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Tau protein quantification in human cerebrospinal fluid by targeted mass spectrometry at high sequence coverage provides insights into its primary structure heterogeneity Nicolas Barthelemy, François Fenaille, Christophe Hirtz, Nicolas Sergeant, Susanna Schraen-Maschke, Jérôme Vialaret, Luc Buee, Audrey Gabelle, Christophe Junot, Sylvain Lehmann, and Francois Becher J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b01001 • Publication Date (Web): 07 Jan 2016 Downloaded from http://pubs.acs.org on January 8, 2016

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Journal of Proteome Research

Tau protein quantification in human cerebrospinal fluid by targeted mass spectrometry at high sequence coverage provides insights into its primary structure heterogeneity

Authors Nicolas R Barthélemy1, 2*, François Fenaille1, Christophe Hirtz2, Nicolas Sergeant3, Susanna SchraenMaschke3, Jérôme Vialaret2, Luc Buée3, Audrey Gabelle2,4, Christophe Junot1, Sylvain Lehmann2,$,, François Becher1,$,*

Affiliations 1. CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, Laboratoire d’Etude du Métabolisme des Médicaments, Gif-sur-Yvette, France. 2. CHU Montpellier, IRMB, hôpital St Eloi, Laboratoire de Biochimie Protéomique Clinique et CCBHM, INSERM-UM1 U1040, Montpellier, F-34000 France. 3. Inserm, UMR-S 1172, Alzheimer & Tauopathies, Centre de Recherche Jean-Pierre Aubert, Lille ; Univ. Lille, Faculté de Médecine, France. 4. Centre Mémoire Ressources Recherche, CHU Montpellier, hôpital Gui de Chauliac, Montpellier. Université Montpellier I, Montpellier, F-34000 France.

$

These authors jointly directed this work.

*To whom correspondence should be addressed. Phone: 1-314-362-3429. E-mail [email protected] Phone: 33-1-69-08-13-15. Fax: 33-1-69-08-59-07. E-mail: [email protected]

Key words Protein absolute quantification, microtubule-associated tau protein, parallel reaction monitoring, cerebrospinal fluid, alternative splicing-dependent peptides, protein fragments, protein precipitation, solid phase extraction.

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Abstract Tau protein plays a major role in neurodegenerative disorders, appears to be a central biomarker of neuronal injury in cerebrospinal fluid (CSF) and is a promising target for Alzheimer’s disease immunotherapies. To quantify tau at high sensitivity and gain insights into its naturally occurring structural variations in human CSF, we coupled absolute quantification with protein standards and the multiplex detection capability of targeted high-resolution mass spectrometry (MS) on a QuadrupoleOrbitrap instrument. Using recombinant tau we developed a step-by-step workflow optimization including an extraction protocol that avoided specific-affinity reagents and achieved the monitoring of 22 tau peptides uniformly distributed along the tau sequence. The lower limits of quantification ranged (LLOQ) from 150 to 1500 pg/mL depending on the peptide. Applied to endogenous CSF tau, up to 19 peptides were detected. Interestingly, there were significant differences in the abundance of peptides depending on their position in the sequence, peptides from the tau mid-domain appearing significantly more abundant than peptides from the N- and C-terminus domains. This MS-based strategy provided results complementary to those of previous ELISA or Western Blot studies of CSF tau and could be applied to tau monitoring in human CSF cohorts.


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Introduction Tau protein is involved in numerous neurological disorders, including Alzheimer’s disease (AD), grouped under the term “tauopathies” and characterized by the sporadic formation of intraneuronal aggregation of the protein into neurofibrils.1 There is increasing evidence that sporadic tauopathies may spread between synaptically connected neurons via extracellular release of pathological tau in a prion-like manner.2,3 However, the molecular mechanisms involved in such tau secretion and diffusion are incompletely understood. Tau is probably one of the most complex proteins in terms of structural modifications. In human brain, tau has six allele-specific isoforms (ranging from 352 to 441 amino acids), many truncated forms, as well as forms extensively modified post-translationally by methylation, glycosylation, ubiquitinylation and phosphorylation at more than 80 sites.4-7 Some of these forms, such as those containing four repetitions (4R) of the microtubule-binding repeat (MTBR) domain, have been implicated in the formation of intraneuronal tau aggregates in several tauopathies.8-10 Given the diversity of expected tau proteoforms, understanding the nature of tau species released from normal and diseased neurons could shed light on the molecular pathways initiating the spread of tauopathies.11 Recent in vitro studies of different cellular models have demonstrated that full-length tau is initially present in neurons and is then mainly released as C-terminally and N-terminally truncated forms.12,13 Since cerebrospinal fluid (CSF) is in continuous exchange with brain interstitial fluid in direct contact with neurons, characterization of corresponding CSF tau proteoforms appears to offer a unique opportunity to relate in vivo molecular events occurring in the central nervous system to in vitro results. Moreover, comparison of CSF tau species in human cohorts could identify alterations specific to distinct tauopathies and refine the molecular mechanisms potentially involved.14 On the other hand, higher CSF tau concentrations can be synonymous with AD and Creutzfeldt-Jakob disease and are assumed to be linked to neuronal death. The latter also supports the relevance of studying tau in this biofluid, although characterization of CSF tau is not trivial given its low abundance and the complexity of the matrix,15,16 as 3 ACS Paragon Plus Environment


