Utilizing Automated Sample Preparation for Sensitive and ......Utilizing Automated Sample Preparation for Sensitive and Reproducible Protein Quantitation with LC/MS Maryann Shen, Ph.D

Utilizing Automated Sample Preparation for Sensitive and Reproducible Protein Quantitation with LC/MS

Maryann Shen, Ph.D.

Automation Marketing Manager

Agilent Technologies, Inc.

Topics

Challenges in peptide quantitation

Automation

Standard flow vs Nanoflow

Software tools

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For Research Use Only. Not For Use in Diagnodtic Procedures.

Challenges in Peptide Quantitation

Efficiently discover the protein of interest

Sample preparation focused on target protein• Specificity• Recovery• Reproducibility

MRM method development that are unique for the protein of interest

Sensitivity of LC/MS detection• Nanoflow vs. regular flow• Mass range

3

AssayMAPAutomated Sample Prep

Quanco

Simplede

R b t

Excellent Reproducibility

Ultra High Sensitivitywide dynamic range

For Research Use Only. Not For Use in Diagnostic Procedures.

AssayMAP Technology Components

Microchromatography CartridgesQuantitative 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• C18Phosphopeptide enrichment:• TiO2• Fe(III)-NTAPositive Displacement Pipetting

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

Simple User InterfaceUses 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

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AssayMAP Technology

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

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Workflow Library

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Workflows

7


Current AssayMAP Application Portfolio

Protein Digestion

Protein/Peptide Clean Up

Fractionation

Antibody Purification

ImmunoaffinityPurification

PhosphopeptideEnrichment

N-glycan Sample Prep

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AssayMAP Application Portfolio-Affinity Purification

Antibody Purification

ImmunoaffinityPurification

Protein A (PA-W) CartridgeProtein G (PG-W) Cartridge

Protein A (PA-W) CartridgeProtein G (PG-W) CartridgeStreptavidin (SA-W) Cartridge

Streptavidin (SA-W) CartridgeImmunoaffinityPurification

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AssayMAP Automation Portfolio-Protein Analysis

Protein Digestion

Peptide Cleanup

Peptide Fractionation

PhosphopeptideEnrichment

No Cartridge used

Reversed-Phase (RP-S) CartridgeReversed-Phase (C18) Cartridge

Strong Cation Exchange (SCX) CartridgeReversed-Phase (RP-S) Cartridge

TiO2 CartridgeFe(III)-NTA Cartridge

Protein Cleanup Reversed-Phase (RP-W) Cartridge

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Purification Reproducibility and Recovery Using PG-W Cartridges

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1

2 34

5 6

Consistent and Robust

Across 96 sample replicates

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

• Affinity purification

• Denaturing• Reduction• Alkylation• Digestion

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All 25 peptides

C18 and RP-W Cartridge: Peptide Recovery

RH = relative hydrophobicity

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Low %CVs for both intra- and interdaydigestion and cleanup for samples prepared using urea or guanidine-based denaturation

Day 1 cleanup = C18Day 2 cleanup = RP-S

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C18 and RP-W Cartridge: Reproducibility


Fe(III)-NTA and TiO2 are the two most common methods for untargeted (discovery) phosphopeptide enrichmentPA-W, PG-W, or SA-W in combination with anti-phosphopeptide antibodies allow more targeted phosphopeptide enrichment

High selectivity• Routinely enrich phosphopeptides (>90%) from complex matrices compared to

starting material having ~1-2% phosphopeptides

Unparalleled reproducibility• %CV of <10%

High Binding Capacity• Capture >95% of phosphopeptides from complex matrices

Phosphopeptide Enrichment Portfolio

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Elution with 1% aqueous NH3 (~ pH 11)

Fe(III)-NTA Cartridge: Small Elution Volume

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150 μg load mass of digested α-casein

Fe(III)-NTA Cartridge: Phosphopeptide Enrichment

92% of identified peptides are phosphopeptides

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Fe(III)-NTA Cartridge: Phosphopeptide Enrichment from Yeast Digests

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Sensitivity: NanoLC vs. Regular Flow LC

Nano LC

Pros: • Ultimate sensitivity

Cons:• Requires more skill to maintain and

use• Column capacity is limited for complex

samples (75 um i.d.)

