was not precipitated (a) or precipitated using perchloric acid (b). The peaks detected corresponding to fragments from cleaved tau are indicated (m/z 1 ) 1393.66, 2 ) 1393.73, 3 ) 1411.76, 4 ) 1420. 78). The m/z values in the mass spectra correspond to the protonated monoisotopic molecular masses (MH+).

of other degenerative dementias, such as FTD or vascular dementia (VaD), the specificity drops to approximately 50–60%, rendering T-tau of little use as a diagnostic marker for distinguishing AD from other neurodegenerative diseases.9 In fact, T-tau seems to be a general marker of axonal damage, a view substantiated by results from studies of stroke, brain trauma, and Creutzfeldt-Jakob disease.11–13 However, by using antibodies recognizing specific phosphorylated motifs in the tau amino acid sequence, phosphorylated tau (P-tau) isoforms were found that appeared to be more specific to AD. For example, P-tau231P and P-tau181P can be used to distinguish AD from control groups and even from FTD and VaD.14–16 Further, we have shown that a combination of T-tau, Aβ42, and P-tau181P can be used to detect incipient AD in patients with mild cognitive impairment with positive and negative predictive values of >80%.17,18 These results imply that a detailed characterization of CSF tau and its modifications might provide even better diagnostic tools as well as insight into the pathogenic mechanisms of AD. In this study, we report on a method for immunoprecipitation (IP) of tau in CSF and subsequent protein identification. The IP method was optimized with regard to several experimental parameters including selection of antibody, antibody immobilization, and CSF pretreatment. ELISA was used to monitor tau recovery in each step. Liquid chromatography (LC) was combined with either matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) or electrospray (ESI) hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometry (QIT-FTICR MS) for analysis of IP samples allowing, for the first time, mass spectrometric identification of tau in CSF. Fragment ion spectra acquired from selected peptides allowed identification of peptides specific to certain tau isoforms.

Experimental Section Cerebrospinal Fluid. The investigation was performed on deidentified CSF samples supplied by the Clinical NeurochemThe Journal of Proteome Research • Vol. 7, No. 5, 2008 2115

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Portelius et al.

Figure 5. LC-MALDI MS/MS analysis of tryptic peptides of tau isolated from CSF by IP. The spectra are annotated with the detected band y-ions and partial sequences of the identified peptides.

istry Laboratory, Sahlgrenska University Hospital. The samples were handled as previously described.19 Sample Preparation. Immunoprecipitation of CSF, crosslinking of antibodies, and the use of the KingFisher magnetic particle processor (polypropylene tubes, Thermo Fisher Scientific, Waltham, MA) was conducted as described earlier19 with some optimized modifications. Briefly, an aliquot (8 µg) of the antibody BT2 (epitope R194-S198), HT7 (epitope P159K163), AT120 (epitope P218-K224), or AT270 (phosphodependent epitope P176-P182)16 was cross-linked using 20 mM dimethyl pimelimidate dihydrochloride (DMP, Pierce Biotechnology, USA) in 0.2 M triethanolamine (pH 8.2, Sigma-Aldrich, St. Louis, MO) to 50 µL Dynabeads M-280 (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s product description with the exception that the beads were washed three times using 1 mL of RotiBlock (Roth, Numberg, Germany) instead of bovine serum albumin and finally once with phosphate-buffered saline (PBS, pH 7.4). CSF was precipitated, except where noted, for 2 h on ice using 2.5% perchloric acid, 2116

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final concentration (Fluka, Buchs, Switzerland), and centrifuged for 10 min (31 180g, +4 °C). The supernatant was neutralized to pH 7.4 using 9 M potassium hydroxide (Eka, Bohus, Sweden). IP was performed on the pretreated CSF using beads with crosslinked antibodies, except where noted. The magnetic beads/CSF solution was transferred to the magnetic particle processor, tube 1. The following four wash steps (tubes 2–5) were conducted for 10 s in 1 mL of each washing buffer: (tube 2) 0.025% Tween-20 (Bio-Rad Laboratories, Inc., Richmond, CA) and 10 mM n-octylglucopyranoside (nOGP) (Roche Molecular Biochemicals, Mannheim, Germany) in PBS, (tube 3) PBS and finally two washes (tubes 4 and 5) with 50 mM ammonium hydrogen carbonate (NH4HCO3, pH 8.0, Riedel-deHaën). The washed beads were transferred to a fresh sample cup (Costar, Microcentrifuge Tube). The supernatant was discarded, and tau was eluted from the beads by adding 25 µL of 0.1% formic acid (FA, Riedel-deHaën) in 2 mM nOGP for 2.5 min. This elution was repeated once, and the collected supernatant (50 µL) was dried in a vacuum centrifuge.