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illustrated by contradictory results from various Western blot (WB) assays.17 Recently, a study combining liquid chromatography (LC) with WB reported the identification of heterogeneous N-terminus and middomain tau fragments in CSF, which is partly in line with in vitro observations.18 A multiple ELISA strategy was subsequently designed to monitor highlighted fragments in CSF.14,18 Importantly, no immunoreactivity was observed when the C-terminal domain and 4R MTBR domain were targeted, which questions the presence in CSF of full-length tau or fragments containing the potentially aggregable domain. The current state of the art, mainly limited by the number, efficiency and specificity of available tau antibodies, paves the way to the development of methods for CSF tau studies to add to antigenic profiling. Mass spectrometry (MS) may be a breakdown technique to address this issue. MS combined with tau immunopurification (IP) was previously applied to the characterization of CSF tau based on the detection of several peptides.19 However, poor sequence coverage in the Cter domain was observed under these conditions. Additionally, the antibodies used in IP-based methods may restrict coverage of tau species.18 Otherwise, recent MS methodologies combining targeted-MS/MS and highresolution/high-mass-accuracy measurement of fragment ions in part addresses the issue of limited MS sensitivity compared with ELISA.20,21 These advances could help to improve the MS coverage of CSF tau. Furthermore, robust and accurate protein quantification by MS has been recently demonstrated by isotope dilution with labeled protein standards.22,23 Full-length labeled protein offers the possibility of absolute quantification of all peptides released after protein extraction and digestion.24 Overall, these properties could potentially be exploited for specific and sensitive comparison of the abundance of peptides along the tau sequence which is currently arduous to perform when multiple ELISA strategies are employed for protein characterization.18 We hypothesized that such quantitative information could be used to estimate the overall proportion of the different tau domains in CSF, in comparison with results from previous studies, and especially to assess the presence and abundance of C-terminus4 ACS Paragon Plus Environment

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containing forms, including the 4R domain. We describe herein an analytical workflow, using 15N-labeled tau and targeted MS/HRMS on a Quadrupole-Orbitrap mass spectrometer operating in parallel reaction monitoring (PRM), for the quantitative monitoring of 22 tau peptides, distributed along the tau sequence. This strategy was then assessed for use in the preliminary characterization and quantification of CSF tau, extracted beforehand using an antibody-free strategy.



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Experimental Section CSF collection Participants gave written consent for participation in the study, which was approved by the ethics committee (CPP Sud Méditerranée IV; number 2011-003926-028). CSF was collected using standardized collection, centrifugation and storage protocols. CSF samples were transferred to the laboratories in less than 4 hours, centrifuged (1000×g, 10 min, at 4–8 °C, without breaks), and aliquoted in polypropylene tubes before storage at −80 °C. CSF tau concentraSon was measured using ELISA INNOTEST® according to the manufacturer’s instructions (Fujirebio Europe NV, formerly Innogenetics NV).

Protein standards 14

N recombinant tau (352, 381 and 441) proteins were from Sigma-Aldrich (Saint Quentin Fallavier,

France) (>90% purity by SDS PAGE) (Supplemental Figure S-1). Lyophilized samples were suspended in water to a final concentration of 100 µg/mL in 5 mM MES (morpholinoethanesulfonic acid) buffer containing 0.05 mM EGTA (ethylene glycol tetraacetic acid) and 1 mM NaCl. The solution of 15N-tau-441 recombinant protein, prepared as detailed elsewhere, was a gift from Guy Lippens (CNRS - Université de Lille 1, UMR8576, Villeneuve-d’Ascq, France).25 Isotopic incorporation of 15N in recombinant protein was estimated at ~99% based on the isotopic profiles of tryptic peptides observed by LC-ESI-high-resolution (HR) MS (not shown). Amino acid analysis was performed on

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N-tau-441 standard reconstituted

solution (SGS M-Scan, Geneva) to determine protein amounts accurately.

Preparation of calibration standards and quality control samples Artificial CSF was obtained by diluting serum to 0.5% in water and was chosen as a surrogate matrix for CSF because of lower cost, greater availability and absence of detectable endogenous tau. All dilutions


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of stock solutions were prepared in artificial CSF. Calibration curves were obtained by using 14N-tau-441 concentrations ranging from 0.05 to 40 ng/mL. Each concentration was prepared independently in triplicate. The accuracy of each dilution step was monitored by weighing spiked sample volumes to minimize error in concentration values during standard preparation (gravimetric method). Quality control (QC) solutions were prepared from three human CSF pools quantified beforehand for t-tau by ELISA, with low, middle and high concentrations of endogenous tau (144, 317 and 639 pg/mL, respectively). Eighteen QC samples were prepared by addition of recombinant

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N-tau-441 at 5

concentrations (final spiked concentrations 0, 200, 600, 1500, 4000 and 10000 pg/mL) to the three pools. Labeled internal standard was incorporated in each sample by addition of 50 µL of 15N-tau-441 solution at 40 ng/mL to a final concentration of 4 ng/mL.

Tau extraction by precipitation-µSPE CSF samples, calibration standards and quality control samples in 0.5% serum were spiked with 15N-tau441 immediately after thawing (50 µL addition to 450 µL sample volume). Twenty-five microliters of 70% perchloric acid was added to precipitate proteins and samples were kept on ice for 15 min before centrifugation (15 min, 4°C and 16000 g). Supernatants were collected and mixed with 50 µL of 1% trifluoroacetic acid (TFA) before µSPE extraction on Oasis HLB sorbent, previously conditioned with 300 µL of methanol and equilibrated with 500 µL of 0.1% trifluoroacetic acid (TFA). Samples were loaded and washed with 500 µL of 0.1% TFA. Protein oxidation was performed on-column with 500 µL of 3% formic acid and 3% hydrogen peroxide solution in water loaded on the cartridge and kept for 12 hours at 4°C. Thereafter, cartridges were washed with 500 µL of 0.1% TFA. Tau was eluted with 100 µL of 27.5% acetonitrile in 0.1% TFA. Extracts were evaporated to dryness with a Turbovap instrument (Biotage, Uppsala, Sweden) (10 psi of nitrogen, 50°C, 2 hours) and digested for 24 hours at 37°C with 40 µL of 1 ng/µL trypsin solution (Sequencing Grade Modified Trypsin, Promega, Madison, WI) in 50 mM



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ammonium bicarbonate. Finally, the digest was acidified with 5 µL of 10% acid formic and stored at 20°C prior to µLC-MS/HRMS analysis.

