Validating ‘PZP’ as a biomarker for preclinicalAlzheimer’s disease

Using a targeted proteomics approach

Diana A.T. Nijholt, PhD

ErasmusMC, Rotterdam, The Netherlands

Skyline User Meeting, San Antonio, Texas

June 5th 2016

Biomarker Development Center

Biomarker research faces several challenges

Disease X

Many potentialbiomarkers

Limited validation in large cohorts

Very few have made it to the clinic

Number of biomarkers

Discovery Clinical validation/

confirmation

Assay development

and application

Bridging the gaps

Aims- Validate previously discovered biomarker candidates in large(r) population cohorts

- Develop assays that can be used in a clinical setting

DiseaseX Y Z

Alzheimer’s diseaseA neurodegenerative disorder with a long preclinical phase

Rel

ativ

e oc

cure

nce

Disease duration (years)

0 5 10 15 20 25 30

Clinical diagnosis

Preclinical phase Clinical phase

Cognitive impairment

Aβ deposits

Neurofibrillary tangles

Ideal window for therapy

No biomarkers available in the clinic

PZP was previously identified as a potential serum biomarker for preclinical Alzheimer’s disease

Pregnancy Zone Protein (PZP/CPAMD6)

Pan-protease inhibitor

73% sequence homology with α2-macroglobulin (α2MG)

Increased abundance in the AD brain

Located to AD amyloid lesions

Preclinical AD - female

Non-demented controls -female

75% sensitivity75% specificity

Nijholt et. al., Journal of Alzheimer’s disease, 2015 Ijsselstijn et. al., Journal of Proteome Research, 2011

PZP/ microglia/ Amyloid β

Targeted quantification of PZP in serum using SRM

Selection of PZP peptides to act as internal reference controls

15 unique PZP peptides observed in a shotgun proteomics approach

ISEITNIVSKAVGYLITGYQR

Preclinical ADControls

Designing and optimizing an SRM assay for targeted PZP quantification in serum

Synthesis of selected peptides with stable isotope label (SIL) at R or K• ≥98% pure

• Internal standard

Method optimization

• Skyline

• Xevo TQ-S Triple Quadrupole

• nanoACQUITY UPLC

• V/M Trap C18 column, 180μm x 20mm

• 1.7μm BEH300 C18 analytical column, 75μm

Protein Peptide sequence

PZP ISEITNIVSK

AVGYLITGYQR

α2MG AIGYLITGYQR

Designing and optimizing an SRM assay for targeted PZP quantification in serum

3. Method optimization

• Transition selection

PZP peptides y6 y7 y8Collision energy

ISEITNIVSK (m/z 552.33 Da, +2) 661.39 774.47 903.52 15VISEITNIVSK (SIL) (m/z 556.33 Da, +2) 669.40 782.49 911.53 15V

y4 y6 y7AVGYLITGYQR (m/z 620.84 Da, +2) 523.26 737.39 850.48 18VAVGYLITGYQR (SIL) (m/z 625.84 Da, +2) 533.27 747.40 860.49 18V

α2MG peptides y6 y7 y9AIGYLITGYQR (m/z 628.33 Da, +2) 738.35 851.44 1071.52 20VAIGYLITGYQR (SIL) (m/z 633.33 Da, +2) 748.36 861.45 1081.53 20V

ISEITNIVSKy8y7y6

y8y7y6

ISEITNIVSK – 1 fmol

Designing and optimizing a selected reaction monitoring assay for targeted PZP quantification in serum

3. Method optimization

• Peptide calibration curves in 0.1% TFA and in biological matrix

Non-matrix (0.1% TFA) Matrix (serum)

Designing and optimizing a selected reaction monitoring assay for targeted PZP quantification in serum

3. Method optimization - Measuring PZP in quality control serum samples

Serum(500x diluted)

Reduction (DTT)Alkylation (IAA)

Acetonprecipitation

Trypsinization

Measurement

Peak pickingQuantification

Skyline

1 fmol/μl SIL peptide

Technical replicates - ISEITNIVSK

Designing and optimizing a selected reaction monitoring assay for targeted PZP quantification in serum

3. Method optimization - Measuring PZP in quality control serum samples

Serum(500x diluted)

Reduction (DTT)Alkylation (IAA)

Acetonprecipitation

Trypsinization

Measurement

Peak pickingQuantification

Skyline

Biological replicates - ISEITNIVSK

1 fmol/μl SIL peptide

Measuring PZP in a population cohort

Serum samples from the Rotterdam Scan Study (RSS)

Subset of the larger Rotterdam Study (15000 participants)

1500 participants, no dementia at baseline

103 persons developed clinical AD (3 – 12 years later)

42 male

61 female

206 controls matched for age and gender

PZP levels are significantly higher in female than in male participants

0

50

100

150

200

250

0 1 2 3 4 5 6 7 8 9 10

controls

preclinical AD

Increased PZP levels in preclinical AD are only apparent in younger female cases

**

p < 0.05

45-50 51-55 56-60 76-8061-65 71-7566-70 81-85 >90

Age dependent increase in PZP was not observed in male preclinical AD cases

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6 7 8 9

controls

preclinical AD

45-50 51-55 56-60 76-8061-65 71-7566-70 81-85 >90

Conclusion/discussion

Functional assay for quantification of PZP in serum

Confirm that levels of PZP are increased in female preclinical AD cases in an age dependent manner

Increase no longer apparent in older female participants

Impacts the usefulness of PZP as a clinical biomarker

Biomarker Development Center

Arfan IkramNakita JainandunsingChristoph StinglTheo Luider

Acknowledgements

Designing and optimizing a selected reaction monitoring assay for targeted PZP quantification in serum

3. Method optimization - Measuring PZP in quality control serum samples

R2 = 0,9801 P = < 0,0001

Strong correlation between PZP peptides ISEITNIVSK and AVGYLITGYQR

ISEITNIVSK used as quantifier