88–126 48%

a The reported molecular mass values are the calculated monoisotopic masses of the matched tryptic peptides. For peptides whose identity was confirmed by MS/MS, the corresponding Mascot ion score is given. The position of the matched peptides in the amino acid sequence is given for each of the six known tau isoforms. b ND ) not detected. c D ) Peptide only assigned by molecular mass.

4R/2N 4R/1N

152–161 202–211 183–192 214–225 355–366 166–180 152–165 183–195 127–141 181–195 98–114 102–121 6–23 6–24 98–119 319–340 68–97 46%

4R/0N

123–132 173–182 154–163 185–196 326–337 137–151 123–136 154–166 98–112 152–166 69–85 73–92 6–23 6–24 69–90 290–311 45–68 48%

3R/2N

181–190 231–240 212–221 243–254 353–364 195–209 181–194 212–224 156–170 210–224 127–143 131–150 6–23 6–24 127–148 317–338

3R/1N

31 Dc 40 56 D 83 38 36 D 19 ND ND 35 ND ND D D 134 ND NDb ND D ND ND 54 27 26 ND D D D 62 33 D ND D 85 D

152–161 183–192 183–192 214–225 324–335 166–180 152–165 183–195 127–141 181–195 98–114 102–121 6–23 6–24 98–119 288–309 68–97 49%

3R/0N

123–132 173–182 154–163 185–196 295–306 137–151 123–136 154–166 98–112 152–166 69–85 73–92 6–23 6–24 69–90 259–280 45–68 52%

ESI ion score MALDI ion score aa sequence Mr

995.49 TPPSSGEPPK 998.54 TPPKSPSSAK 1065.58 TPSLPTPPTR 1308.71 LQTAPVPMPDLK 1330.69 AKTDHGAEIVYK 1392.63 SGYSSPGSPGTPGSR 1410.67 TPPSSGEPPKSGDR 1419.77 TPSLPTPPTREPK 1422.73 GAAPPGQKGQANATR 1662.91 SRTPSLPTPPTREPK 1780.90 MVSKSKDGTGSDDKKAK 1993.01 SKDGTGSDDKKAKGADGKTK 2052.88 QEFEVMEDHAGTYGLGDR 2180.98 QEFEVMEDHAGTYGLGDRK 2209.10 MVSKSKDGTGSDDKKAKGADGK 2291.20 DRVQSKIGSLDNITHVPGGGNK 2423.12 AEEAGIGDTPSLEDEAAGHVTQAR 3009.38 STPTAEAEEAGIGDTPSLEDEAAGHVTQAR 3954.85 QAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQAR sequence coverage:

Table 1. LC-MALDI MS and LTQ FT-ICR MS Analysis of the CSF-Tau Immunoprecipitate Digested with Trypsina

181–190 231–240 212–221 243–254 384–395 195–209 181–194 212–224 156–170 210–224 127–143 131–150 6–23 6–24 127–148 348–369 88–126 45%