Liquid chromatography-mass spectrometry µLC-HRMS was performed on an Ultimate 3000 chromatography system coupled to a Q-Exactive Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Twenty microliters of sample extract was injected. Peptide separation was achieved in 20 min on a custommade Zorbax SB-C18-300A column (1 mm x 150 mm, 5 µm) (Agilent Technologies, Waldbronn, Germany) at a flow rate of 100 µL/min. The mobile phases were (A) 0.1% formic acid in water and (B) 0.1% formic acid in MeOH. After an isocratic step of 2 min at 2% phase B, a linear gradient from 2% to 45% B was run over the next 13 min with a mobile phase flow rate of 100 µL/min. Data were acquired in the positive ion mode with an ion spray voltage of 3700 V (HESI-II Probe, Thermo Fisher Scientific), a capillary temperature set to 320°C, and sheath and auxiliary gases of 35 and 8 psi, respectively. S-lens RF and skimmer voltage were set at 25 and 15 V, respectively. The Orbitrap mass spectrometer was externally calibrated every two days, which guarantees a mass accuracy better than 3 ppm. The AGC target was set to 1e6 and Orbitrap resolution to 70000 at 200 m/z for all experiments.

Peptide selection and MS optimization for quantification Pinpoint software (v.1.3; Thermo Fischer Scientific) was used to extract LC-HRMS peptide signals from a 250 ng/mL 14N-tau-441 digest analyzed in full-scan mode. Peptide identification was performed using a 5 ppm tolerance on the parent ion and the following criteria: specified number of amino acids, 6 to 50; enzyme specificity, 0, 1 and 2 miscleavages; charge states in the positive mode: 2, 3 and 4; unoxidized and oxidized methionine. Parent identification was finally confirmed by subsequent analysis of ion fragments obtained from targeted MS/MS experiments. Proteotypicity of all identified peptides was checked by individual BLAST searches against the UniprotKB/Swiss-Prot Human database 8 ACS Paragon Plus Environment

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(http://blast.ncbi.nlm.nih.gov, taxonomy ID 9606, 7/11/2013). Two semi-tryptic peptides, L.TPPAPK from NOTCH4 and T.VQIINK from HYDIN, were identified as potentially interfering in the human proteome. At this step, we kept these potentially interfering peptides in the global list, before further specificity evaluation. For quantitative experiments, the Q-Orbitrap was operated in the scheduled PRM mode. Best responding charge states (among 2+, 3+ and 4+) were selected, while collision energies were also optimized for each peptide. Quadrupole isolation was typically performed at 10 m/z to isolate simultaneously isotopologue pairs of light and heavy peptides (Supplemental Table S-1). Individual injection times were set from 250 ms to 1200 ms depending on the number of co-eluted peptides in a scheduled retention time window (Supplemental Table S-1).

PRM transition assessment and data analysis Assessment of fragments was performed with Pinpoint Software. PRM spectra of tau peptides from the highest calibration standard concentration (40 ng/mL) in 0.5% serum were used as a reference to select major fragment ions. Briefly, transitions showing an abnormal ion abundance and/or chromatographic profile in CSF and/or 0.5% serum matrices, compared with signals obtained with the reference, were excluded from the final list of selected fragments (Supplemental Figure S-2 and Table S-1). The same procedure was applied to PRM transitions from 15N peptides. A processing method was finally created (QuanBrowser, Thermo Excalibur 2.2, Thermo Fisher Scientific) to extract a composite signal constituted by the sum of selected PRM transitions (3 ppm of isolation range) for each light and heavy peptide monitored. Unlabeled/labeled peak area ratios were calculated for the different peptides and used for further establishment of calibration curves. For specific tau quantification in individual CSF extract, we additionally applied the following inclusion criteria i) co-elution with

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N-tau peptides from internal

standard (+/- 0.05 min), ii) similar peak shape and retention time to spiked

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N peptides from QC

samples (+/- 0.05 min), iii) detection of the most intense PRM transitions, and iv) similar area ratio



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among transitions between endogenous and spiked 14N-tau for signals with enough intensity (Supplemental Figure S-2).


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Results and discussion Tau peptide identification using recombinant tau Selection of relevant peptide candidates is a prerequisite to the implementation of any targeted proteomic method. One possibility is to use results from untargeted discovery experiments in biological samples.26 With regards to tau, this has proven challenging because of the scarcity and unknown characteristics of tau or tau isoforms in CSF. As an alternative, and with the objective of achieving the highest coverage of tau including allele-specific proteoforms, we used recombinant tau proteins to maximize identification of proteotypic peptides.22,27 With a view to detecting as many tau peptides as possible, optimization of the enzymatic digestion conditions was required before all else. Regarding tau digestion by trypsin, peptide release showed peptide-dependent kinetics, as previously described for different proteins.28 Overall, we observed two situations, with rapid to moderately fast release of peptides (5 to 10 hours of digestion) and peptides from more resistant sections, with significantly improved recovery at longer digestion times (Figure 1b and Supplemental Figure S-3). We noted this last category was mainly represented by peptides from the N-terminal domain, as a probable consequence of a particular conformation. However, this observation could also be the consequence of neighboring unfavorable trypsin cleavage sites (i.e., K.D, K.E, R.K).29,30 Thus, one day of digestion time proved to be optimal and was used for the rest of the study. In these conditions, we noted similar peptide yields for the three investigated isoforms of tau, which confirmed digestion efficiency and robustness (Supplemental Figure S-4). Under these conditions, we were able consistently to detect 22 peptides, distributed along the sequence of recombinant tau-441, corresponding to a total sequence coverage of 67% (Supplemental Figure S-5). It is of note that, among the 22 peptides selected, seven were from alternative splicing-dependent (ASD) domains and constituted surrogates of allele-specific isoforms. 11 ACS Paragon Plus Environment


