Mass Spectrometry-Based Quantification of Tau in Human

Subscriber access provided by ALBRIGHT COLLEGE

Article

Mass spectrometry-based quantification of Tau in human cerebrospinal fluid using a complementary tryptic peptide standard Maotian Zhou, Duc M Duong, Erik C.B. Johnson, Jingting Dai, James J Lah, Allan I. Levey, and Nicholas T. Seyfried J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00920 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 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

Journal of Proteome Research

Mass spectrometry-based quantification of Tau in human cerebrospinal fluid using a complementary tryptic peptide standard

Maotian Zhou1,2#, Duc M. Duong1#, Erik C.B. Johnson2, Jingting Dai1,3, James J. Lah2, Allan I. Levey2, and Nicholas T. Seyfried1,2, * 1

Department of Biochemistry, 2Department of Neurology, Emory University School of Medicine, Atlanta, Georgia 30322 3

Department of Neurology, Second Xiangya Hospital, Central South University, Changsha, Hunan, China #

Both authors contributed equally to this manuscript

*Address correspondence to: Nicholas T. Seyfried, Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, USA. Tel. 404.712.9783, Email: [email protected]

Abbreviations: MS: mass spectrometry SRM: selected reaction monitoring PRM: parallel reaction monitoring AD: Alzheimer’s disease CV: coefficient of variation LC: liquid chromatography CSF: cerebrospinal fluid CAA: chloroacetamide TCEP: tris-2(-carboxyethyl)-phosphine MCI: mild cognitive impairment IRB: institutional review board ADRC: Alzheimer’s Disease Research Center BCA: bicinchoninic acid assay AGC: automatic gain control

1 ACS Paragon Plus Environment

Journal of Proteome Research 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

Abstract Here we report a method for the generation of complementary tryptic (CompTryp) isotope labeled peptide standards for the relative and absolute quantification of proteins by mass spectrometry (MS). These standards can be digested in parallel with either trypsin (Tryp-C) or trypsin-N (TrypN), to generate peptides that significantly overlap in primary sequence having C- and N-terminal arginine and lysine residues, respectively. As a proof of concept, an isotope-labeled CompTryp standard was synthesized for Tau, a well-established biomarker in Alzheimer’s Disease (AD), which included both N- and C-terminal heavy isotope labeled (15N and 13C) arginine residues and flanking amino acid sequences to monitor proteolytic digestion. Despite having the exact same mass, the N- and C-terminal heavy Tau peptides are distinguishable by retention time and MS/MS fragmentation profiles. The isotope labeled Tau CompTryp standard was added to human cerebrospinal fluid (CSF) followed by parallel digestion with Tryp-N and Tryp-C. The native and isotope labeled peptide pairs were quantified by parallel reaction monitoring (PRM) in a single assay. Notably, both tryptic peptides were effective at quantifying Tau in human CSF, and both showed a significant difference in CSF Tau levels between AD and controls. Treating these CompTryp Tau peptide measurements as independent replicates also improved the coefficient of variation and correlation with Tau immunoassays. More broadly, we propose that CompTryp standards can be generated for any protein of interest, providing an efficient method to improve the robustness and reproducibility for MS analysis of clinical and research samples.

Keywords: Proteomics, parallel reaction monitoring (PRM), biomarker, Alzheimer’s disease (AD), neurodegeneration


Page 2 of 31

Page 3 of 31 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


Introduction Traditionally, immunoassays, such as ELISAs, have been the primary tool for absolute and relative protein quantitation in tissues and biofluids. However, these immunoassays serve as an indirect approach for protein quantification and do not always meet desired levels of specificity and analytical reproducibility for biomarker studies 1. Moreover, ELISAs typically only measure a single protein biomarker within a given sample. In contrast, mass spectrometry (MS)-based proteomic approaches have been increasingly employed for the direct detection and quantification of multiple peptides from a diverse number of proteins in a single assay 2, 3. Selective reaction monitoring (SRM) and parallel reaction monitoring (PRM) are commonly used techniques for targeted proteomics analysis 4, 5. These MS assays typically utilize stable isotope-labeled (usually 15N or 13C) protein or peptide internal standards, which are added to each sample at a known molar amount to produce peptide precursor and product ion pairs (light and heavy) that can be monitored in a data-independent manner for the relative or absolute quantification of proteins across multiple samples 6. Targeted MS assays are considered the “gold standard” for protein quantification because they are highly reproducible, sensitive, selective and have a broad linear dynamic range of detection in tissues and biofluids

7-9

. However, a limiting

factor for targeted MS assays is the sample preparation required, which can result in batch to batch variability in the efficiency of the proteolytic digestion used to generate diagnostic peptides 10-12. Thus, samples are often analyzed in multiple technical replicates to reduce technical variance, which introduces additional expense, instrument time, and reduced throughput. Thus, new approaches that minimize technical limitations and enhance reproducibility are desired, especially for large scale clinical and research applications. Here we describe a method for the generation of isotope labeled standards that implements a parallel enzymatic digestion scheme. Protein samples of interest are digested in parallel with either trypsin (Tryp-C), which specifically cleaves C-terminal to arginine and lysine, or TrypsinN (Tryp-N) a thermophilic metalloprotease with N-terminal specific for arginine and lysine

13

.

Digestion with both enzymes generates complementary tryptic (CompTryp) peptides that overlap in primary sequence and, in some instances, have the exact molecular mass when flanked by two arginine or lysine residues. However, Tryp-N and Tryp-C generated peptides have unique MS/MS fragmentation profiles for the detection and quantification of proteins in tissue and biofluids 14-16.



Thus, by pooling Tryp-C and Tryp-N digests from the same sample, protein quantification could be reciprocally validated using heavy CompTryp isotope standards by PRM, improving precision and sample throughput by reducing the numbers of technical replicates needed. As a proof of concept, we present the CompTryp digestion of Tau, a well-established biomarker in Alzheimer’s Disease (AD) 17. CompTryp peptides generated from Tau were resolved by liquid chromatography and quantified by PRM in unfractionated CSF samples without the need for prior albumin depletion or Tau enrichment. Notably, both peptides showed a significant difference in CSF Tau levels between AD and controls, and their levels strongly correlated with Tau ELISA immunoassays results. The quantification of both Tryp-N and Tryp-C Tau peptides provided internal, independent technical replicates that enhanced measurement precision by PRM and correlation with Tau levels as measured by ELISA. Overall, the CompTryp method is broadly applicable for the measurement of protein biomarkers.


Page 4 of 31

Page 5 of 31 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


Materials and Methods Materials Trypsin (Tryp-C), mass spectrometry grade was bought from Thermo Fisher Scientific (Waltham, MA). Trypsin-N (Tryp-N) is a thermophilic metalloprotease purchased from Protifi (Huntington, NY) with N-terminal specificity for arginine and lysine as previously described 13. Note, TrypN was aliquoted and stored long term at -80C prior to use as the Tyrp-N enzyme is shipped in working buffer. Purified recombinant Tau-441 (2N4R) was purchased from rPeptide (Watkinsville, GA). CAA (chloroacetamide), TCEP (tris-2(-carboxyethyl)-phosphine) were obtained from Sigma (St. Louis, MO). Acids and organic solvents were MS grade. Heavy labeled AQUA™ Peptides were purchased from Thermo Fisher Scientific (Waltham, MA), note the HPLC purity was determined to be 98.71% and quantification was performed by amino acid analysis. Glass inserts for liquid chromatography auto-sampler were from Wheaton (Millville, NJ). Ethics statement All participants from whom CSF samples were collected provided informed consent under protocols approved by the institutional review board (IRB). Symptomatic individuals with mild cognitive impairment (MCI) or mild AD, received clinical evaluations in the Emory Cognitive Neurology clinic with the CSF collection for clinical reasons. All cognitively impaired individuals were accompanied by spouse or other family member and met additional IRB safeguards for protection of vulnerable populations. Cognitively intact control individuals were recruited through the Goizueta Alzheimer’s Disease Research Center (ADRC) at Emory or the Emory Healthy Brain Study under IRB approved protocols. Human CSF collection and immunoassays CSF samples from 88 individuals, including 20 healthy controls, 37 patients with mild, symptomatic AD (either prodromal AD with mild cognitive impairment or early stage AD dementia with positive AD biomarkers), and 31 with other (non-AD) neurological disease were used (Table 1). The three groups were matched for age and race. Normal controls and subjects with AD were also matched for gender, but a larger proportion of those with non-AD neurological conditions were male. All symptomatic individuals were diagnosed by expert clinicians in the ADRC and the Emory Cognitive Neurology who are subspecialty trained in Cognitive and 5 ACS Paragon Plus Environment