Characterization of Tau in Cerebrospinal Fluid Using MS

research articles The dried sample was redissolved in 10 µL of 50 mM NH4HCO3 followed by the addition of 2 µL of dithiothreitol (25 mM, Sigma-Aldrich, St. Louis, MO) in 50 mM NH4HCO3 and incubated at +37 °C for 30 min. To the reduced sample, 2 µL of 60 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO) in 50 mM NH4HCO3 was added and incubated for 30 min at room temperature. Porcine trypsin (14 ng/µL in 50 mM NH4HCO3) (Promega, Mannheim, Germany) and 34 µL of deionized water were added to the sample for digestion overnight at +37 °C before subsequent MS analysis. MALDI-TOF MS. The peptide calibration standards, Bradykinin (1–7) (MH+ 757.40, Sigma, St. Louis, MO, USA), Angiotensin II (MH+ 1046.54, Sigma, St. Louis, MO, USA), Somatostatin 14 (MH+ 1637.72, Cambridge Research Biochemicals, England), ACTH (18–39) (MH+ 2465.20, Neosystem, Strasbourg, France), and Somatostatin 28 (MH+ 3147.47, Larodan Fine Chemicals AB, Malmö, Sweden) were used for external calibration. Trifluoroacetic acid (TFA, Pierce, Rockford, USA), R-cyano4-hydroxycinnamic acid (CHCA, Fluka Chemie, Buchs, Switzerland), and acetone (Labscan, Dublin, Ireland) were used for the matrix solution: 100 g/L of CHCA in 90% acetone, 0.005% TFA. Thin layers of CHCA were prepared on a MALDI sample support (AnchorChip 400/384, Bruker Daltonics, Bremen) by spreading 30 µL of matrix solution over the target surface with a Teflon rod. The digested tau samples were diluted (1:3) in 0.1% FA, and 1 µL of the sample was spotted on the AnchorChip. The droplet was aspirated after 2 min, and the spot was finally washed with 1 µL of 0.1% TFA for 15 s. Mass spectrometric analysis was performed on a Bruker AUTOFLEX MALDI-TOF (Bruker Daltonics, Bremen, Germany). Positively charged ions in the m/z range 630–3480 Da were analyzed automatically in the reflector mode. Sums of 50 singleshot spectra were acquired from 30 different sample spot positions (1500 in total from each sample). Fixed laser attenuation was used, the optimal value of which was determined prior to analysis by evaluation of a few spots. LC-MALDI TOF/TOF MS. The peptide calibration standards, angiotensin I and ACTH 18–39, were purchased from Bachem (Heidelberg, Germany). Acetonitrile (ACN, HPLC Gradient grade) was purchased from Carl Roth GmbH (Karlsruhe, Germany). TFA, tetrahydrofuran (THF), nOGP, and water used for HPLC solvents and MALDI matrix solutions were purchased from Fluka Chemie (Buchs, Switzerland). Citric acid was purchased from Aldrich (Sigma-Aldrich, St. Louis, MO). LC-MALDI MS was performed as recently described.20 Peptide samples were analyzed on an 1100 Series Nanoflow LC system (Agilent Technologies, Waldbronn, Germany). The mobile phases used for the reversed-phase separation were Buffer A (1% ACN (v/v), 0.05% TFA (v/v)) and buffer B (90% ACN (v/v), 0.04% TFA (v/v)). The digested tau samples, acidified with 0.2% TFA (v/v), were first loaded onto a trapping column (ZORBAX 300 SB C18, 0.3 mm × 5 mm, Agilent Technologies), using buffer A, delivered by the loading pump with a flow gradient according to the manufacturer’s recommendation. After 5 min, the trapping column was connected to the nanoflow path, and the analyte molecules were transferred to the analytical separation column (ZORBAX 300 SB C18, 75 µm × 150 mm, Agilent Technologies), using a binary pump operated at 300 nL/min. The binary gradient was (t(min)/buffer B (%)/flow (nL/min)): 0/3/300; 5/3/300; 8/15/300; 18/30/300; 28/45/330; 33/95/350; 38/95/400; 40/3/350; 42/3/300. The LC effluent was fractionated onto preformed microcrystalline The Journal of Proteome Research • Vol. 7, No. 5, 2008 2117

