Automating MRM-Based Protein Quantification

Tasso Miliotis

AstraZeneca R&D Mölndal

Translational Science iMED CVMD

Webinar - Agilent Technologies Feb 25, 2015

Outline

• Introduction

• Why MRM/MS?

• Sample Prep and Analysis

• Strategy Overview

• Novel Sample Prep Automation via the AssayMAP Bravo Platform

• Development - SPE Optimization (sorbent and elution solvents)

• Implementation in Targeted Quantitative Proteomics Studies

• Results

• Conclusions and Outlook

Limitations of Immunoassays

• Immunoassays exist for~2,000 proteins (10% of proteome) but exhibit:

• extremely variable quality and limited specificity

• degraded performance when multiplexed without extensive optimization

• 50-100 μL plasma per analyte

• Generation of an immunoassay for a new target requires:

• two epitope-matched antibodies

• analyte-specific optimization of components and workflow

• time

• $$

Many Immunoassays Suffer From Unrecognized Interferences

Blocking Complex

False Negatives

Autoantibody Cleaved Ag HAMA

False Positive

Courtesy of Dr. N.L. Anderson

What forms the immunoassay sandwich - or prevents its formation - is not directly visualized:

Immunoassays do not provide a

molecular microscope

Targeted Quant via MRM/MS

• Potential for high specificity, sensitivity, and analyte multiplexing.

Gillette MA et al. Nature Methods 10, 2013, 28.

~200 proteins quantified in one chromatographic run

Benefits of Internal Standards With MRM

• 13C/15N-labeled analogs of the unlabeled analytes help correct for ion suppression and matrix effects.

• 13C/15N labeled standards are chemically identical to unlabeled counterpart and are distinguishable by m/z only.

• Labeled and unlabeled forms behave identically with respect to chromatographic retention, electrospray ionization, and gas-phase fragmentation.

Kuzyk MA et al. Mol. Cell Proteomics 8, 2009, 1860.

Time Time

Inte

nsity

Inte

nsity

Fibrinogen gamma chain Gelsolin

Natural (NAT) Synthetic (SIS)

Experimental Workflow Plasma Matrix

Proteolytic Digest

Plasma Digest with Labeled Peptides

Concentrated and Desalted Peptides

reduce, alkylate, quench, digest

solid phase extraction

13C/15N Labeled Tryptic Peptides

Line Spectra and XICs 1290 Infinity UHPLC with 6490 QqQ (Agilent)

RPLC-MRM/MS

Protein Quantitation

Data Analysis (Standard Curves with Linear Regression)

lower and upper LOQs dynamic range regression info. endogenous protein conc. precision

Afamin (P43652) Beta-2-glycoprotein I (P02749) Haptoglobin (P00738) Alpha-1-antichymotrypsin

(P01011) Ceruloplasmin (P00450) Hemopexin (P02790)

Alpha-1B-glycoprotein (P04217) Clusterin (P10909) Heparin cofactor II (P05546)

Alpha-2-antiplasmin (P08697) Coagulation factor XII (P00748)

Inter-alpha-trypsin inhibitor heavy chain (P19827)

Angiotensinogen (P01019) Complement C3 (P01024) Kininogen-1 (P01042)

Antithrombin-III (P01008) Complement C4-B (P0C0L5) Plasminogen (P00747)

Apolipoprotein A-I (P02647) Complement component C9 (P02748) Retinol-binding protein 4 (P02753)

Apolipoprotein A-II (P02652) Complement factor B (P00751) Serum albumin (P02768)

Apolipoprotein A-IV (P06727) Complement factor H (P08603) Transthyretin (P02766)

Apolipoprotein B-100 (P04114) Fibrinogen alpha chain (P02671) Vitamin D-binding protein (P02774)

Apolipoprotein C-I (P02654) Fibrinogen beta chain (P02675) Vitronectin (P04004)

Apolipoprotein E (P02649) Gelsolin (P06396)

35 Target Protein Panel for Evaluation

Concentration Range: 4 orders of magnitude Protein MW: 7 kDa (Apolipoprotein C-I) to 513 kDa (Apolipoprotein B-100)

Quantotypic Target Peptides

VGYVSGWGR (Haptoglobin, P00738)

General Peptide Details

SIS Peptides

Synthesis ∙ 35 tryptic peptides ∙ C-terminal, 13C/15N-labels ∙ Fmoc chemistry at 5 µmol scale

Purification and Characterization ∙ RP-HPLC-MS ∙ MALDI-TOF-MS & AAA ∙ CZE 95% average purity

QC Standardization Kits

Supplied with each Kit:

• Standards and additional components.

• Detailed SOPs and video.

• LC-MS conditions and parameters.