Behavioral Neurology following extensive clinical evaluations including detailed cognitive testing, neuroimaging, and laboratory studies. For clinical testing, CSF samples from these individuals were all sent to Athena Diagnostics and assayed for Aβ42, total-Tau, and phospho-Tau (CSF ADmark®) using the INNOTEST® assay platform. Healthy control individuals with normal cognition were identified from among participants in the Emory ADRC Clinical Core. Note that only a handful of these individuals (n=4) had been seen clinically for CSF ADmark® testing. Protein digestion of CSF CSF samples were thawed at room temperature and protein concentration measured by the bicinchoninic acid assay (BCA) method (Supplementary Table 1). CSF was aliquoted into two tubes (20 μl each tube) and each sample was reduced and alkylated by adding 5 μl of 50 mM TCEP, 200 mM CAA, 250 mM ammonium bicarbonate, pH 8.0. Each sample was vortexed for 30s and then heated at 90°C for 10 min followed by bath sonication for 10 min. The AQUA™ heavy peptide SGDRSGYSSPGSPGTPGSRSRT with each arginine heavy labeled (13C6,15N4 in bold) and flanking sequence (underlined) was diluted with water from a stock solution (5 pmol/μl, 5% ACN in water) to 5 fmol/μl (working solution). Two microliters were added to the digestion solution at the final concentration 500 amol/μl (equal to CSF volume). Either 80 μl Tryp-C digest solution (enzyme/protein ratio 1:10 in 50 mmol ammonium biocarbonate) or 80 μl Tryp-N digest solution (enzyme/protein ratio 1:10 in 50 mmol ammonium biocarbonate) was added to the CSF samples. Samples were then incubated overnight at 37°C with agitation. Different digestion conditions were assessed, which revealed that 37°C overnight produced the highest digestion. After the incubation, the digestion was quenched by adding trifluoroacetic acid and formic acid (final concentration, 0.1% TFA, 1% FA). At this point, the two digest solutions were mixed and desalted using 50mg tC18 column (Waters, Milford, MA) according to the manufacturer’s protocol and eluates were dried under vacuum. To construct the standard curves for the target Tau peptides, a serial dilution of the AQUATM heavy peptide ranging from 10 amol/μl to 800 amol/μl was added to CSF (pool of all samples in the study), and digested (Tryp-N or Tryp-C) following the same conditions described above. A time course of TrypN or TrypC digestion of the Tau CompTryp standard was also collected at 0.5, 1, 2, 4, 6, 8, 12, 16, 20, and 24 hours, respectively.


Page 6 of 31

Page 7 of 31 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


Protein digestion of recombinant Tau Purified recombinant full-length Tau protein (1-441 residues) was solubilized in water to a final concentration 0.2 μg/μl. For CompTryp digestion, 1μl (0.2 μg/μl equal to 4.1 pmol/μl) of Tau 441 protein

and

1μl

of

stock

solution

(5

pmol/μl)

of

AQUA™

heavy

peptide

SGDRSGYSSPGSPGTPGSRSRT were added into 18μl of 10 mM TCEP, 40 mM CAA, 50 mM ammonium bicarbonate solution, pH 8.0. The CompTryp peptide was also added following protein reduction and alkylation, which had no significant impact on the light/heavy ratio measurements (Fig. S1). Each sample was vortexed for 30s and then heated at 90°C for 10 min followed by bath sonication for 10 min. Either 80 μl Tryp-C digest solution (0.2 μg enzyme in 50 mM ammonium biocarbonate) or 80μl Tryp-N digest solution (0.2 μg enzyme in 50 mM ammonium biocarbonate) was added to the samples. Samples were then incubated overnight at 37°C with agitation. After the incubation, the digestion was quenched by adding trifluoroacetic acid and formic acid (final concentration, 0.1% TFA, 1% FA). At this point, the two digest solutions were mixed and desalted using homemade stage-tip and eluates were dried under vacuum as described

18

. To generate

protein coverage maps, tryptic digests (Tryp-N or Tryp-C) were analyzed by LC-MS/MS on a QExactive mass spectrometer by data-dependent acquisition and searched against the human Uniprot database using MaxQuant 19 as previously described 20. Parallel Reaction Monitoring (PRM) analysis Each sample (equal to 2 µl CSF, or 40 ng of Tau-441 digestion) was analyzed on a Q-Exactive Plus hybrid mass spectrometer (ThermoFisher Scientific) fitted with a Nanospray Flex ion source and coupled to a NanoAcuity liquid chromatography system. The tryptic peptides were resuspended in 40 μl of loading buffer (2% ACN, 0.1% TFA) and 2 µl was loaded onto a selfpacked 1.9 um ReproSil-Pur C18 (Dr. Maisch) analytical column (New Objective, 30 cm × 75 µm inner diameter; 360 µm outer diameter). Elution was performed over a 40-min gradient at a rate of 300 nL/min with buffer B ranging from 2% to 25% (buffer A: 0.1% formic acid in water, buffer B: 0.1% formic in acetonitrile). The column was then washed with 99% B for 20 minutes and reequilibrated with 2% B for 15 minutes. The mass spectrometer was set to collect in PRM mode with an inclusion list consisting of the light and heavy products of each CompTryp peptide (Supplementary Table 2). An additional full survey scan was collected to assess for possible interference. Full scans were collected at a resolution of 70,000 at 200 m/z with an automatic gain 7 ACS Paragon Plus Environment


control (AGC) setting of 1x106 ion and a max ion transfer (IT) time of 100 ms. For PRM scans the settings were: resolution at 17,500 at 200 m/z, AGC target of 5x105 ions, max IT time of 500 ms, loop count of 30, MSX count of 1, isolation width of 1.6 m/z and isolation offset of 0.5 m/z. A pre-optimized normalized collision energy of 24% was used to obtain the maximal recovery of target product ions. The top five product ions from this collision energy optimization were used for downstream peptide quantification. In the case of Tryp-C Tau peptide the top ions were y10, y7, y4, y11 and b3, whereas for Tryp-N Tau peptide the top ions were, y3, b12, b9, a12 and y6 (Supplementary Table S2). Raw files contributing to the described work are also deposited electronically at the Synapse Web Portal (https://doi.org/10.7303/syn18457323). Peptide quantification A spectral library was built using Skyline 21 (Version 3.6) based on tandem mass spectra gathered from the heavy Tau CompTryp standards. A Skyline template was then created to quantify the light endogenous and heavy CompTryp peptides. The template parameters were: Precursor mass analyzer, Centroided; MS1 mass accuracy of 20 ppm; Product mass analyzer, Centroided; MS/MS Mass accuracy of 20 ppm; include all matching scans. All rawfiles were then imported and processed accordingly. The resulting extracted ion chromatograms (XICs) of selected fragments were manually inspected and peak picking adjustments were made accordingly. The sum of all product ion peak areas was calculated by Skyline and extracted for further statistical analysis. Statistical analysis All statistical analyses were performed in GraphPad Prism version 7.00 for Windows (GraphPad Software, San Diego California USA). The light to heavy peptide ratios were directly calculated by dividing the endogenous peptide peak area by the corresponding heavy CompTryp peak area. Standard student’s t-test, or one-way ANOVA test were used to calculate significance.