research articles layers of CHCA prepared on prestructured MALDI sample supports (AnchorChip 600/384, Bruker Daltonics, Bremen). The matrix solution was CHCA (100 g/L) in 90% tetrahydrofuran, 0.001% TFA (v/v), and 50 mM citric acid, containing the two calibration standards angiotensin I (1 pmol/µL) and ACTH 18–39 (2 pmol/µL). Thin layers of CHCA were prepared by spreading 200 µL of matrix solution over the target surface with a Teflon rod.21 Ninety-four fractions were collected over a period of 40 min. Mass spectrometric analysis was performed on an Ultraflex II LIFT MALDI-TOF/TOF (Bruker Daltonics, Bremen, Germany), equipped with a SmartBeam solid-state laser. Positively charged ions in the mass-to-charge-ratio (m/z) range 500–4500 Da were analyzed automatically in the reflector mode. Sums of 50 single-shot spectra were acquired from 14 different sample spot positions (700 in total from each sample). Fixed laser attenuation was used, the optimal value of which was determined prior to analysis by evaluation of a few fractions. Calculation of peptide profiles and selection of precursor ions for MS/MS analysis were assisted by the WarpLC software (Bruker). MS/MS spectra were acquired automatically with fixed laser attenuation and a fixed laser power boost. Automatic detection of the peptide monoisotopic signals was performed using the algorithm SNAP, implemented in the FlexAnalysis software (Bruker Daltonics, Bremen, Germany). Internal mass correction was performed using the signals of two reference peptides (Angiotensin I, MH+ 1296.6853 (monoisotopic mass), and ACTH (18–39), MH+ 2465.1989) included in the MALDI matrix solution. Protein identification was performed using the Mascot software (Matrixscience, London, UK), searching the UniProt/Swiss-Prot and UniProt/Trembl sequence databases. The following settings were used for the searches: mass error tolerance for the precursor ions, 30 ppm; mass error tolerance for the fragment ions, 0.3 Da; fixed modification, carbamidomethylation; variable modification, methionine oxidation and phosphorylation; number of missed cleavage sites, 1; type of instrument, MALDI-TOF-TOF. LC-ESI QIT-FTICR MS. Nanoflow LC-ESI mass spectra were acquired using a hybrid linear quadrupole ion trap-Fourier transform ion cyclotron resonance mass spectrometer equipped with a 7 T magnet (LTQ-FT, ThermoFisher Scientific, Bremen, Germany) coupled to an Ettan MDLC (GE Healthcare, Uppsala, Sweden) multidimensional nanoflow chromatography system as previously described.19 A Zorbax 300 SB-C18 trap column (length 5 mm, i.d. 0.3 mm, particle size 5 µm, Agilent Technologies, Palo Alto, CA, USA) was used for online desalting and sample cleanup, followed by a nanoscale reversed phase column (Zorbax 300 SB-C18, length 150 mm, i.d. 0.075 mm, particle size 3.5 µm, Agilent Technologies) for separation. The separation was performed at a flow rate of approximately 200 nL/min by applying a linear gradient of 0–60% B for 50 min. Mobile phase A consisted of HPLC grade water with 0.1% FA, while mobile phase B was 84% HPLC grade aqueous ACN with 0.1% FA. The peptide samples were redissolved in 50 µL of 0.1% FA. The injected volume was 20 µL. The eluent was electrosprayed (+1.3 kV) from the emitter tip (i.d 15 µm, uncoated, PicoTip Emitter, New Objective, Inc., Woburn, MA, USA) into the heated capillary of the mass spectrometer. The LTQ-FT was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Each scan cycle consisted of one full scan mass spectrum (m/z 350–2000) acquired in the FTICR mode followed by MS/MS in the QIT of the three most abundant ions that met the following criteria. Dynamic exclusion was activated during 30 s with a repeat count of 2 and a 2118

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Portelius et al. repeat duration of 30 s. The resolution was set to 100 000 for the FTICR mode and normal scan in IT (i.e., MS/MS) mode. Evaluation of the LC-MS and MS/MS data was performed with DeCyder 2.0 (GE Healthcare, Uppsala, Sweden). Protein identification was performed using the Mascot software, searching the UniProt/Swiss-Prot and UniProt/Trembl sequence databases. The following settings were used for the searches: mass error tolerance for the precursor ions, 10 ppm; mass error tolerance for the fragment ions, 0.8 Da; fixed modification, carbamidomethylation; variable modification, methionine oxidation; number of missed cleavage sites, 1; type of instrument, ESI-FTICR. ELISA. T-tau in CSF was determined using a sandwich ELISA constructed to measure total tau, i.e., both normal tau and hyperphosphorylated tau, as previously described.22