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Optimized detection of recombinant tau peptides using LC-PRM Scheduled PRM acquisition with the hybrid Quadrupole-Orbitrap (Q-Orbitrap) mass spectrometer was designed for the multiplexed and high-sensitivity targeted analysis of the 22 detected tau peptides along with their 15N isotopologues (Supplemental Table S-1). MS detection was preceded by microbore liquid chromatography (µLC) separation of tau peptides, which was chosen as a good compromise between sensitivity, throughput, and retention time robustness, which is mandatory for effective MS quantification,31 particularly when scheduled experiments are performed.32 MS/MS parameters were thoroughly optimized to yield the best detection sensitivity and multiplexing capacity. High fill times (from 300 to 1200 ms depending on the multiplexing) and simultaneous

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N/15N isotopologue ion

isolation fragmentation and analysis were used for high sensitivity (Supplemental Table S-1).33 Highresolution/accurate mass detection of targeted fragment ions allowed a very high detection specificity devoid of any interference.20,21,33 Finally, the ratios of PRM transitions from unlabeled and labeled peptides were used for establishment of the corresponding calibration curves and their further quantification in CSF samples.

Antibody-free tau extraction Detection of CSF tau by MS at concentrations in the sub-nanogram per milliliter range (100-1200 pg/mL as deduced from ELISA measurements) requires analyte enrichment from CSF samples containing high amounts of proteins with a wide concentration range (0.2-1.2 mg/mL).

34,35

In order to avoid

dependence on IP and to preserve as much of the coexisting protein isoforms as possible, we designed a protein-centered sample preparation based on protein precipitation and micro-solid phase extraction (µSPE). This workflow was initially optimized using recombinant tau in 0.5% serum solution as artificial CSF. This easily available matrix, free of any detectable endogenous tau, was chosen to mimic CSF protein complexity for preliminary method development.36 To consider all possible tau protein losses


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during purification and perform absolute quantification,

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N-labeled protein was routinely added to

artificial CSF and also CSF samples just before their processing. Such entities have until now been regarded as the best source of internal standards, as they behave largely like their endogenous counterparts.23 Thus, our antibody-free enrichment protocol includes first a protein precipitation step with perchloric acid.19,37 It was followed by µSPE with elution conditions thoroughly optimized for concentrating and enriching tau by removing some of the remaining abundant proteins from the matrix (Figure 1a). This last step also efficiently and reproducibly eliminated the strong perchloric acid from the supernatant, which is mandatory for efficient protein enzymatic digestion. During method development, artifactual oxidative modifications of methionine residues were randomly observed. All methionine residues were therefore converted to methionine sulfone residues to make the 4 methionine-containing peptides (6-23, 25-44, 243-254, 406-438) readily amenable to absolute quantification.22,38,39 We took advantage of tau immobilization during µSPE to perform an on-column mild performic acid oxidative treatment. Quantitative methionine conversion to methionine sulfone residues was achieved as previously reported in liquid conditions (Figure 1c).38 Overall, tau recovery, calculated using recombinant standards in artificial CSF, was estimated to be ≈30%, with acid precipitation and µSPE yields being about 70% and 40%, respectively (Figure 1d). Importantly, the recoveries of the different recombinant tau allele-specific isoforms of 352, 381 and 441 amino acids were equivalent, revealing therefore no significant ASD isoform-specific behavior during the extraction procedure (Figure 1d). CSF protein removal by the whole procedure was estimated at about 98%, based on total protein quantification (Supplemental Figure S-6).



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Analytical validation using recombinant tau spiked in artificial CSF and CSF pools As a prerequisite to the quantitative peptide profiling of endogenous tau, detection/quantification limits and quantification accuracy were assessed for each highlighted peptide using recombinant tau spiked in artificial CSF first, then in real CSF pools. The linearity and sensitivity of the 22 peptides were first evaluated by analysis of recombinant tau solutions spiked in artificial CSF (i.e. 0.5% serum). Under these conditions, up to twelve peptides were detected at a tau concentration of 100 pg/mL, illustrating the high sensitivity of the method and its adequacy regarding the expected lowest concentrations in human CSF (Supplemental Table S-2). The lowest limit of quantification (LLOQ), defined by an accuracy between 80-120% and a repeatability below 20% CV, was estimated as ranging from 100 to 600 pg/mL for 20 out of 22 peptides of tau-441 (Supplemental Table S-2). Linearity above the LLOQ typically exceeded two orders of magnitude for all the peptides (Figure 2a and Supplemental Table S-2). No system carry-over was observed after the analysis of recombinant standard extracts at 40 ng/mL. Secondly, method performance for quantification was assessed in CSF using pooled samples spiked with recombinant tau. A decrease of 15N-tau peptide intensity was consistently observed for all 15N-labeled peptides, suggesting a lower extraction efficiency of tau from pure CSF than from 0.5% serum (Figure 2b and Supplemental Table S-3). Therefore, the LLOQs previously determined in 0.5% serum were corrected and correspondingly extrapolated LLOQs in CSF are indicated in Table 1. Then, measurement accuracy was evaluated for each of the 22 peptides using 3 distinct pools of CSF samples containing high, medium and low concentrations of endogenous tau (639, 317 and 144 pg/mL, respectively, as measured by ELISA), and analyzed after being spiked with defined amounts of