Page 8 of 31

Page 9 of 31 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


Results Generation of isotope-labeled complementary tryptic peptide standards for quantitative mass spectrometry Trypsin is the most widely used enzyme in bottom-up proteomic approaches

22

. It cleaves C-

terminal to lysine (Lys) and arginine (Arg) and is robust and specific 23. More recently, enzymes with N-terminal specificity for arginine and lysine have been described and include Tryp-N, which is a thermophilic metalloprotease analogous to the thermophilic proteinase LysargiNase isolated from Methanosarcina acetivorans

13, 16, 24

. Tryp-N essentially generates complementary tryptic

(Tryp-C) peptides with regard to primary sequence. Of note, if the tryptic peptide is flanked by two N- and C-terminal arginine or lysine residues, then Tryp-N and Tryp-C digestion will produce peptides with identical masses (Fig. 1). Therefore, parallel digestion of a given protein or proteome with Tryp-C and Tryp-N generates independent overlapping peptides from the same primary sequence, and when measured simultaneously by MS can serve as independent replicate measurements. A first step for the absolute and relative quantification of these peptides is to synthesize isotope labeled peptide standards with Arg or Lys at both the N- and C-termini. As a proof of concept, we chose the microtubule binding protein Tau. Aggregates of Tau represent a core component of neurofibrillary tangles in Alzheimer’s disease (AD), and measurement of Tau and Aβ42 in CSF serves as a clinically validated diagnostic biomarker for AD

17

. Notably, Tau

accumulation is also a pathological hallmark in corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and frontotemporal dementia (FTD-Tau)

25

, and simple and reliable

measurement of Tau may also prove relevant in these diseases. For our studies, an isotopically labeled peptide standard for Tau (SGDRSGYSSPGSPGTPGSRSRT) was synthesized that harbors “heavy” Arg residues (bold) on both the N-terminus and C-terminus corresponding to residues 194-209 in the full-length tau protein sequence (Fig. 1) along with flanking residues (underlined). This peptide was selected because it was previously described as one of the most proteotypic peptides identified for Tau in CSF 26, 27. We refer to this complementary tryptic peptide standard as a CompTryp standard for its amenability to either Tryp-C or Tryp-N proteolytic digestion.



Page 10 of 31

Monitoring proteolytic digestion of Tau using CompTryp standards Accurate and precise quantitation of peptides is possible using peptide standards, but these measurements may be impacted by variation in efficiency of proteolytic digestion within complex biological samples and differential recovery of peptides during the sample preparation 28. For these reasons, it is preferable to use known quantities of protein standards to calibrate the peptide precursor signal in the mass spectrometer

29, 30

. The major advantage of using a heavy labeled

protein standard is that it undergoes the same sample preparation procedures as the endogenous protein and, thus, can be used to assess digestion efficiency and control other sources of technical variation during sample preparation

28, 31

. However, challenges with this approach include

difficulty and expense in achieving robust expression, complete isotope labeling (15N or SILAC), and purification of the protein standards. To this end, we introduced flanking residues (3 residues each) preceding and following the tryptic cleavage site to provide context for Tryp-N and Tryp-C enzymes and to assess digestion efficiency. This CompTryp peptide is analogous to “winged” peptides described for Tryp-C standards 32-34 and allows monitoring of both complete and partially digested intermediates following Typ-C and Tryp-N digestion (Fig. 2). The complete digestion efficiency for Tryp-C and Tryp-N was calculated as 99.8% and 94.1% respectively. The major partially digested peptide intermediates following Tryp-N was SGDRSGYSSPGSPGTPGS (4.1%). The reduced efficiency of Tryp-N is likely due to the acidic Asp (D) residue immediately preceding the N-terminal Arg residue. Acidic residues adjacent to trypsin sites (Lys/Arg) have been shown to negatively impact both Tryp-N and Tryp-C cleavage 16, 35. Notably, differences in decay rate for AQUA peptide standards have been shown to bias protein quantification

36

.

However, we did not observe a significant decay in signal for either the Tryp-N or Tryp-C peptide up to 24 hrs following digestion (Fig. S2). Thus, both enzymes were efficient at digesting the CompTryp Tau standard into N- or C-terminal tryptic peptides. Previous studies have shown that digestion of isotope labeled peptide standards with flanking residues do not necessarily reflect the intact protein

37, 38

. Thus, we also assessed

CompTryp digestion of purified recombinant Tau protein (441 residues) rather than the standard alone. Following overnight digestion and LC-MS/MS and database searching, Tryp-N and TrypC generated 67 and 69 unique peptides representing 82.2% and 87.8% coverage of the recombinant Tau protein, respectively (Fig. S3). Furthermore, the complete digestion efficiency for Tryp-N and


Page 11 of 31 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


Tryp-C was 96.1% and 99.7%, respectively, which is similar to the Tryp-N and Tryp-C digestion efficiencies of the heavy CompTryp isotope standard (94.1% and 99.8%, respectively).

Detection and quantification of Tau using CompTryp Standards in Human Cerebrospinal Fluid When the isotopically labeled CompTryp peptide is added at known concentration into human cerebrospinal fluid (CSF) samples and digested with Tryp-C, the C-terminal tryptic peptide SGYSSPGSPGTPGSR will be exclusively generated, whereas a separate digestion with Tryp-N will generate the N-terminal tryptic peptide RSGYSSPGSPGTPGS (Fig. 1). Both Tryp-N and Tryp-C peptides have the exact same mass and mass-to-charge ratio (697.3224 m/z, charge +2) because they are flanked by N and C-terminal arginine residues. However, each unique Tau peptide can be distinguished by MS/MS fragmentation and LC retention time (Fig. 3). Having the basic Arg residue on the amino terminus generates b-ions that predominate in the MS/MS spectrum of the Tryp-N peptide compared to the y-ions that dominate the Tryp-C MS/MS spectrum (Fig. 3A). Furthermore, the Tryp-N generated peptide elutes slightly earlier than the Tryp-C generated peptide (Fig. 3B). Notably, both Tryp-N and Tryp-C heavy standards exhibit a nearly identical MS/MS fragmentation pattern to their endogenous Tau peptide counterparts in human CSF samples, confirming the identity of Tau protein in the samples (Fig. 3C). The linearity of quantification of CompTryp standard peptides was determined using a dilution series in human CSF (Fig. 4). To construct a standard curve, the isotopically labeled heavy peptide was diluted in the range from 10 amol/μl to 800 amol/μl followed by digestion with TrypN or Tryp-C. Quantification of the light peptide (endogenous) to heavy peptide (standard) ratio shows excellent correlation with heavy peptide levels for both Tryp-C and Tryp-N digestion (correlations are 0.997 and 0.999 respectively). (Fig. 4A and B). The Lower Limit of Quantification (LLOQ) was determined to be 10 amol/μl even in the presence of highly abundant albumin or immunoglobulins in CSF. Therefore, following parallel digestion, the CompTryp Tau standard produces two unique peptides, which can be distinguished by both LC retention time and MS/MS fragmentation patterns for the relative and absolute quantification of endogenous human Tau in CSF without the need for albumin depletion or Tau enrichment.



