1. 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

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

3. AMYLOID  1-40 CANDIDATE RMP
Josef Pannee 3

4. 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” Aβ N (instead of 14N) Molecular mass = 4380
(expressed in E. coli with 15N as nitrogen source)
m/z 4

5. 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

6. 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

7. 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: – pg/mL 7

8. Institute of Neuroscience and Physiology, University of Gothenburg
Linearity 8

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

10. 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

11. 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

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

13. Candidate reference method for tau
Josef Pannee

14. 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.

15. 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

16. 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): pg/mL

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

18. 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).

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

20. 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).

21. 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!

22. 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

23. 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

24. Pilot commutability study for tau

28. 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)