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N recombinant tau

standards. Eighteen peptides had a near 1:1 correlation between measured and expected tau concentrations obtained after standard addition (Figure 2c and Supplemental Table S-4). Quantification


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accuracy above LLOQs in CSF was excellent for 10 out of the 18 peptides (accuracy in the range 80 to 120%) and acceptable for 8 out of the 18 peptides (65-135%) (Supplemental Table S-4). This result defines these 18 peptides from 2N4R tau isoform for absolute quantification. Nevertheless, the 4 remaining peptides, which did not satisfy these accuracy criteria, were monitored to assess their detectability in CSF, but were not used for absolute quantification purposes. In total, 22 peptides were monitored.

Analysis of CSF samples The method was applied to the analysis of CSF samples from a cohort of 49 samples with variable amounts of endogenous tau (as deduced from ELISA measurements). As illustrated in Figure 4, proportions of monitored tau peptides significantly varied in two dimensions. The first dimension of variation followed the global CSF tau concentrations (as deduced by tau ELISA) and the second was related to peptide position along the tau sequence. Preliminary investigations into tau structural heterogeneity using CSF samples containing high tau levels (>700 pg/mL). In CSF samples with global tau concentrations in the upper range (ELISA > 700 pg/mL typically), the method was able to detect 19 out of the 22 peptides monitored, corresponding to a sequence coverage of 58% of the protein (Table 1, Figures 3, 4 and S2). Among those peptides, 17 were validated for accurate quantification and were included for peptide abundance comparison. When considering molar levels of these 17 peptides in relation to their position along the tau sequence (Figure 4), the peptide profile suggested a higher relative representation of the central part of the endogenous protein (amino acids 156-224 using tau-441 numbering) compared with its N- and C-termini. The most probable explanation for such an observation was the occurrence of tau fragments of different sizes in CSF, which was in line with recently published western-blot characterization of tau.18 In this view, the over-representation of peptides belonging to the protein central core (amino acids 156-224) could reasonably reflect the presence of the already described predominant ~17 kDa and 30 kDa tau 15 ACS Paragon Plus Environment


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proteoforms in CSF (Figure 4).18 The lower concentrations observed for N-terminal peptides (amino acids 6-126) suggested, however, the preponderance of the mid-domain 17 kDa proteoforms in our CSF extracts. This finding appeared to be totally consistent with the trends reported by multiple ELISA strategies.18 Regarding peptide levels specific to the alternative splicing in the N-terminus domain, the two 2N-specific peptides (amino acids 68-87 and 88-126) were dramatically low (Figure 4), unlike the 1N/2N peptide (45-67) and peptides common to all isoforms (6-23 and 25-44). This result suggested that the 1N isoform at the N-terminal side of tau was the one mostly present in CSF (domain corresponding to exon 2 insertion). This assumption was further strengthened by the additional detection of a 1Nspecific ASD peptide that was easily detected but not included in the initial peptide list (since it was not present in the tau-441 used for quantification) (Figure 3 and Supplemental Figure S7). Regarding the Cterminal part of the sequence, peptides corresponding to the sequence from amino acids 243 to 406 were detected, which could be consistent with the occurrence in CSF of ~30-50 kDa tau proteoforms containing C-terminal and microtubule-binding motifs purified using polymerized microtubules.40 Interestingly, we did not detect the peptide from the C-terminal sequence (407-438), which could either reflect its lower detection sensitivity or suggest truncation or modification of this part of the sequence. At the C-terminus, MTBR 4R-specific peptides were either detected below LLOQs or not at all, even though they were among the best responding peptides in standard tau (peptide 299-317 with LLOQ determined at 0.15 ng/mL). Afterward, 3R-specific peptide absent in the initial method was manually searched for and readily detected (Figure 3 and Supplemental Figure S7). Since quantification of this peptide was not performed, it was difficult to conclude that there was overrepresentation of 3Rcontaining isoforms with respect to 4R isoforms. The absence or low detection of the four 4R specific peptides and the 407-438 C-terminal peptide could explain the absence of CSF tau immunoreactivity when these domains were targeted by immunochemistry in other studies.18 Altogether, our results well


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illustrate tau isoform diversity in our CSF extracts and provide a first assessment of the main tau allelespecific proteoforms in this biological fluid. The main limitation of this peptide-based quantitative strategy is the inherent loss of information on full-length tau/fragments that share common peptides. This limitation could in part be addressed by applying the strategy of extended tau peptide quantitative monitoring coupled with CSF tau immunopurification targeting different tau epitopes.

