Introduction

2. Introduction • Clinical Proteomics: a definitionTargeted, multiplexed quantification of known analytes in clinical samples for the diagnosis, prognosis, or therapeutic management of disease • Potential PlatformsImmunoassays (bead methods, arrays) • Mass spectrometry (generally entails denaturing, reducing disulfide bonds, blocking cysteines, digesting proteins with a protease, and then analyzing target peptides with LC-MRM/MS) Multiplexed methods could reduce the cost of clinical testingand provide more markers to improve diagnostic performance

3. Introduction (cont’d) • Immunoassays have problemsEspecially with human serum and plasma samples • Anti-reagent antibodies • Anti-analyte autoantibodies • Poor interplatform concordance • Heteromultimeric protein complexes • Cross-reactivity • Expensive to design and implement

4. Introduction (cont’d) • Can methods using mass spectrometry deliver?Complicated methods that require: • specialized training • large capital investment (reagent rental?) Once established methods would provide: • low reagent cost • direct detection of analyte • destruction of interfering endogenous immunoglobulins Multiplexed LC-MRM/MS has not been previously compared with clinically used immunoassays

5. Introduction (cont’d) • Can methods using mass spectrometry deliver? Trypsin digestion of proteins is variable Trypsin digestion of proteins is variable Hoofnagle, Clin Chem, 56:161-4

6. Introduction (cont’d) • Apolipoprotein B Structural protein of non-HDL lipoproteins Better marker and target than LDL cholesterol More expensive to measure than a lipid panel • Apolipoprotein A-I The structural protein of HDL Highly correlated with HDL cholesterol concentrations Marker of atheroprotective particles The ratio of the two may provide more information than either alone, thus multiplexed assay attractive

7. Questions • How could we pick peptides to analyze? • Why is trypsin digestion so variable? • How could we calibrate mass spectrometric protein assays?

8. Methods • Optimizing digestion with trypsinDifferent denaturing conditions: urea, trifluoroethanol Varied digestion time: 0-21 h • Selection of peptides to monitor by LC-MRM/MS PeptideAtlas (free database of tryptic serum peptides) Correlation of peptides from the same protein Correlation of peptide response with immunoassay Selected two peptides per protein

9. Methods (cont’d) • Comparison of microflow with high-flow HPLCMicroflow to nanoflow standard for research proteomics • Not as robust as high-flow platforms • Correlation with a clinically used immunoassay Nephelometric immunoassays on Siemens BNII Not multiplexed immunoassays (one analyte at a time) Interassay CV 3-5% for the Siemens assays

10. Question • Could serum digestion and direct detection of peptides provide adequate limits of detection for the quantification of low-abundance serum proteins?

11. Results • All peptides are not the same • Peptides are liberated at different rates; peptide selection is important

12. Results (cont’d) • High-flow HPLC correlates with microflow • High-flow platforms are more practical for the clinical laboratory

13. Results (cont’d) • Single point calibrator works well, spiked matrix calibrators do not. This is surprising, and implies that purified, delipidated apolipoproteins do not digest the same as native proteins ineither denaturant.

14. Results (cont’d) • LC-MRM/MS assays correlate with clinical immunoassays

15. Conclusions • Multiplexed LC-MRM/MS for abundant proteins • Works even with two very different serum proteins: • ApoB: large, heavily glycosylated, many disulfide bonds • ApoA-I: small, no glycosylation, no disulfide bonds • Could provide an easy conduit for novel biomarkers to enter the clinical laboratory • The databases and tools to quickly select the right peptides are now in place (PeptideAtlas, Skyline) • Single point calibration could reduce variability in large clinical studies that use LC-MRM/MS quantification clinical studies that use LC-MRM/MS quantification