validating ‘pzp’ as a biomarker for preclinical alzheimer ... · alzheimer’s disease using a...
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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