Application to the whole cohort and impact of structural heterogeneity on absolute tau quantification. Differences in peptide abundances depending on peptide position in the sequence explain why several tau peptides (or epitopes) from the tau mid-domain were more frequently detected and quantified in CSF in previous studies.41,35 This fact also rationalizes the capacity of our method to provide concentrations above LLOQ in all CSF samples from the cohort, even those with low tau concentrations, when using peptides from the most concentrated mid-domain (peptides 156-163, 175180, 181-190, 194-209 and 212-224, Table 1, Figure 4). Quantitative results obtained from these peptides appeared to be highly correlated with ELISA (Table 1) as well, when using the 6 other peptides detected above LLOQ in most CSF samples from the investigated cohort (peptides 6-23, 45-57, 210-224, 243-254, 260-267 and 396-406). This high correlation of MS results with ELISA, whatever the peptide position in the sequence (Figure 5a), suggested that the relative proportions of the co-existing monitored proteoforms could remain constant overall in the cohort, although their respective concentrations vary significantly across the different samples. Similarly, the levels of tau peptides appeared to be correlated with each other, as illustrated for the N- and C-terminal peptides (amino acids 6-23 and 396-406) and the central peptide (amino acids 156-163), (Figure 5b). At first glance, the good correlation between these peptides with the global CSF tau concentration (Figure 5a) suggests similar representations of the corresponding tau species. This trend was observed for almost all peptides



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(Supplemental Table S-5) whose endogenous levels were adequate to provide enough values above the LLOQ (more than 50% according to Table 1) to perform this assessment. One can also consider the discrepancies regarding absolute CSF tau concentrations across all previously published data. First, the majority of the CSF tau data were obtained with ELISA or Luminex-based assays,42 and indicated concentrations ranging from approximately 100 to 1200 pg/mL.41 Previous results obtained using an MS-based assay involving protein immuno-enrichment and quantifying a single peptide from the tau mid-domain suggested up to two-fold higher values (400 to 2500 pg/mL).35 Using immunoassays, Meredith et al. reported concentrations corroborating ours, with values ranging from 167 to 2219 pg/mL and 690 to 8116 pg/mL, for N-terminal/central core and central core assays, respectively.18 In our view, these differences do not challenge the reliability of each of these techniques, but rather reflect variability in the analytical strategy employed to recover CSF tau proteoforms and/or considered in the quantification strategy (i.e. selection of defined proteoforms). Overall, we provide an exhaustive list of peptides detectable which could be used to rationalize tau epitopes targetable in CSF by tau antibodies for analytical or therapeutic purposes. Regarding the application of our MS method to clinical measurements, the 5 peptides from this middomain fully satisfy criteria required for accurate CSF tau quantification across a human cohort, and would be adequate for tau monitoring on instruments with lower resolution and multiplexing capabilities.43 Importantly, almost all of these peptides have been described as phosphorylated in brain and have been the targets of CSF phospho-tau measurement using ELISA. The good MS sensitivity demonstrated for these peptides raises the possibility of targeting corresponding minor modified peptides in future studies using a similar targeted MS strategy. This warrants further investigations. Additionally, our results suggest that a slight improvement of the sensitivity of the method could provide the same level of performance for an additional set of peptides for which the quantification was limited to CSF with middle or high tau concentrations (Table 1) and provide tools for monitoring the 18 ACS Paragon Plus Environment

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abundance of CSF tau isoforms, including minor C-terminal and MTBR-containing species. Future developments leading to improved tau recovery during extraction and more sensitive nanoLC-MS systems would be useful.



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Conclusion We have demonstrated the feasibility of using multipeptide targeted mass spectrometry to quantify CSF tau and deliver new information on sequence heterogeneity. MS sensitivity and specificity allowed quantitative sequence coverage up to 50% in CSF samples. Further improvement with more sensitive detection of low abundance peptides using nanoLC systems would help to target low abundance phosphorylated forms. The ability to cover peptides common or specific to tau isoforms could be extended to the analysis of other tau-containing samples, as brain extracts, cell cultures or other biological fluids. The method appears complementary to current immuno-based assays in assessing protein expression and identifying uncommon sequence modifications that may be associated with a particular disease phenotype. Alternatively, the peptide profiles provided can be used to refine epitopes and antibody selection directed against such protein targets.

Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org Supplemental Figure S-1: SDS-PAGE analysis of 14N-tau-441 Supplemental Figure S-2: Extracted ion chromatograms from PRM experiments on CSF spiked with recombinant tau (quality control), endogenous high tau CSF pool and 15N-recombinant tau Supplemental Figure S-3: Tau-441 digestion kinetic Supplemental Figure S-4 Comparison of peptides digestion yields between recombinant Tau isoforms Supplemental Figure S-5: Sequence coverage of tau isoforms Supplemental Figure S-6: CSF proteins depletion efficiency Supplemental Figure S-7: Extracted ion chromatograms from PRM experiments on CSF extracts targeting 1N and 3R specific peptides. 20 ACS Paragon Plus Environment

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Supplemental Table S-1: PRM transitions Supplemental Table S-2: Validation in artificial CSF Supplemental Table S-3: LLOQs Estimation in CSF Supplemental Table S-4: Validation in CSF Supplemental Table S-5: Inter-peptides correlation



