VIP Call Girl Sector 25 Gurgaon Just Call Me 9899900591
Best practices and challenges for robust Quantitative proteomics of DMEs
1. Best Practices for Robust LC-MS/MS Quantification of Drug
Metabolizing Enzymes and Transporters to Predict Inter-Individual
Variability: Case Examples of Hepatic Cytosolic ADHs and ALDH1A1
Dr. Deepak Kumar Bhatt
Senior Postdoctoral Fellow
Dr. Bhagwat Prasad’s Lab
bhatt81@uw.edu
1
2. LC-MS/MS Quantification of DMEs and transporters
• Applications:
• To predict interindividual variability (effect of age, genotype, gender or disease condition)
• To extrapolate clearance from in vitro to in vivo (IVIVE) (cell lines or microsomes to organs)
Quantification of unique surrogate peptide(s)
Group Age (years)
Neonatal 0.01 to 0.05 (4)
Infancy 0.08 to 0.92 (17)
Toddler/Early
Childhood
1 to <6 (30)
Middle Childhood 6 to <12 (38)
Adolescence 12 to 18 (48)
Adulthood 19 To 87 (57)
• University of Washington liver bank
• Children’s Mercy-Kansas City hospital liver
bank
2
3. Goal: To address technical variability over biological (e.g.,
interindividual) variability in LC-MS/MS protein quantification
Challenges
Surrogate Peptide
Selection
Optimum Stability, Solubility, No PTMs,
No Variants (SNPs)
Peptide Qualification Removing ghost peaks
Sample Preparation
Trypsin digestion efficiency and sample loss in
processing
LC-MS/MS analysis Matrix effect/Ion suppression
Data Analysis
Removing artifacts, peak integration, data
normalization and absolute quantification
3
4. Surrogate peptide selection
Surrogate Peptide
Selection
Peptide Qualification Sample Preparation LC-MS/MS analysis
Data AnalysisCase
Example
Conclusions
•Uniqueness
•Optimum peptide length (6-22)
•No transmembrane regions
•No posttranslational modification (PTM)
•No non-synonymous single nucleotide
polymorphism (SNP)
•Avoid unstable residue (e.g.- C, M, W)
•No splice variants
•No ragged end (RR, KK, RK, KR)
•No missed cleavage sites
•Optimum hydrophobicity (45-155)
MDR3_HUMAN
Unique and stable entry identifier
A compendium of targeted proteomics
assays
PROTTER
MS-Homology
4
Prasad B. and Unadkat J., AAPS J, 16, 1-15 (2014)
6. Surrogate Peptide
Selection
Peptide Qualification Sample Preparation LC-MS/MS analysis
Data AnalysisCase
Example
Conclusions
Surrogate peptide qualification
• Superimposability of multiple transitions
• Elution at predicted retention time
• Validate light transitions with heavy transitions
TVLAVFGK_Light TVLAVFG(K)_Heavy
6
7. Surrogate Peptide
Selection
Peptide Qualification Sample Preparation LC-MS/MS analysis
Data AnalysisCase
Example
Conclusions
Sample preparation
Individual Sample + albumin*
Desalting, enrichment
Trypsin digestion
Reaction quenching, addition of heavy internal
standard
LC-MS sample (multiple fragments and
multiple peptides)$
Denaturation, reduction, alkylation
*Use albumin as a protein internal standard
$Use pooled QC samples in each batch of LC-MS samples
7
8. Surrogate Peptide
Selection
Peptide Qualification Sample Preparation LC-MS/MS analysis
Data AnalysisCase
Example
Conclusions
Ion suppression
TVLAVFGK
2 fold decrease
in the peak height
Internal standard in sample matrixInternal standard in buffer
8
13. Case example: Age-dependent expression of
ADH1A, ADH1B, ADH1C and ALDH1A1 in human
liver cytosol samples
13
Surrogate Peptide
Selection
Peptide Qualification Sample Preparation LC-MS/MS analysis
Data AnalysisCase
Example
Conclusions
16. Addition of heavy peptide internal standard
• Denaturation, reduction, alkylation
• Desalting, enrichment
• Trypsin digestion
4: MS ionization and
measurement reproducibility
2: Trypsin digestion
reproducibility
6: Validation of quantifiable
peptides to establish overall
precision
Addition of protein internal standard (e.g., BSA)
3: Inter-day reproducibility
Analysis of 3 replicates of a pooled HLM sample with
each set of samples
5: Validation of quantifiable
fragments
Robustness parameters
Normalize individual means and standard deviation by QC pool
Do multiple peptides of a protein correlate?
Average of multiple peptide
Yes
No
Select alternate
peptides
Do multiple fragments of a peptide correlate?
No
Average of multiple fragments
Yes
Select
alternate
fragments
Day 1 Day 2 Day 3
Individual sample processed and analyzed
on three different days Fixed total
starting protein
concentration
Conclusions
1: Inter-day assay
variability
Surrogate Peptide
Selection
Peptide Qualification Sample Preparation LC-MS/MS analysis
Data AnalysisCase
Example
Conclusions 16
17. Acknowledgements
University of Washington
• Prasad Lab
• Bhagwat Prasad, Ph.D.
• Neha Saxena, Ph.D.
• Meijuan Xu, Ph.D.
• Abdul Basit Shaikh, Ph.D.
• Marc Vrana
• Haeyoung Zhang
• Kenneth Thummel, Ph.D.
• UW SOP Mass Spec Center
• Funding
• NIH/NICHD; 1R01HD081299-01A1
• Department of Pharmaceutics, UW
Children’s Mercy Hospital, Kansas City
• J. Steven Leeder, Ph.D.
• Andrea Gaedigk, Ph.D.
• Robin E. Pearce, Ph.D.
Special thanks to Division for Drug Metabolism, ASPET for awarding travel grant
17