Quantification of Tau utilizing CompTryp peptides in AD CSF To evaluate the absolute quantification of Tau in AD CSF using CompTryp standards, we selected 88 individual CSF samples from the Goizueta ADRC biospecimen repository representing ADbiomarker confirmed cases of mild cognitive impairment or early stage dementia due to AD (n=37), non-AD neurological disease (n=31), and age- and sex- matched healthy controls (n=20) (Table 1 and Supplementary Table 3). The total Tau protein concentrations determined by ELISA ranged from 100.9 pg/ml to 2204.8 pg/ml, with levels in the AD cases (avg. 1045 pg/ml) higher compared to controls (avg. 391 pg/ml) and non-AD cases (avg. 191 pg/ml), consistent with previously reported levels for Tau

39

. Prior to PRM analysis, the CSF samples were randomized and divided

into 4 batches (22 samples per batch). A pooled standard sample representing an equal volume of all 88 samples was also analyzed at the beginning and end of each batch as shown in Supplementary Fig. 4A. The CompTryp Tau peptide (500 amol/ul) was added to each CSF sample prior to parallel digestion with Tryp-C and Tryp-N as described in Figure 1. Following PRM analysis, Skyline software

21

was used to quantify the fragment ion peptide peak area for

both the light (endogenous) and heavy (standard) in each sample (Supplementary Table 3). The raw peak area of heavy and light peptides for both Tryp-C and Tryp-N digestion from a total of 98 samples, including pooled standards, are provided in Supplementary Fig, 4B-E. The identical pooled standards were analyzed at different injection positions (injection position 1, 2, 25, 26, 49, 50, 73, 74, 97, 98) across the 4 batches, which allowed monitoring of inter-batch differences in signal response and retention time. Notably, the sensitivity for the both N- and C-terminal tryptic Tau peptides (light and heavy) decreased proportionally to run order. This drift in intensity is typically caused by reduced LC or MS performance over time 40. However, the retention times of the N-term peptide RSGYSSPGSPGTPGS (Mean. 25.87, ±1.38/1.58 min), and C-term peptide SGYSSPGSPGTPGSR (Mean. 26.04, ±1.37/1.54 min) across all standards was highly stable (Fig. S5A, B). The peak width of heavy/light peptides pair in both Tryp-N and Tryp-C digestions were also nearly identical (Fig. S5C). Importantly, the light/heavy ratio did not significantly differ for the pooled standard samples analyzed at different injection positions (P > 0.05) across the batches (Fig. S4C). Thus, the loss of signal sensitivity over time is likely due to the buildup of contaminating species (e.g., lipids) within the ion transfer tube. Because the both N- and Cterminal CompTryp Tau standards are equivalent in all samples, they adjust for the signal drift


Page 12 of 31

Page 13 of 31 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


across batches (Fig. S4C), allowing for the relative and absolute quantification of endogenous Tau in CSF. The absolute level of Tau quantified by Tryp-N or Tryp-C approaches is shown in Table 1 for AD patients (avg. 8.11 ng/ml, 10.02 ng/ml respectively), normal controls (avg. 3.49 ng/ml, 3.68 ng/ml respectively), and other control (i.e., non-AD neurodegenerative disease) patients (avg. 2.41 ng/ml, 2.40 ng/ml respectively) (see Table 1 and Figure 5A, B). The absolute level of Tau quantified by an average of Tryp-N or Tryp-C approaches is shown in Fig. 5C. As expected, the absolute levels of endogenous Tau measured by Tryp-C and Tryp-N were extremely well correlated (R2 = 0.962) across all 88 samples, highlighting the high reproducibility of the complementary digestion approach (Fig. 5D). However, the difference in absolute levels quantified by Tryp-N and Tryp-C from AD CSF samples were larger than those from normal or other neurodegenerative disease samples groups. For example, the average absolute difference for AD group samples was 2.05 ng/ml, with an average percentage of difference 17.2%. In contrast, the average absolute difference in controls and other neurodegenerative disease CSF samples was 0.44 ng/ml (11.4% difference) and 0.35 ng/ml (14.7% difference), respectively. No significant difference was observed across Tryp-N and Tryp-C across the various outgroups (Figure 5E). These differences could be due to a technical limitation of the single isotope dilution approach or alternatively reflect sample-specific biological differences that may influence enzymatic cleavage at either the N- or C-terminal tryptic site. To address this question, we measured the absolute levels of purified recombinant full-length Tau (expressed in E. Coli) using the CompTryp approach and found that the absolute measurements of Tryp-N and Tryp-C Tau strongly agreed with each other (0.175 ±0.09 μg and 0.183 ±0.01 μg, respectively; average percentage of difference 5.2%, not significant) (Figure 5F). Furthermore, the CV was 5.4% and 5.1% for Tryp-N and Tryp-C in this assay, respectively (n=6 replicates). Therefore, it is likely that patient-specific differences in Tau, such as post-translational modifications (PTMs), could underlie the difference in absolute levels of Tau measured by Tryp-N and Tryp-C in CSF.

Combining Tryp-N and Tryp-C Tau measurements reduces the coefficient of variation and improves correlation with Tau immunoassays Another advantage of the CompTryp quantification approach is that both N- and Cterminal Tau peptides generated from the CompTryp standard can be considered unique 13 ACS Paragon Plus Environment


independent measurements. To this end, the coefficient of variation (CV) determined from the 10 pooled standards for the Tryp-C generated peptide was 11%, whereas the CV for the Tryp-N generated peptide was slightly lower at 8%. Averaging the CV for Tau measurement from both Tryp-N and Tryp-C Tau peptides reduced the CV to ~7% (Fig. 6). The slightly higher CV for Tryp-C compared to Tryp-N is largely explained by a co-eluting ion in the Tryp-C MS/MS isolation window, which interferes with the quantification (Fig. S6). A subset of samples (n=53) also had total tau levels quantified using a traditional plate ELISA

41

(Fig. 6A). Absolute levels of endogenous Tau quantified by either Try-N or Tryp-C

peptide showed excellent correlation with ELISA (Tryp-N, R2 = 0.959; Tryp-C, R2 = 0.962) (Fig. 6B, C), and, as expected, averaging both Tryp-N and Tryp-C measurements improved correlation (R2 = 0.970) (Fig. 6D). The strong correlation between Tau ELISA and PRM measurements likely reflects the specific Tau epitopes for the capture and detection antibodies used for immunoassays, which are adjacent (AT120 and HT7) or directly overlap (BT2) with the CompTryp Tau peptides (residues 194-208) generated 42, 43. Collectively, these results highlight the utility of the replicate CompTryp peptide MS measurements, which reduce the variance compared with any single enzyme digestion alone.

Discussion Here we provide a new method for generating isotopically labeled peptide standards that includes a parallel enzymatic digestion approach utilizing two separate enzymes (Tryp-C and Tryp-N) to produce internal replicates for detection and quantification of protein biomarkers in tissue and biofluids. Following digestion, both tryptic peptides were effective at quantifying Tau in CSF within the same sample. Furthermore, each peptide showed a significant difference in Tau levels between AD cases and healthy controls. Of note, these standards allow the assessment of proteolytic digestion efficiency, and each had high sensitivity and broad linear range of detection in CSF. Averaging Tryp-N and Tryp-C measurements as technical replicates also improved the CV of CSF Tau measured to ~7%, which is much lower than the CVs (~20%) reported for Tau ELISA measurements 44. A key strength of this approach is that the Tau peptide standards can be directly detected in CSF samples without pre-fractionation or immunodepletion of high abundant proteins such as albumin. Interestingly, the level of Tau measured by the CompTryp method was substantially 14 ACS Paragon Plus Environment

Page 14 of 31

Page 15 of 31 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


higher (~10 fold) than that obtained with immunoassays, which is consistent with previous studies 45, 46

and suggests that ELISAs may underestimate the total amount of Tau present in CSF

45

.

Notably, ELISA is an indirect assay that utilizes antibodies and purified standards to quantify the amount of protein in a complex mixture compared to the MS-based quantitation of a known amount of peptide (CompTryp), which may account for these observed differences. It is also unclear which Tau proteoforms are most abundant in CSF, especially since there are a number of alternatively spliced, proteolytically cleaved and post-translationally modified species of Tau 47, 48. Although we selected for these studies a CompTryp Tau standard in the same region of the protein targeted by current CSF ELISA assays, in principle, any portion of the protein with appropriate flanking Arg or Lys residues could be targeted and quantified 46, 49. Since Tau is known to play a central role in multiple neurodegenerative diseases, it is possible that a more thorough identification of tau peptide fragments and modifications will reveal novel and selective biomarkers for other tauopathies. PTMs on Tau likely differ between AD and other neurodegenerative disease, which could influence the absolute levels of tau protein in CSF. Despite the advantages of using CompTryp to generate overlapping peptide measurements in a single MS assay, a clear limitation of the method is increased mass spectral complexity (i.e., the number of ion species within a chromatographic time window) that is introduced following pooling of peptides from two distinct digests. Although this did not affect measurements for Tau in our study, the Tryp-C Tau peptide did have another co-eluting ion contaminant that increased the CV compared to Tryp-N Tau quantification (Fig. S6). Co-eluting ions are a larger limitation in PRM compared to SRM, where SRM assays on triple-quadrupole mass spectrometers are better suited to filter and minimize co-eluting contaminating ions

