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Biomarker Assays for Alzheimer’s Disease

- An update for the GBSC Workgroup

Malcolm Ward , Emma Schofield, Claire Russell, Christopher Lößner,

Sasa Koncarevic, Stephan Jung, Hans Dieter Zucht and Ian Pike

13th July 2013, Boston, MA.

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Biomarkers for AD

Ideally, we require the ability to measure biochemical markers

within peripheral biofluids to:

• Assist early diagnosis and stratify patients for PET imaging

• Discriminate between MCI convertors and non convertors

• Predict fast/slow rate of decline for individual patients

• Correlate with response to experimental medicines during clinical trials

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Novel Assays for AD biomarkers

We have developed several new mass spectrometrybased multiplexed assays to measure key analytes associated with Alzheimer‘s disease.

Abeta isoforms

CSF16plex

Total CSF Tau

TMTcalibrator IV

(Hybrid)

NN

NH

O

O

O

O

O

Mass reporter Massnormalisationgroup

Cleavablelinker

Reactivegroup

NN

NH

O

O

O

O

O

Mass reporter Massnormalisationgroup

Cleavablelinker

Reactivegroup

Thompson et. al

Anal. Chem. 2003, 75, 1895-1904

TMT is covered by granted and pending patents in Eu rope, USA and JapanTMT is licensed to and distributed by Pierce Biotec hnology a Thermo Fisher Scientific Company

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CSF Sample Cohort

• CSF samples have kindly been provided by Dr. Henrik Zetterburg, Mölndal (Sweden)

• 31 non AD but memory impaired and 31 AD CSF samples

• Following metadata has been provided– Diagnosis, gender, age, and concentration of tau, beta-amyloid

and phospho-tau

• Additional Metadata such as vial type and protein concentration after Bradford assay has also been considered

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ABeta Isoforms Assay

CSF samples were processed according to the Abeta-I P-MALDI process (adapted from Portelius et al with slight modifications)

200 µl of CSF spiked with 10 ng/ml heavy 1-40 and used for IP MALDI preparation in triplicate

Referencing of endogenous peptides to spike enables comparison of spectra in each batch of Immunoppts.

Aβ-IP-MALDI workflow. Enrichment from CSF andsimultaneous MS-detection of different A β-peptidesand heavy internal standard A β 1-40.

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1-42 1-40 1-39 1-38 1-37 1-34 1-33 1-19 1-17

ratio of ratios [AD/non AD]

ratio of ratios [AD/ctrl]Up in AD

Mean AD/non AD ratios of 4 IP -batches

A mean ratio of [AD/non AD] and each endogenous pep tide was calculated for each IP batch and the mean of these 4 ratios is displayed above for e ach of the peptides (error bars display the variation of the relative ratio between the 4 IP-ba tches.

• A clear upregulation of a factor up to ~2 is seen f or peptides 1-40, 1-39, 1-38 and 1-37 in ADs.

• An upregulation trend is less pronounced but visibl e in 1-34, 1-33 and 1-19 in ADs.

• These IP-MALDI data do not demonstrate differences in 1-17 and 1-42 between AD cases and non AD subjects (1-17 has high variations, 1-42 is a very weak signal, close to background)

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PCA Scores/Loadings Plots

Measurement of A β-peptides inthe IP-MALDI assay enables aclear separation between ADcases and non AD samples.

The PCA Scores Plot shownrelates to single IP batch (8 vs 8)to illustrate typical outcome.

Overall the separation is mainlydriven by higher levels of A β1-37,Aβ1-38, Aβ1-39 and Aβ1-40isoforms in AD cases as indicatedin the Loadings Plot.

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p[2]

p[1]

Simca_L2Ratios_bySasa_collapsed_korrigiert.M1 (PCA-X)p[Comp. 1]/p[Comp. 2]

R2X[1] = 0,862467 R2X[2] = 0,0885625

1_17

1_19

1_331_34

1_371_381_39

1_40

1_42

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A subset of A β-peptides deliver excellent p-values for thediagnosis of 31 vs 31 AD cases and non AD subjects.