• Reference values.

• Performance quality guide.

• Analysis software – QUALIS.

Monthly QC Daily QC

LC-MS Platform Assessment Kit (#1)

LCMSPKM-AG6490S-1 mo

www.mrmproteomics.com

LCMSPKD-AG6490S-1 wk

UHPLC-MRM/MS LC separation

∙ 10 µg column load

∙ RP-UHPLC column (2.1 x 150 mm) ∙ 0.4 mL/min

∙ 27 min gradient

MRM Detection ∙ dynamic mode

∙ 3 transitions/peptide

∙ 1 min detection window

∙ 1 acquisition method/run

10 µg plasma digest on-column 100 fmol balanced SIS mix on-column

NAT peptides SIS peptides

Overview of Data Analysis Strategy

Protein Quantitation via Peptide Std Curves

L1 L2 L3 L4 L5 L6 L7

~1:1 of SIS:NAT

∙ Use balanced SIS mixes since give lower analytical variation.

∙ Protein concs (NATconc) determined from regression equation of control.

∙ RR is measured from the sample, while SIS concentration, slope, and y-intercept are fixed values.

Percy AJ et al. Biochim. Biophys. Acta 1844, 2014, 917.

Automated Analysis via Qualis-SIS

[Calculated SIS] [Actual SIS]

x 100%

Qualification Criteria

Mohammed Y et al. J. Proteome Res. 2015 (PMID: 25546269).

Automated extraction of attributes and performance

metrics in just seconds.

Target Customer AssayMap Technology Components

Microchromatography Cartridges quantitative binding & elution

Positive Displacement Pipetting Syringes interface directly with cartridges and enable precise, controlled liquid flow through cartridges with no air bubbles to disrupt binding

Automated workflows designed for analytical chemists Simple User Interface Uses customer language - not automation language

15 Author | 00 Month Year Set area descriptor | Sub level 1

AssayMAP Technology

Each AssayMAP cartridge is slurry packed, back-pressure tested, and inspected for voids

Manual vs Automated Protocol Analyzing 50 samples

1 • Sodium Deoxycholate (Na-DOC)

2 • TCEP

3 • Iodoacetamide

4 • DTT

5 • Trypsination

6 • Addition of SIS peptides

7 • Precipitate Na-DOC

8 • Centrifugation

9 • Supernatant Manual SPE

10 • Lyophilization

11 • Rehydrate LC-MS/MS analysis

Man

ual w

ork:

4

h, D

ay 1

1 • Urea

2 • TCEP

3 • Iodoacetamide

4 • TCEP + Dilution

5 • Trypsination

6 • Addition of SIS peptides

6 • SPE LC-MS/MS analysis

Assa

yMAP

: 1

.5h,

Day

1

Man

ual w

ork:

2

.5h,

Day

2

Overnight: 16h, Day 1

Assa

yMAP

: 1

h, D

ay 2

Overnight, Day 2

1h, Day 3

Preliminary SPE Optimization

• C18 cartridges (part no. G5496-60013) • stationary phase: silica-based C18 resin • 150 Å pore size, 16% carbon load, pH 2-8 stability

• RP-S cartridges (part no. G5496-60034) • stationary phase: underivatized polystyrene-divinylbenzene • 100 Å pores, 15-20 µm particles, pH 1-13 stability

Evaluated AssayMap-Compatible Cartridges

18 Author | 00 Month Year Set area descriptor | Sub level 1

Elution volumes (µL)

Acetonitrile (%)

Trifluoroacetic acid (0.1 %)

Formic acid (0.1%)

10/15/20 50 X

10/15/20 50 X

10/15/20 60 X

10/15/20 60 X

10/15/20 70 X

10/15/20 70 X

Overview of tested elution conditions (~100 µg of plasma digest loaded onto cartridges)

Comparison of C18 and RP-S cartridge Performance Elution conditions: 10 µL of 60% AcN, 0.1% FA (100 µg loaded)

Hydrophilic proteotypic peptides

Hydrophobic proteotypic peptides

0

0.2

0.4

0.6

0.8

1

1.2

Rel

ativ

e R

espo

nse

C18RP-S

Action Solvent Volume (µL) Flow rate (µL/min) Activation 50% AcN 100 300

Equilibration 0.1% TFA 50 10

Loading Plasma digest (0.25 µg/µL) 400 10

Cup wash 0.1% TFA 50 10

Internal cartridge wash 0.1% TFA 50 10

Elution 70% AcN, 0.1% TFA 13* 5

The wells of the collecting plate wer already filled with 90 µL of acidified water (0.1% FA), final protein concentration typically ~1 µg/µL

Optimised SPE protocol C18 cartridges

*3 µL is discarded prior collection, i.e. 10 µL is collected

1 • Urea

2 • TCEP

3 • Iodoacetamide

4 • TCEP + Dilution

5 • Trypsination

6 • Addition of SIS peptides

6 • SPE

Automated MRM Workflow

LC-MRM analysis

20 identical samples subjected to fully automated MRM workflow

Automated MRM Workflow Repeatability

0

2

4

6

8

10

12

14

0 - 3 3 - 6 6 - 9 9 - 12 12 - 18

# of

Pro

tein

s

CV Range (%) for NAT conc.