50

. Nonetheless, Tryp-N ions may

rescue quantitation of a target peptide when the corresponding Tryp-C ion is subject to significant interference. Another limitation is the robustness of Tryp-N itself. Although this enzyme has the desired specificity and comparable digestion efficiency for the Tau CompTryp standard, we noted that the rate of digestion of Tryp-N is slower than Tryp-C, even though no difference in product peptide decay was observed up to 24 h (Fig. S2). Thus, it will be prudent to allow ample time— preferably overnight—to ensure complete digestion of the protein and standards when using TrypN. Since each CSF sample is digested in parallel, twice the sample volume is needed to complete the assay. However, the volume required for digestion (20 μl) is consistent with the per-sample volumes required for ELISA. 15 ACS Paragon Plus Environment


Page 16 of 31

In summary, the CompTryp method is an integrated and robust approach for MS-based peptide and protein quantification that generates two independent tryptic peptides for quantification measurements. We show that this approach can be used to robustly measure the Tau protein in CSF. More broadly, we propose that the CompTryp method can be applied to any protein of interest to improve the robustness and reproducibility of targeted MS quantitative protein analysis.

Acknowledgement This study was provided by grants from the Accelerating Medicine Partnership AD (U01AG046161;

U01AG061357),

the

National

Institute

on

Aging

(R21AG054206,

5R01AG053960, RF1AG057470, and RF1AG057471), the NINDS Emory Neuroscience Core (P30NS055077), and the Emory Alzheimer's Disease Research Center (P50AG025688). N.T.S. was supported in part by a Biomarkers Across Neurodegenerative Diseases grant (11060) funded by the Alzheimer's Association (ALZ), Alzheimer's Research UK (ARUK), The Michael J. Fox Foundation for Parkinson's Research (MJFF), and the Weston Brain Institute.

Conflict of interest The authors declare no competing financial interest. A provisional patent related to this work has been filed by Emory University.


Page 17 of 31 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


Legends to Figures: Figure 1. Complementary trypsin digestion method to measure Tau in cerebrospinal fluid. (A) CompTryp standard peptide mapping to residues 195-209 in the proline-rich domain of Tau (MAPT isoform 2N4R) with two heavy labeled arginine residues (red bold, right) as well as flanking residues (underlined) for monitoring protein digestion. When the Tau CompTryp standard is digested with Tryp-C (purple), the C-terminal tryptic peptide will be predominately generated, whereas a separate digestion with Tryp-N (green) will generate the N-terminal tryptic peptide. (B) Typically for targeted quantification (top) an isotope labeled AQUA standard is added after trypsin digestion followed by MS analysis to quantify light and heavy product ions from the peptide. In the CompTryp approach (bottom), the sample is first split and the CompTryp standard added prior to parallel digestion with Tryp-C and Tryp-N. The peptides are then mixed from both digests followed by PRM analysis to quantify light and heavy product ions from both digests. Figure 2. Monitoring proteolytic digestion of Tau using CompTryp standards. The CompTryp Tau standard peptide has two isotopically heavy arginine residues (red) on both the Nand C-terminus and corresponding flanking sequences. Fully Tryp-C (A) and Tryp-N (B) cleaved peptide (#1) as well as any partial tryptic products (#2-5) were detected by mass spectrometry. All peptide products, including the undigested peptide, were monitored by PRM and used to calculate proteolytic efficiency for each enzyme. Tryp-C and Tryp-N were 99.8% and 94.1% efficient, respectively, in digesting the Tau CompTryp standard in buffer. Figure 3. Retention time and product ion discriminate Tryp-C from Tryp-N Tau peptides with the exact same precursor mass. (A) MS/MS spectrum of the Tryp-N generated isotopelabeled peptide RSGYSSPGSPGTPGS (m/z 702.3249, charge +2) or the Tryp-C generated isotope-labeled peptide SGYSSPGSPGTPGSR (m/z 702.3249, charge +2). The top five product ion patterns used for targeted PRM are colored. (B) MS1 and MS/MS chromatograph of a mixture of both Tryp-N and Tryp-C digested heavy isotope-labeled peptide RSGYSSPGSPGTPGS and SGYSSPGSPGTPGSR, respectively. The x-axis reflects peptide retention time (min) and the yaxis indicates the signal intensity. (C) The top five product ions pattern for the heavy standard (H) and endogenous light (L) Tryp-N peptide produced in human CSF (left). The top five product ion patterns for the heavy (H) and endogenous light (L) Try-C peptide produced in human CSF (right). For both Tryp-N and Tryp-C Tau peptides, the ratio dot-product values (rdopt) indicate the similarity of product ion pattern between endogenous peptide and heavy peptide standard. Figure 4. Standard curves for Tryp-N and Tryp-C Tau CompTryp standards. To construct a standard curve, the CompTryp standard was diluted in the range from 10 amol/μl to 800 amol/μl followed by digestion with Tryp-N (A) or Tryp-C (B). Quantification of the light peptide (endogenous) to heavy peptide (standard) ratio shows excellent correlation for both Tryp-N and Tryp-C digestion (R2 = 0.997 and 0.999 respectively). Figure 5. CompTryp standards provide reproducible measurements of Tau in control and AD CSF. (A and B). Absolute quantification of the Tryp-N and Tryp-C Tau standard showed significantly different levels in patients with early stage dementia due to AD (n=37) from other non-AD neurological disease (n=31) and age- and sex- matched normal controls (n=20). The average absolute values are provided Table 1. The y-axis indicates the calculated Tau protein 17 ACS Paragon Plus Environment


concentration (ng/mL) in each sample, which is significantly higher in AD patient CSF compared to control and non-AD patients (ANOVA, ***p 0.05). The results are shown as the mean ± S.D. ***, P < 0.001. n.s, non-significant. Figure 6. Tau levels by CompTryp PRM and ELISA (A). The absolute levels of Tau quantified by ELISA and CompTryp methods for 53 CSF samples across AD (n=27), normal (n=4) and other non-AD neurodegenerative controls (n=22). The absolute levels are shown as the mean ± standard deviation. (B and C) Correlation between Tau measured by ELISA and Tau quantified by Tryp-N or Tryp-C. (D) Correlation between Tau level measured by ELISA and Tau level measured by the average of both Tryp-N and Tryp-C peptides in the same sample.


Page 18 of 31

Page 19 of 31 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


Table 1: Clinical characteristics and absolute Tau CSF levels by PRM#

AGE (±SD) %Female %Caucasian Tryp-N(ng/ml) (±SD) Tryp-C(ng/ml) (±SD)

Normal (n=20) 70.0±7.8 57% 86% 3.49(±1.29) 3.68(±1.68)

AD (n=37) 72.7±8.9 60% 70% 8.11(±4.99) 10.02(±7.02)

#Age and Tau absolute levels are shown as mean ± Standard Devia�on (SD).


Non-AD (n=31) 72.8±9.1 35% 81% 2.41(±1.15) 2.40(+1.51)


Figure 1

A MAPT (2N4R) N1 N2

AQUA Proline-rich

R1 R2 R3 R4

1

CompTryp Heavy Standard Peptide

441

(6C13, 4N15)

…….. SGDRSGYSSPGSPGTPGSRSRT …….. TrypsinC TrypsinN

SGYSSPGSPGTPGSR (195-209, 1392.63Da) RSGYSSPGSPGTPGS (194-208, 1392.63Da)

SGDRSGYSSPGSPGTPGSRSRT TrypsinC TrypsinN

SGYSSPGSPGTPGSR (195-209, 1402.64Da) RSGYSSPGSPGTPGS (194-208, 1402.64Da)

B Traditional Approach Parallel reaction monitoring (PRM)

AQUA pep t ide

Tryp-C

C18 Desalt

Heavy

Intensity

Endogenous

Retention time

CompTryp Approach AQUA peptide

PRM

Tryp-C

Tryp-N

Mix

C18 Desalt Heavy Intensity

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

Page 20 of 31

Endogenous Retention time


Page 21 of 31

Figure 2

A Un-digest

SGDRSGYS SPG SP G TPG S R SR T

Tryp-C Digestion 100%

#1 .