Statistics: Linear Mixed Effect Modelling

IP Batch 1

Diagnosis AD/non AD

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TMT-SRM CSF 16plex Assay

Content: 16 proteins represented by 31 peptides with 236 transitions

– Amyloid-like protein 1 – Amyloid beta A4 protein– Beta-2-microglobulin – Complement C3 alpha and beta– Chromogranin A – Complement factor H – Cystatin C – Serum amyloid P-component– Clusterin alpha chain– Clusterin beta chain– Apolipoprotein E – Alpha-2-macroglobulin – Secretogranin-2 – Gelsolin– Fibrinogen gamma chain

Isotopic TMTduplex workflow• TMTzero: Individual samples (50 µL)• TMT6-127: Universal reference • (Seralab #CSF-123-S-26975) �• Volumetric 1:1 mixture• QC samples: Universal reference

– TMTzero and TMT6-127 1:1

• 1h nLC gradient• TSQ Vantage• Triplicate analysis• 2 µL CSF o/c• PinPoint 1.1 including manual editing

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CV

[%]

Assay Precision - All CV’s below 15% except three pe ptides

Decreased performance of peptides GLEVTITAR, ALQEYR and AVEVLPK due to reduced quality of SRM transitio ns

TMT-SRM CSF 16plex Assay

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Statistical Analysis

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p[2]

p[1]

Proteine_SimcaStyleb.M1 (PCA-X)p[Comp. 1]/p[Comp. 2]

R2X[1] = 0,741894 R2X[2] = 0,140655

Amyloid-like protein 1

Amyloid beta A4 protein

B2M Beta-2-microglobulin

Complement C3 alphaComplement C3 beta

Chromogranin A

CFH

Cystatin C

Serum amyloid P-component

Clusterin alpha

Clusterin beta

APOE Apolipoprotein E

A2M Alpha-2-macroglobulin

Secretogranin-2

Gelsolin

Fibrinogen gamma chain

� For this cohort, the 16plex CSF assay gives remarka ble separation of AD cases and non AD subjects with most analytes havi ng a strong influence on the differentiation of the clinical gr oups

PCA Scores Plot

PCA Loadings Plot

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t[2]

t[1]

Proteine_SimcaStyleb.M1 (PCA-X)t[Comp. 1]/t[Comp. 2]

Colored according to Obs ID (Sample_short)

R2X[1] = 0,741894 R2X[2] = 0,140655 Ellipse: Hotelling T2 (0,95)

ADControl

AD 1

AD 2

AD 3 AD 4

AD 5

AD 6

AD 7

AD 8

AD 9

AD 10AD 11

AD 12

AD 13

AD 14

AD 15

AD 16

AD 17

AD 18

AD 19

AD 20

AD 21

AD 22 AD 23AD 24

AD 25

AD 26

AD 27

AD 28

AD 29AD 30

AD 31

Control 1

Control 2

Control 3

Control 4

Control 5

Control 6Control 7

Control 8

Control 9

Control 10

Control 11

Control 12Control 13Control 14

Control 15

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Control 19

Control 20Control 21

Control 22

Control 23Control 24

Control 25Control 26

Control 27

Control 28

Control 29

Control 30

Control 31

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TMTcalibrator CSF-Tau

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TMTcalibrator for CSF Tau

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TMTcalibrator affords more scope

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TPPSSGEPPK

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SGYSSPGSPGTPGSR

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SPVVSGDTSPR

Several Tau peptides as surrogates for Total Tau measurement each with afour-point internal calibration curve

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Illustration of TMTcalibrator results

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Our hunt for Tau phospho-peptides in CSF continues…

TMTcalibrator for CSF pTau

• Further exploration of Tau/pTau enrichment prior to MS– Immuno-ppt and TiO2

• Create optimal TMTcalibrator standard– Post mortem AD brain or RecTau+Kinase(s) + ATP

• Use of additional TMT related fragment ions for quantitation purposes (TMTC ions Wühr & Gygi 2012)

• We are still looking for additional partners to help– Access to CSF cohorts– Advice on tau fragments for inclusion– Funding

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TMTcalibrator IV

A hybrid targeted and non targeted screening approa ch

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TMTcalibrator IV

Input A: Samples (n=90)

Input B: Include List Content

Output: New Biomarker Panels

2089 distinct peptides correspondingto 199 identified protein groups

Reference for GMDH: Alexey G. Ivakhnenko 1968

Group modeling of data handling (GMDH)

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To Conclude

• Use of MS combined with TMT facilitates rapid and inexpensive set up and assay validation

• Multiple analytes are measured with high quantitative precision and accuracy

• Several AD Biomarker Assays are “Ready to Use”

• Further details on our posters at AAIC tomorrow

# P1-141: Quantitative mass spectrometry assays for Amyloid beta, tau and phospho-tau in CSF

# P1-233: Further exploration of Plasma Biomarkers for Alzheimer’s disease using isotopic Tandem Mass Tags and a combined targeted /non-targe ted LC/MS/MS method