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References

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(34) Gabelle, A.; Dumurgier, J.; Vercruysse, O.; Paquet, C.; Bombois, S.; Laplanche, J. L.; Peoc'h, K.; Schraen, S.; Buee, L.; Pasquier, F.; Hugon, J.; Touchon, J.; Lehmann, S. Impact of the 2008-2012 French Alzheimer Plan on the use of cerebrospinal fluid biomarkers in research memory center: the PLM Study. J. Alzheimers Dis. 2013, 34, 297-305. (35) McAvoy, T.; Lassman, M. E.; Spellman, D. S.; Ke, Z.; Howell, B. J.; Wong, O.; Zhu, L.; Tanen, M.; Struyk, A.; Laterza, O. F. Quantification of tau in cerebrospinal fluid by immunoaffinity enrichment and tandem mass spectrometry. Clin. Chem. 2014, 60, 683-689. (36) Lame, M. E.; Chambers, E. E.; Blatnik, M. Quantitation of amyloid beta peptides Abeta(1-38), Abeta(1-40), and Abeta(1-42) in human cerebrospinal fluid by ultra-performance liquid chromatography-tandem mass spectrometry. Anal. Biochem. 2011, 419, 133-139. (37) 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. (38) Simon, R.; Girod, M.; Fonbonne, C.; Salvador, A.; Clement, Y.; Lanteri, P.; Amouyel, P.; Lambert, J. C.; Lemoine, J. Total ApoE and ApoE4 isoform assays in an Alzheimer's disease case-control study by targeted mass spectrometry (n=669): a pilot assay for methionine-containing proteotypic peptides. Mol. Cell. Proteomics 2012, 11, 1389-1403. (39) Pesavento, J. J.; Garcia, B. A.; Streeky, J. A.; Kelleher, N. L.; Mizzen, C. A. Mild performic acid oxidation enhances chromatographic and top down mass spectrometric analyses of histones. Mol. Cell. Proteomics 2007, 6, 1510-1526. (40) Zemlan, F. P.; Rosenberg, W. S.; Luebbe, P. A.; Campbell, T. A.; Dean, G. E.; Weiner, N. E.; Cohen, J. A.; Rudick, R. A.; Woo, D. Quantification of axonal damage in traumatic brain injury: affinity purification and characterization of cerebrospinal fluid tau proteins. J. Neurochem. 1999, 72, 741-750. (41) Kang, J. H.; Korecka, M.; Toledo, J. B.; Trojanowski, J. Q.; Shaw, L. M. Clinical utility and analytical challenges in measurement of cerebrospinal fluid amyloid-beta(1-42) and tau proteins as Alzheimer disease biomarkers. Clin. Chem. 2013, 59, 903-916. (42) Olsson, A.; Vanderstichele, H.; Andreasen, N.; De, M. G.; Wallin, A.; Holmberg, B.; Rosengren, L.; Vanmechelen, E.; Blennow, K. Simultaneous measurement of beta-amyloid(1-42), total tau, and phosphorylated tau (Thr181) in cerebrospinal fluid by the xMAP technology. Clin. Chem. 2005, 51, 336-345. (43) Bros, P.; Vialaret, J.; Barthelemy, N.; Delatour, V.; Gabelle, A.; Lehmann, S.; Hirtz, C.; Antibodyfree quantification of seven tau peptides in human CSF using targeted mass spectrometry. Front. Neurosci. 2015, 9, 302-310.



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FIGURES:

Figure 1: Protocol for CSF protein depletion and tau recovery. A) Experimental workflow for the antibody-independent extraction of tau proteoforms from CSF and mass spectrometry analysis. B) Impact of the digestion time on the recovery of two tau peptides exhibiting different kinetic patterns (all kinetic results are shown in Supplemental Figure S-4). C) Methionine oxidation of LQTAPVPMPDLK peptide obtained after on-column oxidation and monitored by PRM (sum of y7+, y9+ and y10+ fragments). d) Extraction recoveries for three recombinant tau isoforms.


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1.0

30

B

LLOQ

12

1.2E+5

Intensity (cps)

0.5

25 0.0

20

C

1.4E+5

0.0

0.5

1.0

1.5

15 10

y = 0.6322x - 0.2026 R² = 0.9987

5

Predicted (ng/mL)

A 35 R(14N/15N) x C15N (ng/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.0E+5 8.0E+4 6.0E+4 4.0E+4

0

10

20

C14N (ng/mL)

30

8 6 4 2

2.0E+4

0

0.0E+0

0

y = 0.909x + 0.3103 R² = 0.9978

10

CSFCSF (n=24) Serum 0.5% (n=42) Serum 0.5%

(n=24)

(n=42)

0

2

4

6

8

10

12

Measured (ng/mL)

Figure 2: Method assessment for the quantification of SGYSSPGSPGTPGSR (195-209) peptide. A) Linearity, dynamic range and lowest limit of quantification (LLOQ) obtained in 0.5% serum extracts. B) Mean intensity of 15N-SGYSSPGSPGTPGSR peptide from spiked 15N-tau-441 obtained on replicates in CSF or 0.5% serum. Response difference between the two series can be attributed to the matrix effect and was used to estimate LLOQ in CSF. C) Quantification accuracy assessment in low-concentration tau CSF extract spiked with known quantities of 14N-tau-441. Results from all monitored tau peptides can be found in Supplemental Tables S-2, S-3 and S-4.



40000 20000

406-438 (nd)

60000

243-254 (unOx, nd)

80000

68-87 (2N) 88-126 (2N)

100000

VQIVYKPVDLSK (3R)

243-254 (Ox) 299-317 (4R) 243-254 (Ox2)

120000

STPTAEAEEAGIGDTPSLEDEAAGHVTQAR (1N)

212-221 6-23 354-369

156-163*

140000

181-190* 175-180* 260-267 396-406 195-209* 25-44 386-395 275-280 (4R) 281-290 (4R, nd) 210-224* 282-290 (4R, nd) 212-224* 45-67 (1N+2N)

160000

Intensity (cps/s)

0

70000

3

4

5

6

7

8

9

10 11 Time (min)

12

13

3

4

5

6

7

8

9

10 11 Time (min)

12

13

14

15

16

17

15

16

17

QAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQAR

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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65000 60000 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0

14

Figure 3: µLC-MS/HRMS extracted ion chromatograms: comparison between CSF tau (top) from a CSF extract quantified at 996 pg/mL by tau ELISA, and recombinant tau (bottom) from an extract of 0.5% serum spiked at 4 ng/mL with 14N-tau-441 (isoform 2N/4R). Two additional peptides from 1N- and 3Rcontaining isoforms, not present in tau-441, were subsequently detected after additional targeted PRM experiments in the CSF extract (sequences boxed). Asterisks indicate peptides from the central domain (156-224).


