Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list...
Transcript of Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list...
![Page 1: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/1.jpg)
Targeted proteomics (and Skyline) to characterize an in vitro model of human neuronal development Skyline User Meeting May 31st 2015
Tom Dunkley Roche Innovation Center Basel
![Page 2: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/2.jpg)
Neurons in a dish to model disease Enabled by induced pluripotent stem cells & genome editing
iPSCs NPCs Patients Blood Neuronal culture
NPC – day 0 Neurons - day 41 day 14
Genome editing
![Page 3: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/3.jpg)
Protein regulation in synapse function …and dysregulation in neurodevelopmental disease
Kelleher R. J. & Bear M. F. The Autistic Neuron: Troubled Translation? Cell (2008) 135, 401–406
Understanding protein dysregulation in neurodevelopmental disease enables: • Target identification
• Mechanism-of-action biomarker ID
• Identification of endpoints for phenotypic screens
![Page 4: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/4.jpg)
Testing of protein hypotheses:
• Antibody independent
• Precise quantification
• High multiplex
Hypothesis generation:
• Proteins in disease biology
• Proteins as predictive biomarkers
• Proteins in pharmacodynamics & pharmacokinetics
Heavy peptide on col. (amole)
1e5
1e6
1e7
100 1000 10000
proteomics
genomics
bioinformatics
literature
Targeted proteomics (SRM/MRM/PRM)
![Page 5: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/5.jpg)
eIF5
RPL7A
RPS20
Delorme R. et al. Nature Medicine 19, 685–694 (2013)
246-protein neuroSRM panel
• Multiplexed SRM method
• Internal standards for all peptides
• 246 proteins, 478 peptides, 2,868 tran.
• 2 h per sample
![Page 6: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/6.jpg)
Targeted - SRM, Vantage (2868 tran.) Discovery – MS1, QE (70k res.)
analyte
IS
analyte
IS
UBE3A - VDPLETELGVK
WT mutant WT mutant
Targeted proteomics: precise quant, confident ID
![Page 7: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/7.jpg)
… but don’t miss the bigger picture Combine targeted and discovery proteomics
Targeted proteomics (SRM/PRM)
• Precise quantification
• High confidence in specificity with internal standards
Discovery proteomics
VALIDATE
Test hypotheses Generate new hypotheses
![Page 8: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/8.jpg)
Targeted proteomics (SRM) workflow
method build
target list spectral library
target species proteome
data acquisition data processing
data archive
data analysis
![Page 9: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/9.jpg)
Retention time scheduling enables 2,868 transitions/run Simplified using iRT
Escher C. et al., Using iRT, a normalized retention time for more targeted measurement of peptides. PROTEOMICS (2012)
![Page 10: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/10.jpg)
batch start
batch end
Dynamic scheduling adjusts for RT drift Enables 2 min windows (and weekends off)
Gallien S. et al., Highly multiplexed targeted proteomics using precise control of peptide retention time. PROTEOMICS (2012)
54 hours
![Page 11: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/11.jpg)
IS dotp= 1
library dotp= 0.98
LIGHT
HEAVY
Peak review Chromatogram libraries (Panorama) provide a useful reference
![Page 12: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/12.jpg)
Tracking instrument performance Panorama QC folder
Poster #134 (Tue): Performing quality control on targeted proteomics assays using Skyline and Panorama.
![Page 13: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/13.jpg)
NPC – day 0 Neurons - day 41 day 14
Characterization of the neuronal development model Experimental design
SA001 SA001 GE1 SA001 GE2 • 3 hPSC-derived neural precursor cell (NPC) lines
• Parental (SA001) & two lines with ‘silent’ genome editing (GE)
• 4 GE lines with disease-relevant mutations also analyzed (not reported here)
• 3 developmental stages
• 5 to 9 independent replicate differentiations
• Pooled QC prepped and analyzed 3-5 times / batch
• Assess technical variation for whole process (except cell lysis)
QC
• 177 samples
• 8 batches analyzed over ~ 2 months
![Page 14: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/14.jpg)
Technical performance of the SRM assay Robust, reproducible measurements over a 2-month experiment
‘All peptides’
• Skyline output after manual peptide + transition exclusion
‘Filtered peptides’
• In >70% samples from any day:
• <30% intra-batch CV
• Within linear range (<30% relative error)
Methods for automated feature selection needed
![Page 15: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/15.jpg)
Clustering of samples based on peptide data (PCA) Protein regulation over time is the most significant source of variation
Principal component 1
Princi
pal co
mpon
ent 2
![Page 16: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/16.jpg)
Protein regulation during neuron differentiation Majority of NeuroSRM proteins regulated between 3 developmental stages
![Page 17: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/17.jpg)
Correlation between the isogenic cell lines Pattern of protein regulation consistent across the WT & 2 ‘silent’ GE lines
![Page 18: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/18.jpg)
Regulation of key developmental marker proteins In vitro hESC model recapitulates in vivo neurodevelopment
Dunkley T. et al. Proteomics Clinical Applications (2015)
• Comparison Human Brain Transcriptome (HBT) mRNA data:
• Significant match (p-value = 3.8e-11)
• 104/165 mRNAs/proteins (63%) having identical modulation
• 17 proteins (10%) modulated in opposite directions in human brain (mRNA) & hPSC-derived neuronal development model (protein)
![Page 19: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/19.jpg)
QC
Summary hESC-derived neurons characterized through targeted proteomics batch start
batch end
data archive
246 proteins, 478 peptides
![Page 20: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/20.jpg)
Acknowledgements • Arno Friedlein • Peter Jakob • Sabine Kux van Geijtenbeek • Axel Ducret • Carine Steiner • Hanno Langen • Paul Cutler • Michel Petrovic • Ignacio Fernandez Garcia
• Kristin Wildsmith
• Josh Eckels
• Ravi Jagasia • Veronica Costa • Sebastian Lugert • Stefan Aigner • Martin Ebeling
• Meghan T. Miller • Christoph Patsch • Paolo Piraino
• Olga Vitek • Lin-Yang (Mike) Cheng
• Mike MacCoss • Brendan MacLean • Jarrett Egertson • Vagisha Sharma
![Page 21: Tom Dunkley Roche Innovation Center Basel proteomics (SRM) workflow method build target list spectral library target species proteome data acquisition data processing data archive](https://reader031.fdocuments.in/reader031/viewer/2022022012/5b2559097f8b9a56678b6413/html5/thumbnails/21.jpg)
Doing now what patients need next