Sample Preparation Automation and QQQ Workflows for

Peptide Quantitation in DMPK

Alex Zhu & Yanan Yang

LCMS Applications Scientist

Agilent Technologies

Why is Agilent Workflow a Good Choice for DMPK?

AssayMAP

Automated Sample Prep

Quantitation with confirmation

Simple MRM method development

Great Robustness

Excellent Reproducibility

Ultra High Sensitivity

wide dynamic range

2

Sample Prep. Method Dev. Data

Acq.

Data

Ana.

Agilent Workflow for DMPK

Skyline

AssayMAP Bravo MH Quant 6495 QQQ

tMRM Skyline

6495 QQQ

App Library

All Ions MSMS

3

AssayMAP Technology Components

Microchromatography

Cartridges Quantitative binding & elution

Protein purification

• PA-W (protein A)

• PG-W (protein G)

• SA-W (streptavidin)

Reversed-phase cleanup:

• C18 (peptide)

• RP-S (peptide)

• RP-W (denatured mAbs)

Peptide Fractionation:

• SCX

• RP-S

• C18

Phosphopeptide enrichment:

• TiO2

• Fe(III)-NTA

Positive Displacement Pipetting Syringes interface directly with cartridges and

enable precise, controlled liquid flow through

cartridges with no air bubbles to disrupt binding

Simple User Interface Uses customer language - not

automation language

• Affinity (protein) Purification

• In-Solution Digestion

• Peptide Cleanup (desalting)

• Protein Cleanup (desalting)

• Phosphopeptide Enrichment

• IMAC Cartridge Customization

• Fractionation

• Sample Normalization

• Liquid handling utilities

Target Customer

Automated workflows designed for analytical chemists

4

Workflow Library

5

Workflows

6

Purification Reproducibility and Recovery Using

PG-W Cartridges

7

1

2 3

4

5 6

Consistent and Robust

Across 96

sample replicates

EIC overlays of mAb peptides from a single row of samples (n = 12)

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• Affinity

purification

• Denaturing

• Reduction

• Alkylation

• Digestion

AssayMAP Power Users

Daniel Spellman (Merck, West Point, USA)

Field: large molecule pharmacokinetics

AssayMAP Application: enrichment, digestion, cleanup

Dan has made AssayMAP a critical part of a workflow used at Merck. AssayMAP

provides significant reductions in both consumable cost and time to results. No

more messy mag beads!

Jacob Jaffe, The Broad Institute in Cambridge and MIT, USA

Field: High-Throughput Proteomics

AssayMap Application: mAb & phosphopeptide enrichment, digestion, cleanup

"Using the combination of extremely consistent, parallelized digestion with

automated reverse-phase cleanup via AssayMAP, at a scale appropriate for

ultrasensitive proteomics applications, has enabled us to contemplate collaborative

studies of previously unheard-of scales and throughput.”

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Kevin Bateman (Merck-WP, European Bioanalysis Forum, Barcelona Nov 2014

Magnetic Bead AssayMAP

Comparison Standard vs. High Throughput

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6495 QQQ LC/MS - Premium Performance

Improved sensitivity (IDL / MDL) – Average 3x in S/N for peptides comparing to 6490

Improved precision and excellent accuracy at the lowest levels

Proven 6 orders of linear dynamic range

Proven robustness in complex matrix –biological matrix (plasma)

Improved mass range (2250), fast scan speed and MRM acquisition rate

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Enhancing Sensitivity for Higher Flow LC Using

More Efficient Ionization of Peptides

2 x10

0.2

0.4

0.6

0.8

1.0

Acquisition Time (min) 3 3.5 4 4.5 5 5.5 6 6.5 7

1 2 3 4 5 6 7 AJS normalized response

ESI relative response

MS Inlet

Heated Sheath Gas

Thermal Gradient Focusing Region

Heat Sink with Active Cooling

Agilent JetStream interface:

• Thermal gradient focusing electrospray

• Usable with flow rates from 10 µL/min and up

• Yields 3-5x increase in sensitivity for peptides

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Agilent Jet Stream

Off On

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Enhancing Sensitivity by Increasing Ion Sampling

and Transmitting

Samples

more ions

Removes

neutrals

Gives

higher

ion yield

High

Pressure

Stage 1

Low

Pressure

Stage 2

Existing

RF Ion

Guide

6 Bore

Capillary

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6495 QQQ Technologies

Continued Development

1 2

3

• New Detector with High Energy Conversion Dynode

• Improved ion detection with low noise 3

• New Tapered Hexapole Collision Cell

• Effective ion collection and transmission 2

Proven iFunnel Technology • Agilent Jet Stream

• Hexabore Capillary

• Dual Ion Funnel

• Increased ion generation

• Enhanced ion sampling

• New Enhanced Q1 Ion Optics

• Improved ion transmission 1

15

Collision Energy Optimization Using Skyline SW

CE

Replicate

0

10

20

30

40

50

60

P e

a k

A r e

a (

1 0

3

)

CE

Replicate

0

10

20

30

40

50

60

P e

a k

A r e

a (

1 0

3

)

Opt CE

Skyline SW will export MRM list or acquisition method to run on Agilent QQQ

with optimal CE for each transition

Agilent QQQ and Skyline Software

Automation Tool for Automatic CE optimization

Automation tool automatically

creates QQQ methods and

acquires data for CE

optimization, reloads and

analyzes the results, exports

the final optimized QQQ

method (MRM, dMRM, tMRM)

and use it to runs any real

samples queued up in the

worklist

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Agilent QQQ and Skyline Software

Automation Tool for Automatic CE optimization

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Challenges

No prediction for representative peptides/preferred charge

state.

Numerous runs for collision energy optimization based on the

number of proteins and peptides.

Questionable prediction for modified peptides.

Thursday, September 03, 2015

Mayo CMSL Mass Spec Overview

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y3+

y5+ y12++

y14++

y8+ y9+

y10+ y11+ y7+

CE=0

CE=25 V

mAb Tryptic Digest

p++

P 3+

P 2+

Simplified MRM Method Development: Agilent All Ions MS/MS on a QTOF + Skyline SW

Thursday, September 03, 2015

Mayo CMSL Mass Spec Overview

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Method Development for Multiple Proteins

Method development for multiple proteins can be done from the same All Ions result, simultaneously or at a later time.

Import a different protein sequence and load the same All Ions data. Same procedure followed.

Thursday, September 03, 2015

Mayo CMSL Mass Spec Overview

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Skyline Prediction Workflow vs. All Ions-Skyline Workflow

mAb Tryptic Digest Skyline Prediction Workflow All Ions – Skyline Workflow

Number of peptides for CE

optimization

40 3

(Selected by All Ions run)

Injections required for CE

optimization

40

(One inj. for one peptide)

3

Total time required for MRM method

development

40×10 = 400 min (3+1)×10=40 min

(1 is for All Ions run)

Save up to 90% of time and 90% of samples needed for method development

Thursday, September 03, 2015

Mayo CMSL Mass Spec Overview

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Triggered MRM

Threshold

Peptide 3 triggers

Peptide 3 triggers secondary transitions,

collects ‘n’ cycles & goes back to primary cycle

Primary cycle (below threshold)

Triggered cycle (above threshold)

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Quantitation with Confidence

Qualifier to Quantifier Ratio Reference vs. experiment

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Ultra High Sensitivity: Low Attomole LLOD 2.1 mm ID column w AJS and Hexabore = Cap LC Detection

blank

3.0 amol

on-column

5.0 amol

on-column

7.5 amol

on-column

Instrumentation: 1290 UHPLC + Agilent JetStream + 6495 QQQ

Sample: synthetic peptide standard (LVNEVTEFAK) spiked into enolase tryptic digest