SGDRSGYS SPG SP G TPG S R

#2 .

SGDRSGYS SPG SP G TPG S R

#3 .

SGDRSGYS SPG SP G TPG S R SR

40%

#4 .


20%

#5 .

SGDRSGYS SPG SP G TPG S R SR

99.8%

80% 60%

0

#1

B Un-digest


#2

#3

#4

#5

Un-digest

Tryp-N Digestion 100%

#1 .

SGDRSGYS SPG SP G TPG S

80%

#2 .

SGDRSGYS SPG SP G TPG S

60%

#3 .

SGDRSGYS SPG SP G TPG S R S

40%

#4 .


20%

#5 .

SGDRSGYS SPG SP G TPG S R S

0

94.1%

#1


#2

#3

#4

#5

Un-digest

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



Figure 3

A Tryp-N

260.12 y3

80

b12 1144.53

60

b9 889.40

40 20 0

155.08 y1 y2

y6 a9 b6 515.25 861.41 648.30 497.24 343.16 702.34y9 620.30 y4 b8y10 b2 b4 b5 b13++

200

RSGYSSPGSP G TPG S R

y10 922.46

100

Relative Abundance

Relative Abundance

100

Tryp-C

RSGYSSPGSP G TPG S

400

600

800

a12 1116.53 b11 1043.47

1000

b13 1219.54

1200

1400

80 60

y7 681.35

136.08 40 20 0

y4 b3 426.23 663.34 y10++ 308.12 y11++ y6 242.11 505.25 b4 b7 b2 y1 b5 y5 y2 y3 200

400

C 3× 10 06

Intensity

TrypN

TrypC

TrypN

6× 10 05

4× 10 05

2× 10 06

TrypC 2× 10 05

1× 10 06

0

26.0

26.5

y131332.67

b11 800

1000

1200

1400

26.0

26.5

min

0


TrypC

TrypN

MS/MS Chromatograph Precursor m/z 702.32

y11 y12 1009.49 1096.53

825.41

m/z

B MS1 Chromatograph

y8 768.39 y9

600

m/z

100%

Intensity

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

Page 22 of 31

rdotp 0.99

a12

100%

rdotp 0.98

b3

b12

y4

b9

y7

y3

y10 y11

y6 50%

50%

0.0

0.0

Page 23 of 31

Figure 4 A

B

Tryp-N digested CSF Tau

Tryp-C digested CSF Tau 2

2.50

LLOQ

LLOQ

1.25 0

20

0

2.2(50)

4.5(100)

15 10 5

R2=0.999

0 9.1(200) 18.3(400) 27.5(600) 36.7(800)

0

20

0

2.2(50)

4.5(100)

15 10 5 0

0

1

25 Heavy/Light Ratio

25 Heavy/Light Ratio

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


R2=0.997 0

9.1(200) 18.3(400) 27.5(600) 36.7(800) Heavy peptide ( ng/ml, (amol/μl))

Heavy peptide ( ng/ml, (amol/μl))



Figure 5 B

10

0

30

20

10

10

Tryp-N vs. Tryp-C CSF Tau

Other

10

10

20

Endogenous (ng/ml) TrypN

30

Recombinant Tau

Tryp -N

30

Normal

20

F

n.s

AD R2=0.962

***

0

E

30

***

20

0

Tryp-N vs. Tryp-C CSF Tau

0

***

0.5

Tryp -C 20

n.s

n.s

10

0.4 Tau441 (μg)

20

0

***

30 Endoge nous (ng/ml)

***

***

Average Tryp-N and Tryp-C CSF Tau

Endoge nous (ng/ml)

30

Tryp-C CSF Tau

Endogenous (ng/ml)

Endogenous (ng/ml) TrypC

C

Tryp-N CSF Tau

Endoge nous (ng/ml)

1 2 3 4 5 A 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 D 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

Page 24 of 31

0.3 0.2 0.1 0.0

0 AD

Normal


Other

n.s

Page 25 of 31

Figure 6 Normal (n=4)

AD (n=27)

Non-AD (n=22) 191.03(±69.26)

391.39(±131.85) 1045(±577.72) 4.33(±1.12)

9.41(±5.07)

2.02(±0.74)

5.02(±1.52)

11.76(±7.14)

1.90(+1.05)

C

D

Tryp-C vs. ELISA

R2=0.962

1.5 1.0

Normal Other

R2=0.970

1.5 1.0 0.5

0.5 0

AD

2.0 ELISA (ng/ml)

ELISA (ng/ml)

2.0

20

Average Tryp-N and Tryp-C vs. ELISA 2.5

2.5

ELISA (ng/ml)

1 2 3 4 5 6A 7 8 9 ELISA, Total-Tau (pg/ml) (±SD) 10 11 CompTryp,Tryp-N(ng/ml) (±SD) 12 CompTryp,Tryp-C(ng/ml) (±SD) 13 14 15 16 17 18 19B Tryp-N vs. ELISA 20 21 2.5 22 R2=0.959 23 2.0 24 1.5 25 26 1.0 27 0.5 28 0 29 0 5 10 15 30 Endogenous (ng/ml) 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


0

10

20

30

Endogenous (ng/ml)


0

0

5

10

15

Endogenous (ng/ml)

20

25


Supporting information The following supporting information is available free of charge at ACS website http://pubs.acs.org

Supplementary Figure S1: Impact of cysteine reduction and alkylation Supplementary Figure S2: Rate of digestion and decay for CompTryp Tau peptide Supplementary Figure S3: Tryp-N or Tryp-C digestion of Tau-441 protein Supplementary Figure S4: Peak area of CompTryp peptides across all samples Supplementary Figure S5: Retention time of CompTryp peptides across all samples Supplementary Figure S6: Co-elution ion with Tryp-C Tau Peptide Supplemental Table S1: Protein concentration of CSF samples Supplemental Table S2: CompTryp Peptide information Supplemental Table S3: Sample information and PRM measurements


