IFCC Project Update on New Projects Henrik Zetterberg, MD, PhD Professor of Neurochemistry University of Gothenburg and University College London Ingrid Zegers, PhD Joint Research Centre, the European Commission's science and knowledge service

New projects Candidate reference method for A40 Candidate reference method for tau Pilot commutability study for tau

AMYLOID  1-40 CANDIDATE RMP Josef Pannee 3

Stable isotope internal standards Institute of Neuroscience and Physiology, University of Gothenburg Stable isotope internal standards Endogenous Aβ1-40 Regular (14N) Molecular mass = 4328 “heavy” Aβ1-40 15N (instead of 14N) Molecular mass = 4380 (expressed in E. coli with 15N as nitrogen source) m/z 4

Solid Phase Extraction Institute of Neuroscience and Physiology, University of Gothenburg 180 µL CSF Internal standard GdnHCl H3PO4 Solid Phase Extraction Reversed Phase Liquid Chromatography Dry & resuspend eluate MS/MS 5

Sample preparation SPE (Waters Oasis MCX µElution 96-well plates) 180 µL CSF Add isotope labeled internal standard (20 µL) 200 µL 5 M guanidine hydrochloride 200 µL 4% phosphoric acid SPE 6

LC-MS/MS MS: Thermo Scientific Q Exactive LC column: ProSwift RP-4H 1.0 mm x 250 mm LC flow: 0.3 mL/min Run-time: 15 min (including re-equilibration) Measurement range target: 1 500 – 40 000 pg/mL 7

Institute of Neuroscience and Physiology, University of Gothenburg Linearity 8

Institute of Neuroscience and Physiology, University of Gothenburg Measurement range Relative errors < 15% in the whole measurement range (1 500-40 000 pg/mL) with a goodness-of-fit of R2 >0.99 9

Precision Sample Average concentration (pg/mL) sr (pg/mL) CVr (%) Institute of Neuroscience and Physiology, University of Gothenburg Precision Sample Average concentration (pg/mL) sr (pg/mL) CVr (%) sRW (pg/mL) CVRw (%) HIGH 4197 172 4.1 252 6.0 LOW 2738 93.5 3.4 166 6.1 CV, coefficient of variation. s, standard deviation. r, repeatability. Rw, intermediate precision. 10

Institute of Neuroscience and Physiology, University of Gothenburg Sample stability A sample can go through up to five freeze/thaw cycles with no effect on the measured concentration of the analyte. Storage in -80ºC is preferred, storage in -20ºC is acceptable. 11

The method has just been submitted for evaluation to the Joint Committee for Traceability in Laboratory Medicine (JCTLM)

Candidate reference method for tau Josef Pannee

tau-441 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHV PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L Conserved sequence (352, 381, 410, 383, 414 & 441) No S, T or M (phosphorylation, glycosylation & oxidation) Since the entire protein is to big to be measured using MS, a tryptic peptide is selected. Criteria: Short, should fly well in the ion soruce. It has to have a conserved sequence common for tau 352, 381, 410, 383, 414 & 441. Preferably without Serine, Threonine and Methionine since they can be modified.

Sample preparation SPE (HLB) of 340 µL CSF Add isotope labeled tau to CSF Partial protein precipitation (2.5% perchloric acid) of 340 µL CSF Centrifuge & transfer supernatant to new tube Acidify sample (0.1 % TFA) SPE Dry eluate Digest with trypsin over night

LC-MS/MS MS: Q Exactive Column: C18, 3.6µ, 2.1x100 mm Flow: 0.3 mL/min Run-time: 13 min Precursor ion isolation: GAAPPGQK [2+] Product ions: several fragments for quantification selected post acquisition. Currently 3. Measurement range (target): 100-2500 pg/mL

Endogenous t-tau in CSF Very good signal to start with (e.g. compared to starting developing the Ab42 method).

Endogenous tau in 340 µL CSF 14 replicates from a CSF pool to study precision. 7 were prepared as usual (Digested), 7 were without trypsin (Undigested) to see that it actually is digested tau we are seeing (i.e. and not a contaminant or an endogenous peptide).

Native tau in CSF 340 µL CSF CV% = 4.6 n = 7 t-tau ≈ 650 pg/mL The 7 replicates from a CSF pool.

Calibration Recombinant tau 150 – 2500 pg/mL Isotope labeled as IS Recombinant full length tau-441 was used to construct a calibration curve. Isotope labeled tau was used as internal standard (IS).

Three levels of t-tau Endogenous tau A rough test to see if the signal in the MS increases with increasing tau concentration as measured by ELISA. Again to see that we are measuring the right analyte. Good signal at low tau!

Next steps Decide if full length proteins or surrogate analytes should be used for calibration improve recovery full length protein standard OR determine actual concentration of aliquots using amino acid analysis. Skip protein precipitation (2.5% perchloric acid) for SPE Use guanidine instead? Validation

Pilot commutability study for tau The 3 candidate CRMs for A42 were assayed for T-tau 34 individual CSF samples were also included Samples were analyzed by the respective ”originating lab” ULF ANDREASSON

Pilot commutability study for tau

Conclusions The CSF A42 project almost completed Promising data on candidate reference methods for A40 and tau Promising commutability data on human CSF-based tau reference materials (spiked artificial CSF may work, would be much easier and should be evaluated in a formal commutability study of different candidate reference materials)