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Establishing Quality Control Metrics for Immunodepletion of High- Abundance Plasma Proteins and Applying to a Large Clinical Cohort Meredith Turner ; Erik J. Soderblom; J. Will Thompson; Laura G. Dubois; M. Arthur Moseley Duke Proteomics Core Facility, Institute for Genome Sciences & Policy, Duke University School of Medicine, Durham, NC INTRODUCTION Human plasma is a commonly interrogated biological matrix to identify candidate protein biomarkers because of its wide range of expressed protein functionalities. These samples are also relatively easy to obtain from large patient cohorts with routine sample collection procedures. However, measuring changes in biologically relevant low-abundance proteins by LC-MS strategies is hindered by the large dynamic range of these biological matrices. In human plasma, the concentrations of proteins span over ten orders of magnitude with the fourteen most abundant proteins making up about 94% of the total protein mass. This large dynamic range of protein concentrations represents a challenge to the identification and characterization of biologically significant low-abundance proteins due to a “masking” effect. To address these challenges, immunodepletion of these high-abundance proteins is often employed and results in a subsequent increase in protein identification depth of coverage. As variations in immunodepletion efficiencies can have consequences on downstream LC-MS based protein quantitation, we have developed a series of quality control metrics which allow us to assess the efficiency of immunodepletions using Agilent’s Multi-Affinity Removal System (MARS-14) LC column. The metrics focus on bound versus unbound peak area ratios from UV chromatograms, pre- and post- depletion protein concentration ratios, and image analysis of 1D SDS-PAGE gels. These metrics provide unique insights into basic sample preparation variables and importantly allow the identification of potential outliers in the cohort prior to LC- MS/MS analysis. Potential outliers can then be flagged for statistical consideration in the downstream analysis or excluded from further analysis all together. METHODS Fifty uL aliquots of EDTA human plasma from a 243 patient chronic hepatitis C clinical cohort were subjected to immunodepletion using a 4.6x100 mm Agilent MARS14 HPLC column (part number 5188-6558) on an Agilent 1100 series LC system. Before immunodepletion, the aliquots were diluted with 200 uL of Agilent Buffer A , and then 100 uL of the 5x dilution samples was injected on the column. The manufacturer’s recommended LC gradient protocol was followed. Areas under the curve for bound and unbound fractions (automated peak integration) were recorded from each UV chromatogram (280 nm) within Agilent ChemStation (v10.02). and their ratios were utilized for one depletion metric. Bradford assays (Bio-Rad) were performed on plasma samples pre- and post-depletion (post-depletion done after a buffer exchange and ~20x concentration into 50 mM ammonium bicarbonate) and the values were plotted as the total plasma protein concentration versus the percentage of total protein in the unbound fraction. Finally, 5 ug of each depleted plasma sample was run on an Invitrogen NuPAGE 4-12% Bis-Tris gel, stained with Colloidal Blue (Invitrogen) and subjected to semi-automated densiotometery measurements within TotalLab Quant (Non-Linear Dynamics). 20000 40000 60000 80000 100000 120000 140000 160000 0 20000 40000 60000 80000 AUC Unbound Fraction AUC Bound Fraction Flow Cell 2 Unbound AUC vs. Bound AUC – 243 sample HCV study MARS14 Depletion min 10 15 20 25 30 mAU -100 0 100 200 300 400 500 600 700 DAD1 A, Sig=280,16 Ref =360,100 (042009\004-0201.D) 11.018 Area: 2387.19 22.150 Area: 647.967 23.455 DAD1 A, Sig=280,16 Ref =360,100 (042009\003-0101.D) QC Metric 1 – Characterizing Depletion Chromatogram QC Metric 2 – Bradford Assay QC Metric 3 – SDS-PAGE Gel Image Analysis QC METRIC 1 - Characterizing Depletion Chromatogram 100 80 60 40 20 0 [Total Serum Protein] (ug/uL) 35 30 25 20 15 10 5 Percentage of Total in Unbound Fraction (%) Unbound (non-depleted) proteins Bound (depleted) proteins **The samples showing the extra peak were previously noted to be cloudy. Early eluting peak possible lipid (micelle) contamination. QC METRIC 2 – Bradford Assay 0 10 20 30 40 50 60 70 80 0 5 10 [Pre-Depletion Total Serum Protein] (ug/uL) Percentage of Total Protein in Unbound Fraction (%) Depletion QC Based on Bradford Assays *Samples highlighted are more than two standard deviations away from the average. QC METRIC 3 - SDS-PAGE Gel Image Analysis The three sets of depletion QC metrics described are effective at identifying potential outliers to assess the efficiency of the depletion protocol. The flagged samples can then be set aside in statistical analyses so as not to affect the biological variability calculations. The QC metrics were applied to a large clinical cohort and successfully found samples that were potential outliers. Features of UV (A280) Chromatogram of Depleted Human Plasma Integration of Unbound Peak 20000 40000 60000 80000 100000 120000 140000 160000 0 20000 40000 60000 80000 AUC Unbound Fraction AUC Bound Fraction Unbound AUC vs. Bound AUC –179 sample HCV study Raw AUC Intensities 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 50.0 55.0 60.0 65.0 70.0 Unbound/Bound AUC Ratio % of Total AUC in the Unbound AUC Depletion QC Based on AUC Ratios from 243 sample HCV Study The area under both curves is included in the AUC determination. www.genome.duke.edu/proteomics/ 450 Research Drive, Durham, NC 27710 Flow Cell 1 Unbound/Bound AUC Ratio Unbound AUC / Bound AUC % of Total AUC in Unbound AUC [Unbound AUC / (Unbound AUC + Bound AUC)] * 100 Red Boxes Samples highlighted in red are not within two standard deviations of the average point, so are flagged for further consideration. Pre-Depletion Total Serum Bradford Post-Depletion Bradford Percentage of Total Protein in Unbound Fraction (%) Seven bands monitored across all samples Representative gel from 243 patient HCV cohort min 5 10 15 20 25 mAU -100 0 100 200 300 400 500 600 DAD1 A, Sig=280,16 Ref =360,100 (040909\019-1501.D) 1-P2-E-07 1-P2-E-08 1-P2-E-09 1-P2-F-01 1-P2-F-02 1-P2-F-03 1-P2-F-04 0.00 0.10 0.20 0.30 0.40 1 2 3 4 5 Protein Concentration Bradford Assay (A 595 ) [ug/ul] QC 1 • Characterizing Depletion Chromatogram • Samples not within two standard deviations of the average point are flagged for further examination QC 2 • Bradford Assay • Samples with an extremely low post-depletion concentration are not normalized with the other samples QC 3 • SDS-PAGE Gel Image Analysis • Samples showing a hemoglobin band are run at the end of the queue [(Volume of plasma depleted* pre-depletion concentration) / (Volume of depleted plasma obtained * post-depletion concentration)] * 100 OVERVIEW Analysis of Atypical Chromatogram Features CONCLUSIONS