Page 26 of 31

Page 27 of 31 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


References 1. Hoofnagle, A. N.; Wener, M. H., The Fundamental Flaws of Immunoassays and Potential Solutions Using Tandem Mass Spectrometry. Journal of immunological methods 2009, 347, (1-2), 3-11. 2. Gillette, M. A.; Carr, S. A., Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nature methods 2013, 10, (1), 28-34. 3. Zhang, G.; Ueberheide, B. M.; Waldemarson, S.; Myung, S.; Molloy, K.; Eriksson, J.; Chait, B. T.; Neubert, T. A.; Fenyö, D., Protein Quantitation Using Mass Spectrometry. Methods in molecular biology (Clifton, N.J.) 2010, 673, 211-222. 4. Peterson, A. C.; Russell, J. D.; Bailey, D. J.; Westphall, M. S.; Coon, J. J., Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol Cell Proteomics 2012, 11, (11), 1475-88. 5. Lange, V.; Picotti, P.; Domon, B.; Aebersold, R., Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol 2008, 4, 222. 6. Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P., Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proceedings of the National Academy of Sciences 2003, 100, (12), 6940-6945. 7. Seyfried, N. T.; Gozal, Y. M.; Dammer, E. B.; Xia, Q.; Duong, D. M.; Cheng, D.; Lah, J. J.; Levey, A. I.; Peng, J., Multiplex SILAC analysis of a cellular TDP-43 proteinopathy model reveals protein inclusions associated with SUMOylation and diverse polyubiquitin chains. Mol Cell Proteomics 2010, 9, (4), 705-18. 8. Seyfried, N. T.; Gozal, Y. M.; Donovan, L. E.; Herskowitz, J. H.; Dammer, E. B.; Xia, Q.; Ku, L.; Chang, J.; Duong, D. M.; Rees, H. D.; Cooper, D. S.; Glass, J. D.; Gearing, M.; Tansey, M. G.; Lah, J. J.; Feng, Y.; Levey, A. I.; Peng, J., Quantitative analysis of the detergent-insoluble brain proteome in frontotemporal lobar degeneration using SILAC internal standards. J Proteome Res 2012, 11, (5), 272138. 9. Ronsein, G. E.; Pamir, N.; von Haller, P. D.; Kim, D. S.; Oda, M. N.; Jarvik, G. P.; Vaisar, T.; Heinecke, J. W., Parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) exhibit comparable linearity, dynamic range and precision for targeted quantitative HDL proteomics. Journal of proteomics 2015, 0, 388-399. 10. Hustoft, H. K.; Malerod, H.; Wilson, S. R.; Reubsaet, L.; Lundanes, E.; Greibrokk, T., A Critical Review of Trypsin Digestion for LC-MS Based Proteomics. Integrative Proteomics 2012, 73-92. 11. Calderon-Celis, F.; Encinar, J. R.; Sanz-Medel, A., Standardization approaches in absolute quantitative proteomics with mass spectrometry. Mass Spectrometry Reviews 2018, 37, (6), 715-737. 12. van den Broek, I.; Smit, N. P.; Romijn, F. P.; van der Laarse, A.; Deelder, A. M.; van der Burgt, Y. E.; Cobbaert, C. M., Evaluation of interspecimen trypsin digestion efficiency prior to multiple reaction monitoring-based absolute protein quantification with native protein calibrators. J Proteome Res 2013, 12, (12), 5760-74. 13. John P. Wilson, J. J. I., Samantha N. Peacock, Keith D. Rivera, Katharine H. Dusenbury, Darryl J.C. Pappin, Tryp-N: a thermostable, N-terminal arginine and lysine specific protease for ≤ 1 hr digestion, simplified peptide fragmentation and increased MS/MS sensitivity. 62nd Annual Conference of American Society for Mass Spectrometry (Baltimore, Maryland) 2014. 14. Boersema, P. J.; Taouatas, N.; Altelaar, A. F.; Gouw, J. W.; Ross, P. L.; Pappin, D. J.; Heck, A. J.; Mohammed, S., Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase. Mol Cell Proteomics 2009, 8, (4), 650-60. 15. Tsiatsiani, L.; Giansanti, P.; Scheltema, R. A.; van den Toorn, H.; Overall, C. M.; Altelaar, A. F.; Heck, A. J., Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X)n and (X)nK/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics. J Proteome Res 2017, 16, (2), 852-861.



16. Huesgen, P. F.; Lange, P. F.; Rogers, L. D.; Solis, N.; Eckhard, U.; Kleifeld, O.; Goulas, T.; Gomis-Ruth, F. X.; Overall, C. M., LysargiNase mirrors trypsin for protein C-terminal and methylationsite identification. Nat Methods 2015, 12, (1), 55-8. 17. Hampel, H.; Frank, R.; Broich, K.; Teipel, S. J.; Katz, R. G.; Hardy, J.; Herholz, K.; Bokde, A. L. W.; Jessen, F.; Hoessler, Y. C.; Sanhai, W. R.; Zetterberg, H.; Woodcock, J.; Blennow, K., Biomarkers for Alzheimer's disease: academic, industry and regulatory perspectives. Nat Rev Drug Discov 2010, 9, (7), 560-574. 18. Rappsilber, J.; Mann, M.; Ishihama, Y., Protocol for micro-purification, enrichment, prefractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2007, 2, (8), 1896-906. 19. Cox, J.; Neuhauser, N.; Michalski, A.; Scheltema, R. A.; Olsen, J. V.; Mann, M., Andromeda: A Peptide Search Engine Integrated into the MaxQuant Environment. Journal of Proteome Research 2011, 10, (4), 1794-1805. 20. Seyfried, N. T.; Dammer, E. B.; Swarup, V.; Nandakumar, D.; Duong, D. M.; Yin, L.; Deng, Q.; Nguyen, T.; Hales, C. M.; Wingo, T.; Glass, J.; Gearing, M.; Thambisetty, M.; Troncoso, J. C.; Geschwind, D. H.; Lah, J. J.; Levey, A. I., A Multi-network Approach Identifies Protein-Specific Coexpression in Asymptomatic and Symptomatic Alzheimer's Disease. Cell systems 2017, 4, (1), 60-72.e4. 21. MacLean, B.; Tomazela, D. M.; Shulman, N.; Chambers, M.; Finney, G. L.; Frewen, B.; Kern, R.; Tabb, D. L.; Liebler, D. C.; MacCoss, M. J., Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 2010, 26, (7), 966-968. 22. Zhang, B.; Gaiteri, C.; Bodea, L. G.; Wang, Z.; McElwee, J.; Podtelezhnikov, A. A.; Zhang, C.; Xie, T.; Tran, L.; Dobrin, R.; Fluder, E.; Clurman, B.; Melquist, S.; Narayanan, M.; Suver, C.; Shah, H.; Mahajan, M.; Gillis, T.; Mysore, J.; MacDonald, M. E.; Lamb, J. R.; Bennett, D. A.; Molony, C.; Stone, D. J.; Gudnason, V.; Myers, A. J.; Schadt, E. E.; Neumann, H.; Zhu, J.; Emilsson, V., Integrated systems approach identifies genetic nodes and networks in late-onset Alzheimer's disease. Cell 2013, 153, (3), 707-20. 23. Olsen, J. V.; Ong, S.-E.; Mann, M., Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues. Molecular & Cellular Proteomics 2004, 3, (6), 608-614. 24. Yang, H.; Li, Y.; Zhao, M.; Wu, F.; Wang, X.; Xiao, W.; wang, Y.; Zhang, J.; Wang, F.; Xu, F.; Zeng, W.-F.; Overall, C. M.; He, S.-M.; Chi, H.; Xu, P., Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for large-scale proteomics. Molecular & Cellular Proteomics 2019, mcp.TIR118.000918. 25. Iqbal, K.; Liu, F.; Gong, C.-X.; Grundke-Iqbal, I., Tau in Alzheimer Disease and Related Tauopathies. Current Alzheimer research 2010, 7, (8), 656-664. 26. Barthelemy, N. R.; Gabelle, A.; Hirtz, C.; Fenaille, F.; Sergeant, N.; Schraen-Maschke, S.; Vialaret, J.; Buee, L.; Junot, C.; Becher, F.; Lehmann, S., Differential Mass Spectrometry Profiles of Tau Protein in the Cerebrospinal Fluid of Patients with Alzheimer's Disease, Progressive Supranuclear Palsy, and Dementia with Lewy Bodies. J Alzheimers Dis 2016, 51, (4), 1033-43. 27. Bros, P.; Vialaret, J.; Barthelemy, N.; Delatour, V.; Gabelle, A.; Lehmann, S.; Hirtz, C., Antibody-free quantification of seven tau peptides in human CSF using targeted mass spectrometry. Front Neurosci 2015, 9, 302. 28. Brun, V.; Dupuis, A.; Adrait, A.; Marcellin, M.; Thomas, D.; Court, M.; Vandenesch, F.; Garin, J., Isotope-labeled protein standards: toward absolute quantitative proteomics. Mol Cell Proteomics 2007, 6, (12), 2139-49. 29. Scott, K. B.; Turko, I. V.; Phinney, K. W., Quantitative Performance of Internal Standard Platforms for Absolute Protein Quantification Using Multiple Reaction Monitoring-Mass Spectrometry. Analytical Chemistry 2015, 87, (8), 4429-4435. 30. Stergachis, A. B.; MacLean, B.; Lee, K.; Stamatoyannopoulos, J. A.; MacCoss, M. J., Rapid empirical discovery of optimal peptides for targeted proteomics. Nat Meth 2011, 8, (12), 1041-1043. 31. Singh, R.; Crow, F. W.; Babic, N.; Lutz, W. H.; Lieske, J. C.; Larson, T. S.; Kumar, R., A liquid chromatography-mass spectrometry method for the quantification of urinary albumin using a novel 15Nisotopically labeled albumin internal standard. Clin Chem 2007, 53, (3), 540-2. 28 ACS Paragon Plus Environment