Results and Discussion We have developed a method for IP of tau in CSF, with subsequent detection by MS. The method was implemented on a robotic magnetic-bead purification system (KingFisher, Thermo), in which we have previously developed a method for IP of the AD related peptide β-amyloid.19 The low tau concentration in CSF and the high structural heterogeneity of the protein makes IP-MS challenging and necessitates optimization of several steps in the IP. Antibody Cross-Linking. A general problem in IP-MS is bleeding of the immobilized capture antibody into the antigen eluate, which may impair downstream analyses. If the immunoprecipitated sample is subjected to trypsin digestion prior to MS, the multitude of proteolytic peptides produced from the antibody may obscure detection of antigen derived peptides. By chemically cross-linking the antibody to the stationary phase this problem can be avoided. However, cross-linking may in some cases lead to loss of binding strength. To evaluate these potential losses for IP of tau, we determined the tau concentration in a CSF sample using ELISA before and after the affinity binding step in two parallel experiments: one in which the capture antibody (BT2) was only immobilized noncovalently to the sheep antimouse affinity beads, and one in which BT2 was subsequently covalently cross-linked to the sheep antimouse antibodies using DMP. The tau concentration measured in the CSF sample before immunoprecipitation was 1140 ng/ L. After incubation with the affinity beads, the tau concentration in the tau-depleted CSF sample was 65 ng/L with crosslinking and 445 ng/L without. This corresponds to a 95% capture efficiency of the beads with cross-linked antibodies, while without cross-linking it was only 61%. Thus, the BT2 antibody can be cross-linked without any significant loss of capture efficiency. One should note that the measured capture efficiency, for the noncross-linked sample, may be artificially high: if the affinity beads bleed the capture antibody into the CSF sample, it will compete with the secondary antibody in the ELISA, which is also BT2, resulting in a too low measured tau concentration. Cross-linking of the antibody also improved MS detection of tau (Figure 2). With cross-linking (Figure 2a), three tau-derived peptides were detected. Without cross-linking (Figure 2b), the background signals were significantly stronger, and only a single tryptic tau peptide was detected. Antibody Selection. There exists several different antibodies directed toward tau which are raised against different epitopes of the protein. While the binding strength of an antibody is reflected by the extent to which it can be diluted in an ELISA, the performance of an antibody in IP-MS experiments is

research articles

Characterization of Tau in Cerebrospinal Fluid Using MS unpredictable. We evaluated the IP-MS suitability of four different antitau antibodies: HT7, AT120, AT270, and BT2. In parallel experiments, each antibody was immobilized and cross-linked to magnetic beads and used to immunoprecipitate tau in 3 mL samples of CSF. Figure 3 shows a region of the MALDI TOF mass spectra acquired of the tau immunoprecipitates obtained with the different antibodies. The best results were obtained with BT2, for which three signals with m/z values that matched tryptic peptides of tau were detected. MS/MS analysis of these peptides performed in later experiments (see below) confirmed their identity. Weak signals of the same peptides were detected using the antibody HT7 but not with the other two antibodies. The phospho-specific antibody AT270 resulted in detection of three other signals, which might be related to cross-reactivity of AT270 with neurofilaments.16 CSF Protein Precipitation. High-abundant CSF proteins such as albumin and IgG compete with the antigen for binding to the capture antibodies and may bind nonbiospecifically to the affinity beads. This impairs detection of the antigen by MS and complicates data interpretation. It was previously reported for the analysis of tau in protein extracts from brain tissue that the tau protein remains in the supernatant upon protein precipitation with perchloric acid.2 We tested if this procedure could be used to enrich tau in CSF prior to IP-MS. A 3 mL CSF sample was subjected to protein precipitation. The tau concentration was determined by ELISA in the supernatant, in the redissolved protein pellet, and in an aliquot of the original CSF sample. The measured tau concentrations were the same in the supernatant as in the original CSF sample, and the concentration of tau in the pellet was below the detection limit of the ELISA, implying that the loss of tau by perchloric acid–protein precipitation was very low. Implementing the protein precipitation step prior to IP improved the quality of the resulting mass spectra, as is documented in Figure 4. In the sample, which was subjected to perchloric acid treatment (Figure 4b), the abundance of background signals in the m/z range of 900–1600 is much reduced, and three tau tryptic peptides were detected instead of a single one. Total Yield of the IP Method. The total yield of the optimized IP method was determined by ELISA. The tau concentration in an IP sample was 893 ng/L, which compared to the tau concentration in the original CSF sample (1140 ng/L) which corresponds to a yield of 78%. The efficiency of the binding step was above 90% (see above). Measuring the loss of tau during the washing steps is difficult because of the large combined volume of washing buffer used, which results, after concentration by vacuum centrifugation, in a high salt concentration which may affect ELISA results. However, when the solution of the first washing step (out of four) was analyzed, no tau was detected, suggesting that analyte losses during washing are low. LC-MS. While direct MALDI MS analysis allowed detection of tau in affinity purificates and was valuable for optimization of the IP method, only three tau-derived peptides were detected, which provides insufficient sequence coverage for indepth characterization of the protein, for example, discriminating between different isoforms and determining the status of tau’s many potential phosphorylation sites. For peptide samples of biological origin, the detection sensitivity is often improved by implementing a reversed-phase chromatographic separation step prior to MS.20 Furthermore, reducing the sample complexity by fractionating the analytes decreases the occurrence of