Regular Flow LC

Pros: • Sub-2 micron columns provide

outstanding chromatographic performance (speed, resolution, loading capacity)

• Rapid, robust analysis

Cons:• Less sensitive than nanoLC

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Enhancing Sensitivity for Higher Flow LC Using More Efficient Ionization of Peptides

2x10

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 34 5 6 7AJS normalized response

ESI relative response

MS Inlet

Microflow LC/MS

NebulizerHeated 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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Proven iFunnel Technology

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Agilent Jet Stream Hexabore Capillary Dual Ion Funnel

• Thermal gradient focusing• Efficient desolvation • Creates an ion rich zone

• Six capillary inlets• Samples x10 times more ion

rich gas

• Removes the gas but captures the ions

• Removes neutral noiseFor Research Use Only. Not For Use in Diagnostic Procedures.

Comparison of Standard (0.3 mL/min), Capillary (17 µL/min) and Nanospray (600 nL/min)

100 amol on-column2x10

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11

2.1 mm column 0.5 mm

column

75 µm column

3x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11

2.1 mm column 0.5 mm

column

75 µm column

1 fmol on-column

575.5937.5 575.5937.5

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Jet Stream Proteomics

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Agilent’s unique solution for proteomics that uses the 1290+Jet Stream source with ion funnel MS systems Jet Stream proteomics is• Enables near nanoflow sensitivity for proteomics• Intended for scientists who are not sample limited (plasma, cell

cultures, plants, food, etc.)• Offers the ease-of-use, robustness and reproducibility associated

with standard flow separations

Combined with sensitivity of ion funnel systems, makes standard flow feasible!


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MRM Analysis of INDISHTQSVSSK in Mouse Plasma

3x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1 11.7415833

12.053839

Counts vs. Acquisition Time (min)11 11.2 11.4 11.6 11.8 12 12.2

3x10

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 4.4285092

Counts vs. Acquisition Time (min)3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9

10x more injected on Agilent Jet Stream system

Jet Stream Proteomics Nanoflow

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Stability of Standard Flow LC/MS

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Retention time stability

Peak area stability

High retention time stability

Narrow RT windows in DMRM

Fewer overlapping transitions

Longer dwell times

Better data


Outstanding Sensitivity with Standard Flow LC

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Levels %RSD(n = 10)

%Accuracy

RT %RSD(n = 100)

5 amol 14.0 109.8

0.12

7.5 amol 16.0 108.715 amol 9.4 105.030 amol 9.0 87.1300 fmol 1.6 85.23 fmol 1.2 81.430 fmol 0.6 86.4300 fmol 0.7 87.43 pmol 2.1 105.65 pmol 10. 97.5

Zoom-in 5 – 30 amol

LVNEVTEFAK 5 amol – 5 pmol6 OrdersR2 = 0.998

• Low attomole sensitivity for synthetic peptide spiked in tryptic digest using the 6495 with standard flow chromatography

• Six orders of linear dynamic range• Excellent precision and accuracy are observed at all levels including the LLOQ

level

Blank

3 amol

5 amolArea %RSD = 14.0, n = 10%Accuracy = 109.8

7.5 amol

LOD

LLOQ

Injection volume = 1 µL


Proven System Robustness in Complex Matrix: Protein Quantitation in Plasma

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• 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

Response Normalized to Day 1 Response


Software Tools

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MRM Method Development: Manual Chromatographic Process

• Create Skyline document

• Add proteins/peptides• Export MRM method• Run MRM analyses

Determine RT

• Import MRM results (determine RT)

• Export CE optimization method

• Run CE optimization

Optimize CE• Import CE optimization

results• Export final RT-

scheduled method• Run final method

Create final method

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Skyline Software: Agilent Automation Tool for Automatic CE Optimization