Page 28 of 31

Page 29 of 31 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


32. Cao, Z.; Mou, R.; Cao, Z.; Lin, X.; Ma, Y.; Zhu, Z.; Chen, M., Quantitation of glutathione Stransferases in rice (Oryza sativa L.) roots exposed to cadmium by liquid chromatography-tandem mass spectrometry using isotope-labeled wing peptides as an internal standard. Plant Methods 2017, 13, 64. 33. Zhang, J.; Lai, S.; Zhang, Y.; Huang, B.; Li, D.; Ren, Y., Multiple reaction monitoring-based determination of bovine alpha-lactalbumin in infant formulas and whey protein concentrates by ultra-high performance liquid chromatography-tandem mass spectrometry using tryptic signature peptides and synthetic peptide standards. Anal Chim Acta 2012, 727, 47-53. 34. Kushnir, M. M.; Rockwood, A. L.; Roberts, W. L.; Abraham, D.; Hoofnagle, A. N.; Meikle, A. W., Measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies. Clin Chem 2013, 59, (6), 982-90. 35. Slechtova, T.; Gilar, M.; Kalikova, K.; Tesarova, E., Insight into Trypsin Miscleavage: Comparison of Kinetic Constants of Problematic Peptide Sequences. Anal Chem 2015, 87, (15), 7636-43. 36. Shuford, C. M.; Sederoff, R. R.; Chiang, V. L.; Muddiman, D. C., Peptide Production and Decay Rates Affect the Quantitative Accuracy of Protein Cleavage Isotope Dilution Mass Spectrometry (PCIDMS). Molecular & Cellular Proteomics 2012, 11, (9), 814-823. 37. Barnidge, D. R.; Hall, G. D.; Stocker, J. L.; Muddiman, D. C., Evaluation of a cleavable stable isotope labeled synthetic peptide for absolute protein quantification using LC-MS/MS. J Proteome Res 2004, 3, (3), 658-61. 38. Arnold, S. L.; Stevison, F.; Isoherranen, N., Impact of Sample Matrix on Accuracy of Peptide Quantification: Assessment of Calibrator and Internal Standard Selection and Method Validation. Analytical Chemistry 2016, 88, (1), 746-753. 39. Yamamori, H.; Khatoon, S.; Grundke-Iqbal, I.; Blennow, K.; Ewers, M.; Hampel, H.; Iqbal, K., Tau in cerebrospinal fluid: a sensitive sandwich enzyme-linked immunosorbent assay using tyramide signal amplification. Neurosci Lett 2007, 418, (2), 186-9. 40. Bourmaud, A.; Gallien, S.; Domon, B., A quality control of proteomic experiments based on multiple isotopologous internal standards. EuPA open proteomics 2015, 8, 16-21. 41. Andreasen, N.; Minthon, L.; Davidsson, P.; Vanmechelen, E.; Vanderstichele, H.; Winblad, B.; Blennow, K., Evaluation of CSF-tau and CSF-Abeta42 as diagnostic markers for Alzheimer disease in clinical practice. Arch Neurol 2001, 58, (3), 373-9. 42. Meredith, J. E., Jr.; Sankaranarayanan, S.; Guss, V.; Lanzetti, A. J.; Berisha, F.; Neely, R. J.; Slemmon, J. R.; Portelius, E.; Zetterberg, H.; Blennow, K.; Soares, H.; Ahlijanian, M.; Albright, C. F., Characterization of novel CSF Tau and ptau biomarkers for Alzheimer's disease. PloS one 2013, 8, (10), e76523-e76523. 43. Vandermeeren, M.; Mercken, M.; Vanmechelen, E.; Six, J.; Voorde, A.; Martin, J.-J.; Cras, P., Detection of Proteins in Normal and Alzheimer's Disease Cerebrospinal Fluid with a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay. Journal of Neurochemistry 1993, 61, (5), 1828-1834. 44. Mattsson, N.; Andreasson, U.; Persson, S.; Carrillo, M. C.; Collins, S.; Chalbot, S.; Cutler, N.; Dufour-Rainfray, D.; Fagan, A. M.; Heegaard, N. H. H.; Robin Hsiung, G.-Y.; Hyman, B.; Iqbal, K.; Kaeser, S. A.; Lachno, D. R.; Lleó, A.; Lewczuk, P.; Molinuevo, J. L.; Parchi, P.; Regeniter, A.; Rissman, R. A.; Rosenmann, H.; Sancesario, G.; Schröder, J.; Shaw, L. M.; Teunissen, C. E.; Trojanowski, J. Q.; Vanderstichele, H.; Vandijck, M.; Verbeek, M. M.; Zetterberg, H.; Blennow, K.; Alzheimer's Association, Q. C. P. W. G., CSF biomarker variability in the Alzheimer's Association quality control program. Alzheimer's & dementia : the journal of the Alzheimer's Association 2013, 9, (3), 251-261. 45. Pottiez, G.; Yang, L.; Stewart, T.; Song, N.; Aro, P.; Galasko, D. R.; Quinn, J. F.; Peskind, E. R.; Shi, M.; Zhang, J., Mass-Spectrometry-Based Method To Quantify in Parallel Tau and Amyloid beta 1-42 in CSF for the Diagnosis of Alzheimer's Disease. J Proteome Res 2017, 16, (3), 1228-1238. 46. Barthelemy, N. R.; Fenaille, F.; Hirtz, C.; Sergeant, N.; Schraen-Maschke, S.; Vialaret, J.; Buee, L.; Gabelle, A.; Junot, C.; Lehmann, S.; Becher, F., Tau Protein Quantification in Human Cerebrospinal Fluid by Targeted Mass Spectrometry at High Sequence Coverage Provides Insights into Its Primary Structure Heterogeneity. J Proteome Res 2016, 15, (2), 667-76. 29 ACS Paragon Plus Environment


47. Caillet-Boudin, M. L.; Buee, L.; Sergeant, N.; Lefebvre, B., Regulation of human MAPT gene expression. Mol Neurodegener 2015, 10, 28. 48. Meredith, J. E., Jr.; Sankaranarayanan, S.; Guss, V.; Lanzetti, A. J.; Berisha, F.; Neely, R. J.; Slemmon, J. R.; Portelius, E.; Zetterberg, H.; Blennow, K.; Soares, H.; Ahlijanian, M.; Albright, C. F., Characterization of novel CSF Tau and ptau biomarkers for Alzheimer's disease. PLoS One 2013, 8, (10), e76523. 49. Sato, C.; Barthélemy, N. R.; Mawuenyega, K. G.; Patterson, B. W.; Gordon, B. A.; JockelBalsarotti, J.; Sullivan, M.; Crisp, M. J.; Kasten, T.; Kirmess, K. M.; Kanaan, N. M.; Yarasheski, K. E.; Baker-Nigh, A.; Benzinger, T. L. S.; Miller, T. M.; Karch, C. M.; Bateman, R. J., Tau Kinetics in Neurons and the Human Central Nervous System. Neuron 2018, 97, (6), 1284-1298.e7. 50. Rauniyar, N., Parallel Reaction Monitoring: A Targeted Experiment Performed Using High Resolution and High Mass Accuracy Mass Spectrometry. International Journal of Molecular Sciences 2015, 16, (12), 28566-28581.


Page 30 of 31

Page 31 of 31

TOC Figure Tau Protein N1 N2

Proline-rich

CompTryp Heavy Standard Peptide

R1 R2 R3 R4

(6C13, 4N15)

SGDRSGYSSPGSPGTPGSRSRT

…….. SGDRSGYSSPGSPGTPGSRSRT …….. TrypsinC TrypsinN

AQUA pep tide

CSF

SGYSSPGSPGTPGSR RSGYSSPGSPGTPGS

Tryp-C Tryp-N

TrypsinC TrypsinN

SGYSSPGSPGTPGSR RSGYSSPGSPGTPGS

PRM MS

Heavy Intensity

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


Endogenous Retention time

ACS Paragon Plus Environment

Mass Spectrometry-Based Quantification of Tau in Human

Recommend Documents