23 Author | 00 Month Year Set area descriptor | Sub level 1

Standard Curve for Apolipoprotein A-1 in Reference Plasma

LOQ: 16 fmol/µL

Dynamic range: 10000

Results for [Apolipoprotein A-1] in Patient Plasma

CV = 4.5%

Summary Results LC-MRM measurements of 35 Human Plasma Proteins

Automated Workflow AssayMAP

Proteins quantified 35

Dynamic range 10000

Average coefficients of determination (r2) 0.99

LOQ range 5 fmol/µL – 11500 fmol/µL

CV* range (35 proteins measured across 20 identical samples) (1-12) %

*Alpha-1B-glycoprotein (CV = 15%)

Conclusions and Outlook • MRM with SIS peptide approach has emerged as a popular and precise

technique for protein quant of candidate protein biomarkers

• Bravo AssayMAP platform merits:

• Simple automation (no programming skills required)

• Frees time for skilled labor

• High-throughput (96-384 samples)

• Unique liquid-driven elution enabling 100x enrichment

• Excellent precision and repeatability

• Automated sample preparation for automated MRM protein quant:

• Biomarker Assessment → discovery and verification

• Future work:

• Investigation of the intra-/inter-assay precision

• Final comparison to the manual prep workflow will also be conducted

Acknowledgements

UVic-Genome BC Proteomics Centre Andrew Percy, PhD Nicole Sessler, PhD Christoph Borchers, PhD

MRM Proteomics

Christoph Borchers, PhD

Gary Kruppa, PhD

Gothenburg University

Martin Uhrbom Master Thesis Student

Agilent Technologies

Jason Russell, PhD

Steve Murphy, PhD Zach Van Den Heuvel, PhD

AssayMAP Bravo Platform

Steve Murphy, Ph.D.

AssayMAP technology components Target Customer

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AssayMAP Bravo

AssayMAP Cartridge

Simple User Interface

AssayMAP Bravo Liquid Handler

• Features • 96 probe syringes • No air gap between probes and cartridge bed • Precision flow control across cartridge bed • Chromatographic behavior

• Benefits • Highly reproducible results • Quantitative binding and elution • Highly efficient washing • Very small elution volumes • Process 8-96 samples in parallel

AssayMAP Bravo Liquid Handler Head

AssayMAP Cartridges

Packed Bed

Top Frit

Bottom Frit

• Features • 5 µL packed bed • Each cartridge is back pressure tested • Cartridges are packed with a variety of resins

• Benefits • Highly reproducible results • Consistent capacity • Address multiple workflows

AssayMAP User Interface

• Features • Easy-to-use interface • Designed for scientists doing sample preparation not automation experts • Minimal user inputs required • Harmonized interfaces for different applications

• Benefits • Allow highly skilled scientist to do more value added work • Minimal training required • Rapid Adoption • Simple protocol transfer between people and sites.

Protein Sample Prep Workbench

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Workflow Library

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App Library

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Utility Library

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In-Solution Digestion user interface

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Peptide Cleanup user interface

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Peptide Cleanup user interface

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AssayMAP digestion and cleanup workflow

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Each step in the workflow was performed using the AssayMAP Bravo

AssayMAP tools for antibody purification: PA-W (Protein A) & PG-W (Protein G)

Input: • Particulate-free solutions

containing antibodies • Up to 96 samples (1 plate) • Up to 1 mL

Output: • Highly purified antibodies in

aqueous buffer • Elution volume as little as

10 µL Up to 100x

concentration factor

PA-W PG-W

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Each step in the workflow can be performed with an AssayMAP Bravo

AssayMAP mAb quantification workflow

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AssayMAP Bravo Platform Low variability • Minimize total workflow variability by more reducing sample prep • Reduce replicate number to increase throughput

Decrease labor • Minimize hands on time • Allow highly skilled scientist to do more value added work

Easy-to-use software control • Designed for non-automation experts • Minimal training required enabling rapid adoption • Simple person to person or site to site transfers

Single platform for a wide variety of sample prep needs

Solutions from Samples to Answers

Separation Preparation Analysis

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