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MassHunter Quant Software

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Quantifier/Qualifier ratiosFlag outliersCalculate absolute concReporting


SIS Peptides Analysis in Human Plasma12 SIS peptides standard mixture

Biomarker information Peptide Label Compound nameAdiponectin IFYNQQNHYDGSTGK K+6 Adiponectin_IFY_hAntithrombin‐III DDLYVSDAFHK K+6 Antithrombin‐III_DDL_hApolipoprotein A‐II precursor SPELQAEAK K+6 Apolipoprotein A‐II_SPE_hApolipoprotein C‐III GWVTDGFSSLK K+6 Apolipoprotein C‐III_GWV_hCeruloplasmin EYTDASFTNR R+6 Ceruloplasmin_EYT_hHeparin cofactor II TLEAQLTPR R+6 Heparin cofactor II_TLE_hHistidine‐rich glycoprotein DGYLFQLLR R+10 Histidine‐r‐g_DGY_h Kininogen‐1 TVGSDTFYSFK R+6 Kininogen‐1_TVG_hL‐selectin AEIEYLEK K+6 L‐selectin_AEI_hPlasminogen LFLEPTR R+6 Plasminogen_LFL_hVitamin D‐binding protein THLPEVFLSK K+6 Vitamin D‐binding_THL_hvon Willebrand Factor ILAGPAGDSNVVK K+6 von Willebrand_ILA_h

Sample preparation

• SIS peptides are spiked into 2.5 ug/uL non-depleted human plasma tryptic digest solution with final peptide concentrations as 10, 5, 1, 0.75, 0.5, 0.1, 0.075, 0.05, 0.025, 0.01 and 0.005 fmol/uL.

The samples were provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre

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Reproducibility for 110 Injections (10 fmol SIS Peptides and 2.5 µg Plasma Digest On-column)

Protein Response %RSD

Ret. Time %RSD

Adiponectin:IFYNQQNHYDGSTGK 9.8 0.13

Antithrombin-III :DDLYVSDAFHK 4.7 0.16

Apolipoprotein A-II precursor:SPELQAEAK 6.7 0.12

Apolipoprotein C-III:GWVTDGFSSLK 2.3 0.08

Ceruloplasmin :EYTDASFTNR 9.6 0.14

Heparin cofactor II:TLEAQLTPR 6.1 0.15

Histidine-rich glycoprotein:DGYLFQLLR 3.4 0.02

Kininogen-1:TVGSDTFYSFK 3.3 0.13

L-selectin:AEIEYLEK 9.5 0.15

Plasminogen:LFLEPTR 2.2 0.13

Vitamin D-binding protein:THLPEVFLSK 3.0 0.12

von Willebrand Factor:ILAGPAGDSNVVK 9.5 0.15

Plasminogen_LFL_h - 6 Levels, 6 Levels Used, 15 Points, 15 Points Used, 110 QCs

Relative Concentration5 10 50 100 500 1000 5000 10000

Rel

ativ

e R

espo

nses

0.001

0.0025

0.005

0.00750.01

0.025

0.05

0.0750.1

0.25

0.5

0.751

2.5

5 y = 3.602471E-004 * x - 4.854952E-004R^2 = 0.99909469

2.2% RSD n=110

4.7% RSD, n=4

7.9% RSD, n=4

12.3% RSD, n=4

Plasminogen LFLEPTR

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Summary

Agilent peptide quantitation workflow enables • Automated and reproducible

sample enrichment, digestion and clean up

• Software tools to easily develop protein specific quantitation methods

• Sensitive LC/MS instrument to deliver accurate quantitation at ultra low levels

AssayMAPAutomated Sample Prep

Quanco

Simplede

R b t

Excellent Reproducibility

Ultra High Sensitivitywide dynamic range

35


Documents

Utilizing Automated Sample Preparation for Sensitive and ......Utilizing Automated Sample Preparation for Sensitive and Reproducible Protein Quantitation with LC/MS Maryann Shen, Ph.D