Calibrated Quantification. Christopher M. Shuford Laboratory Corporation of America Holdings

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1 Calibrated Quantification Christopher M. Shuford Laboratory Corporation of America Holdings

2 Why Calibrate reduce magnitude of analytical variance (and increase accuracy of relative quantification) Apolipoprotein E Un-calibrated Calibrated to Internal Standard Courtesy of Andrew Hoofnagle, UW A.N. Hoofnagle et al., Clin. Chem., 2012, 8(4),

Internal Intensity 1 nmol known amount of labeled signature peptide sample Proteolysis (digestion) Enrichment LC-MS Elution time target protein unlabeled

3 Internal Intensity 1 nmol known amount of labeled signature peptide sample Proteolysis (digestion) Enrichment LC-MS Elution time target protein unlabeled signature peptide (NAT) = labeled internal calibrator peptide (SIL) = incomplete proteolysis product = contaminants [NAT] = [SIL] I NAT I SIL I NAT [NAT] = [SIL] I SIL

Internal Hierarchy 2 Concatamer 1 Protein Protein-Level Enrichment 3 Peptide Peptide-Level Enrichment LC-SRM

Full-length Protein: Desiderio, D. M. and co-workers, Biol. Mass Spectrom. 1991, 2(2), 149-16 Brun, V.

Methods 200, 2(8), 87-89 Beynon, R. J. and co-workers, Nat.

4 Internal Hierarchy 2 Concatamer 1 Protein Protein-Level Enrichment 3 Peptide Peptide-Level Enrichment LC-SRM Detection Digestion NAT:SIL (optional) (optional) time [Protein] [Peptide NAT ] = [Peptide SIL ] I NAT I SIL Full-length Protein: Desiderio, D. M. and co-workers, Biol. Mass Spectrom. 1991, 2(2), Brun, V. and co-workers, Mol. Cell. Proteomics 2007, 6(12), Peptide Concatemer: Beynon, R. J. and co-workers, Nat. Methods 200, 2(8), Beynon, R. J. and co-workers, Nat. Protocols 2006, 1(2), (Partial) Peptide: Barr, J. R. and co-workers, Clin. Chem. 1996, 42(10), Gygi, S. P. and co-workers, P. Natl. Acad. Sci. USA 2003, 100(12),

5 Internal 2 Concatamer 1 Protein Protein-Level Enrichment 3 Peptide Peptide-Level Enrichment LC-SRM Detection Digestion NAT:SIL (optional) (optional) time [Protein] [Peptide NAT ] = [Peptide SIL ] I NAT I SIL Pitfalls: Assume digestion is stoichiometrically complete (peptide and concatamers) Results vary by digestion/denaturation conditions Reproducibility between digestions/days is critical Internal Calibrator must be stable Response assumed to be linear

NAT Intensity : Multipoint NAT:SIL Intensity Ratio Qualified protein calibrator added to blank matrix known amounts of Protein 1 10 0 Test constant amount of internal standard

6 NAT Intensity : Multipoint NAT:SIL Intensity Ratio Qualified protein calibrator added to blank matrix known amounts of Protein Test constant amount of internal standard Without Internal Standard With Internal Standard y=(m x)+b x=(y b)/m y=(m x)+b x=(y b)/m known Protein Amount known Protein Amount

7 : Multipoint Qualified protein calibrator added to blank matrix known amounts of Protein Test constant amount of internal standard Pitfalls: Blank Matrix must be essentially identical to sample matrix (requires verification) Qualified protein should have same structure as endogenous protein (size, conformation, PTMs, etc) calibrators should be processed in parallel with unknown samples (added cost/time). calibrators must be stable

8 NAT Intensity : Single-point NAT:SIL Intensity Ratio Qualified protein calibrator added to blank matrix known amount of Protein Replicates Test constant amount of internal standard Without Internal Standard With Internal Standard y=(m x)+0 x=y/m y=(m x)+0 x=y/m known Protein Amount known Protein Amount

9 : Single-point Qualified protein calibrator added to blank matrix known amount of Protein Replicates Test constant amount of internal standard Pitfalls: Blank Matrix must be essentially identical to sample matrix (requires verification) Qualified protein should have same structure as endogenous protein (size, conformation, PTMs, etc) calibrators should be processed in parallel with unknown samples (added cost/time). calibrators must be stable Response assumed to be linear

10 Additional Reading Source of Analytical Variance in Proteomics H.D. Cox et al., Clin. Chem. 2016, 60 (3), Peptide Degradation during Digestion C.M. Shuford et al., Mol. Cel. Proteomics, 2012, 11 (9), More Peptides = More Confidence Q. Fu et al., Clin. Chem. 2016, 62 (1), Single-point with Sample Pool S.A. Agger et al., Clin. Chem. 2010, 6 (12), Analytical Verification in Translational Research R.P. Grant and A.N. Hoofnagle, Clin. Chem. 2014, 60 (7),

Application Note # LCMS-89 High quantification efficiency in plasma targeted proteomics with a full-capability discovery Q-TOF platform

Application Note # LCMS-89 High quantification efficiency in plasma targeted proteomics with a full-capability discovery Q-TOF platform Abstract Targeted proteomics for biomarker verification/validation