overlapping signals in the mass spectra, which otherwise complicates peak detection and impairs precursor ion selection for MS/MS. LC-MALDI MS of affinity purified tau of a CSF sample from an Alzheimer patient resulted in detection of a total of 13 peptides with m/z values that matched tryptic cleavage peptides of tau within the mass accuracy of the analysis (30 ppm). The identities of six of these were confirmed by MS/MS data (Figure 5a-f). Ten of the detected peptides are present in all six tau isoforms. Thus, these data do not allow us to determine which of the isoforms are present in the CSF sample. Of the other three tryptic peptides, one (Mr 2423.12 (monoisotopic mass)) is specific to the 0N isoform, one (Mr 3009.38) to the 1N isoform, and one (Mr 3954.85) to the 2N isoforms. Peptides raging from amino acid 6 to 395 (4R/2N isoform) have been identified, indicating that the isolated tau isoforms are fulllength proteins. The results obtained by LC-MALDI MS were largely confirmed by LC-ESI MS. Analysis of affinity purified tau of a CSF sample from an Alzheimer patient resulted in 14 tau peptides detected. Of these, nine were confirmed by MS/MS (Table 1).

Conclusion The combined approach of using immunoprecipitation and LC followed by tandem mass spectrometry allowed, for the first time, characterization of tau in human CSF. The sensitivity needed for successful MS analysis of tau was gained by optimization of several experimental parameters including selection and immobilization of antibodies in the IP-procedure and pretreatment of the CSF. By the optimized IP-LC-MS method, we were able to detect 19 tryptic tau fragments, 10 of which were verified by MS/MS analysis. Three of the fragments were specific to different isoforms (0N, 1N, and 2N), which may hold diagnostic information. An interesting application of the IP-MS method is to characterize the phosphorylation of tau. This might require the use of different antibodies and additional purification steps. The utilization of tau phosphorylation sites may be useful both as a diagnostic tool and as a biomarker for treatment efficacy of novel drugs that target ADassociated tau hyperphosphorylation, e.g., GSK-3β inhibitors.

Acknowledgment. This work was supported by grants from the Swedish Medical Research Council (projects 2006– 2740 and 2006–6227), the Sahlgrenska University Hospital, the Inga-Britt and Arne Lundberg Research Foundation, the Göteborg Medical Society, Swedish Brain Power, Stiftelsen Gamla Tjänarinnor, Alzheimer Foundation, Sweden, Tore Nilsons Stiftelse för Medicinsk Forskning, the National Genome Research Network 2 (NGFN2), and SMP Protein of the German Ministry for Education and Research (BMBF). The authors acknowledge Beata Lukaszewska-McGreal for technical assistance. References (1) Blennow, K.; de Leon, M. J.; Zetterberg, H. Alzheimer’s disease. Lancet 2006, 368 (9533), 387–403. (2) Lindwall, G.; Cole, R. D. The purification of tau protein and the occurrence of two phosphorylation states of tau in brain. J. Biol. Chem. 1984, 259, 12241–12245. (3) Kosik, K. S.; Joachim, C. L.; Selkoe, D. J. Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 1986, 83 (11), 4044–4048. (4) Nukina, N.; Ihara, Y. One of the antigenic determinants of paired helical filaments is related to tau protein. J. Biochem. (Tokyo) 1986, 99 (5), 1541–1544.

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