Injection volume: 1 µL

LLOD

LLOQ

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Excellent Precision at LLOQ,

2.1 mm ID column w AJS + Hexabore = Cap LC

DDetection

Amount measured Replicates %RSD t (99%) IDL

5.0 amol (LLOQ) n = 10 injections 14.0 2.821 2.0 amol

MDL = t x (%RSD/100) x Amount = 2.821 x (14.0/100) x 5.0 amol = 2.0 amol

Inj # Peak

Area

1 30.5

2 28.7

3 30.4

4 33.91

5 35.1

6 31.5

7 34.5

8 20.7

9 26.7

10 26.6

%RSD 14.0

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Six Orders of Dynamic Range

Zoom-in 5 – 500 amol

LVNEVTEFAK

5 amol – 5 pmol on-column

6 orders of linear dynamic range

R2 = 0.998

LVNEVTEFAK

Calibration Standards (amount on-column; 1 µL injected)

5.0

amol

7.5

amol

15

amol

30

amol

300

amol

3

fmol

30

fmol

300

fmol

3

pmol

5

pmol

%Accuracy 109.8 108.7 105.0 87.1 85.2 81.4 86.4 87.4 105.6 97.5

Reproducibility

(%RSD, n=10) 14.0 16.0 9.4 9.0 1.6 1.2 0.6 0.7 2.1 1.0

RT (%RSD, n=100) 0.12

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Peptide X - 8 Levels, 8 Levels Used, 47 Points, 47 Points Used, 0 QCs

Concentration (ng/ml)

-5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105

R e

s p

o n

s e

s

4 x10

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

y = 141.655763 * x + 1.897223

R^2 = 0.99805733 Type:Linear, Origin:Ignore, Weight:1/x

mAb Peptide in Serum Matrix

Peptide External Calibration Concentration (1 µl injection)

10 amol 25 amol 50 amol 100 amol 250 amol 500 amol 1 fmol 5 fmol

Accuracy (%, n=6) 90.70 106.43 92.83 105.93 96.68 103.63 103.07 99.15

Cal. Conc. %RSD (n=6) 14.38 10.22 16.89 4.36 4.92 3.93 3.09 2.73

Retention Time %RSD (n=48) 0.17%

Range: 10 amol to 5 fmol

R2>0.998

Type: Linear, Origin: Ignore, Weight:1/x

Zoom in: 10-250 amol

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MassHunter Quant: Compound-at-a-glance

Blank

10 amol

25 amol

50 amol

100 amol

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Increased Mass Range Useful for Peptides

QQQ MRM spectrum for glycopeptide: EEQYN[+1606.6]STYR (G1F)

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Proven System Robustness in Complex Matrix:

Protein Quantitation in Plasma Hexabore with six 0.6 mm ID Capillaries is more Robust

and has better peak shape Vs a single large Diameter orifice

• Selected peptides from 42 peptides in the QC sample – normalized to Day 1 response

• Peptide QC samples analyzed daily after every ~25 plasma digest injections

• No significant signal degradation observed after 853 injections of 40 µg plasma

digest per injection and 3.5 weeks of continuous operation

• Response %RSD: 6 - 15

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

140.00%

01/06/14 01/11/14 01/16/14 01/21/14 01/26/14 01/31/14 02/05/14

Apolipoprotein E

L-selectin

Plasminogen

Albumin_serum

Kininogen

Transthyretin

Hemopexin

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Summary

• Automated sample prep: Automated sample cleanup, enrichment

and protein digestion using AssayMap platform

• Sensitive: low attomole LLOQ for peptides with standard flow

• Wide dynamic range: six orders

• Reproducible: small CV at low levels

• Robust: less down time and less need for maintenance

• Simple MRM method development: automated optimization of CE;

All Ions MSMS-Skyline workflow

• Quant with confidence: triggered MRM

• Easy to use quantitative analysis software

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