Tau ELISA:

440

109 pg/mL 144 pg/mL

317 pg/mL 639 pg/mL 966 pg/mL

20

330

15

*

*

4R

4R

4R

4R

10

2N

110

2N

1N+2N

220

5

*

Measured Measuredconcentration concentration (ng/mLeq. eq.tau441) tau441) (ng/mL

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Measuredconcentration concentration(fmol/mL) (fmol/mL) Measured

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* 0

0

0N 1

1N

3R

2N

Exon2 Exon3

4R

Exon10

1N >> 2N and 0N

’’17kD’’ proteoforms ’’30kD’’ proteoforms

Low 4R

3R > 4R

441

undetected Cter

Figure 4: CSF tau peptide abundance as a function of position in the tau sequence. Five distinct CSF samples with increased concentrations of CSF tau according to ELISA are shown, illustrating the relative release of the detected peptides from endogenous tau in comparison with the recombinant tau standard. The limits of quantification for each peptide monitored in this study are indicated (black boxes). Asterisks indicate peptides not satisfying accuracy criteria during method assessment for quantification. Concentrations are reported as fmol/mL and ng/mL of tau-441. Below, inferred fragments and allele-specific tau proteoforms found in CSF: inference of fragments previously described by Meredith et al. (in blue); part of tau sequence detected by MS but not previously targeted or identified in CSF by immunoblot (in red). 0N = no exons 2/3 insert, 1N = exon 2 insert, 2N = exons 2/3 insert. 4R and 3R = with or without exon 10 insert, respectively.


R² = 0.8626

2 LLOQ

0

30

500

R² = 0.8722 20 10 LLOQ

0 500

2

R² = 0.8857

1 LLOQ

0 0

500

LLOQ

R² = 0.939

20 10

0

2

4

156-163

30

LLOQ

R² = 0.8909

20 10 LLOQ

0 0

1

396-406 2

6-23 4

LLOQ

R² = 0.8108

2 LLOQ

0 0

1000

ELISA concentration (pg/mL)

6-23

LLOQ

0

1000

396-406

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156-163

30

1000

156-163

0

MS concentration (ng/mL)

4



B

6-23


A

0


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



1

396-406 2


Figure 5: A) Example of correlation curves of N-terminus (6-23), central (156-163) and C-terminus (396406) peptide values determined by MS with ELISA tau values obtained across the cohort of 49 patients. B) Interpeptide correlation. Results from all monitored tau peptides can be found in Supplemental Tables S-4 and S-5.


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Peptide sequence

Residues

(R) QEFEVMox2EDHAGTYGLGDR (K) (K) DQGGYTMox2HQDQEGDTDAGLK (E) (K) ESPLQTPTEDGSEEPGSETSDAK (S) (K) STPTAEDVTAPLVDEGAPGK (Q) (K) QAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQAR (M) (R) GAAPPGQK (G) (K) TPPAPK (T) (K) TPPSSGEPPK(S) (R) SGYSSPGSPGTPGSR (S) (R) SRTPSLPTPPTREPK (K) (R) TPSLPTPPTREPK (K) (R) TPSLPTPPTR (E) (R) LQTAPVPMox2PDLK (N) (K) IGSTENLK (H) (K) VQIINK (K) (K) KLDLSNVQSK (C) (K) LDLSNVQSK (C) (K) HVPGGGSVQIVYKPVDLSK (V) (K) IGSLDNITHVPGGGNK (K) (K) TDHGAEIVYK (S) (K) SPVVSGDTSPR (H) (R) HLSNVSSTGSIDMox2VDSPQLATLADEVSASLAK (Q)

6 25 45 68 88 156 175 181 194 210 212 212 243 260 275 281 282 299 354 386 396 406

-

23 44 67 87 126 163 180 190 209 224 224 221 254 267 280 290 290 317 363 395 406 438

Isoforms all all 1N/2N 2N 2N all all all all all all all all all 4R 4R 4R 4R all all all all

Proportion of CSF Correlation CSF samples (%, n=49) with t-tau LLOQ 2 (ng/mL) Detected >LLOQ ELISA (R ) 1.20 1.54 0.92 0.16 0.31 0.29 0.26 0.47 0.62 0.63 0.52 0.59 0.27 0.30 0.31 0.94 1.03 0.15 0.59 1.03 0.30 1.21

98 69 100 90 100 100 100 100 100 100 100 100 100 100 69 0 0 65 72 81 95 0

63 12 57 12 2 100 100 100 100 73.5 100 100 80 96 20 0 0 6 2 35 73 0

0.86 nd 0.78 nd nd 0.87 0.81 0.77 0.86 0.81 0.82 nd 0.83 0.83 nd nd nd nd nd nd 0.89 nd

Table 1: Assay performance for each targeted tau peptide for the monitoring of endogenous concentration ranges found in human CSF. For each peptide, the determined lower limit of quantification (LLOQ) is shown. The usefulness of each peptide for CSF tau monitoring in human CSF is illustrated by the percentage of samples found above LLOQ or detected. 49 CSF samples ranging from the lowest to highest tau concentrations according to ELISA were used for this assessment. Correlation trends between ELISA and MS values obtained for each peptide, having at least 50% of the values above LLOQ across the CSF cohort, are indicated with the respective correlation coefficient (R2). -nd: not determined.



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Tau Protein Quantification in Human ... - ACS Publications

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