Definition of the Measurand: CRP

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1 A Reference Measurement System for C-reactive Protein David M. Bunk, Ph.D. Chemical Science and Technology Laboratory National Institute of Standards and Technology Definition of the Measurand: Human C-reactive Protein: Non-covalent pentamer with identical subunits Subunit consist of a single 206 amino acid chain Relative molecular mass of subunit Minimal known post-translational modifications N-terminal pyroglutamic acid Normal serum concentration 1 mg/l Serum concentration slightly elevated above normal in atherosclerosis Serum concentrations significantly elevated above normal in cases of infection and inflammation 1

2 Status of the Reference Measurement System 1) Measurand: Well Defined 2) Primary Reference Material: NMIJ CRM 6201-a 3) Secondary Reference Material: IRMM ERM-DA472/IFCC 4) Reference Measurement Procedure:??? Metrological Traceability Chain Materials Measurement Procedure SI Unit (moles) NMIJ CRM 6201-a Amino Acid Analysis + Nitrogen Determination IRMM ERM-DA472/IFCC Manufacturer s Assay Calibrators Manufacturer s Value Transfer Method Patient Serum Sample Routine Clinical Assay 2

3 Development of a Reference Measurement Procedure: MS-based Proteomics Approach for Protein Quantification Protein Analyte Peptide Measurand(s) ID-MS Quantification Enzymatic Digestion (typically with trypsin) Isotopically labeled peptide(s) used as internal standards Limitations of a MS-based Proteomics Approach to Protein Quantification No internal standard present during sample preparation (including disulfide reduction, cysteine alkylation, and enzymatic digestion) High possibility for interferences Specificity of the peptide measurand(s) for the analyte protein Measurand stability 3

4 A Higher Order Method: MS-based Proteomics + Affinity Purification + Standard Addition Measurement Approach: 1. Spike sample with a known amount of purified 2. Perform monoclonal antibody-based purification of spiked and un-spiked sample 3. Digest isolated in spiked and unspiked samples with trypsin 4. Add isotopically labeled synthetic peptides 5. Quantify using MRM-based isotope dilution LC-MS/MS A Higher Order Method: MS-based Proteomics + Affinity Purification + Standard Addition Benefits: 1. Spiked experiences the same affinity purification conditions and digestion condition as endogenous and acts as internal standard 2. Affinity purification improves measurand stability and reduced likelihood for interferences Standard Addition Results* from [ ] (Area Ratio)(V/Vo) 3.5 Sample A 3.0 Sample B 2.5 Sample C 2.0 Sample D [ Spike](Vs/Vo) 4

5 Limitations of Proteomics + IP + Standard Addition Treatment of spiked sample may not be identical to that of unspiked sample Use of multiple data points to determine analyte concentration increases measurement uncertainty A Higher Order Method (part 2): Complete Protein ID-MS Measurement Approach: 1. Spike sample with isotopically-labeled r 2. Perform monoclonal antibody-based purification spiked sample 3. Digest isolated in spiked sample with trypsin 4. Quantify using MRM-based isotope dilution LC-MS/MS on multiple tryptic peptides using calibrators prepared from labeled and unlabeled (i.e., double isotope dilution mass spectrometry) Digest Wash Elute LC-MS/MS and Spiked 15 N in serum mab to conjugation with bead -mab-bead complex Affinity Purified Tryptic peptides of 5

6 Preparation of the Internal Standard Recombinant 15 N-labeled human C-reactive protein: Expressed in E. coli with media containing 15 N-ammonium chloride as the sole nitrogen source Expressed with a hexahistidine affinity tag on leader sequence Purified by sequential immobilized metal and phosphatidylcholine affinity chromatography purifications Hexahistidine affinity tag enzymatically cleaved leaving 4-amino acid leader sequence on the N-terminus of Assessment of the Internal Standard Protein purity of 15N-r was greater than 99% as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis MW (kda) A

7 Assessment of the Internal Standard Analysis by matrix-assisted laser desorption ionization mass spectrometry indicates that the principle product has a mass consistent with with 15 N incorporation along with the hexahistidine leader sequence 15 N- 15 N- m/z Assessment of the Internal Standard Incorporation as a percent of total nitrogen was estimated to be greater than 99% as determined by isotopic profile analysis m/z m/z 7

8 Assessment of the Internal Standard Equivalence of 15 N-labeled r to purified human under digestion conditioned assessed by comparing expected to observed tryptic peptide ratios in blends of labeled and unlabeled Equivalence of 15N-labeled to purified human under affinity purification conditioned assessed by comparing expected to observed tryptic peptide ratios in affinity purified blends of labeled and unlabeled Equivalence of 15N-labeled to endogenous human under digestion and affinity purification conditions assessed by comparing expected to observed tryptic peptide ratios in affinity purified 15 N-r spiked serum samples Assessment of the Internal Standard Linearity analysis of mixed labeled- and unlabeled- following affinity purification, tryptic digestion, and LC- MS/MS. A mixture of labeled r and purified human with specific mass ratios (0.2:1, 0.3:1, 1:1, 3:1, 5:1) were digested with trypsin and analyzed by LC-MS/MS. Experiment was performed with 3 replicates and error bars represent standard deviation. 8

9 The Complete RMP for 1. Spike serum sample with 15 N- labeled r 2. Perform monoclonal antibodybased affinity purification (on magnetic beads) of 3. Digest isolated in spiked sample with trypsin 4. Quantify using MRM-based isotope dilution LC-MS/MS on 6 tryptic peptides using calibrators prepared from labeled and unlabeled (i.e., double isotope dilution mass spectrometry) Intensity, cps 1.19e5 1.15e5 1.10e5 1.05e5 1.00e5 9.50e4 9.00e4 8.50e4 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 1.50e4 1.00e Time, min A Dream Realized: The Reference Measurement System for Materials Measurement Procedure SI Unit (moles) NMIJ CRM 6201-a IRMM ERM-DA472/IFCC Amino Acid Analysis + Nitrogen Determination Complete Protein ID-MS RMP Manufacturer s Assay Calibrators Manufacturer s Value Transfer Method Patient Serum Sample Routine Clinical Assay 9

10 Future Applications of Complete Protein ID-MS Albumin in Urine (with 15 N-labeled rhsa) Cardiac Troponin I (with 15 N-labeled recombinant troponin complex) Prostate Specific Antigen (with 15 N-labeled recombinant PSA + 15 N-labeled recombinant PSA- ACT complex) Acknowledgements Eric Kilpatrick Illarion Turko Wei-Li Liao Johanna Camara Nathan Dodder Thank You 10

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