Establishing Quality Control Metrics for Immunodepletion ...

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Establishing Quality Control Metrics for Immunodepletion of High-

Abundance Plasma Proteins and Applying to a Large Clinical CohortMeredith 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).

100000

120000

140000

160000

0 20000 40000 60000 80000AU

AUC Bound Fraction

Flow Cell 2

Unbound AUC vs. Bound AUC

– 243 sample HCV study

MARS14 Depletion

min10 15 20 25 30

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 AssayQC Metric 3 – SDS-PAGE

Gel Image Analysis

QC METRIC 1 - Characterizing Depletion Chromatogram

3530252015105

Percentage of Total in Unbound Fraction (%)

55 HCV Samples (1797)

34 DARPA Samples (1871)

MARS14 "Typical" Depletion "Average" Serum Protein Conc (55ug/ul)

Unbound (non-depleted)

proteinsBound (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 5 10

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

100000

120000

140000

160000

0 20000 40000 60000 80000

AUC Bound Fraction

Unbound AUC vs. Bound AUC

–179 sample HCV study

Raw AUC Intensities

50.0 55.0 60.0 65.0 70.0

% 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

min5 10 15 20 25

DAD1 A, Sig=280,16 Ref =360,100 (040909\019-1501.D)

1 2 3 4 5

• Characterizing Depletion Chromatogram• Samples not within two standard deviations of the average point are flagged

for further examination

• 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

Establishing Quality Control Metrics for Immunodepletion ...

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