targeted protein and peptide quantitation using agilent lc ......but it is not protein-centric!!...

Yanan Yang, Ph.D.

LC/MS Application Scientist

Agilent Technologies

Santa Clara, CA

Targeted Protein and Peptide Quantitation Using Agilent

LC/MS and Skyline Software

Outlines

Skyline Workflow for Targeted Protein Quantitation

- Skyline based MRM method development

- MassHunter and Skyline automation workflow

- All ions DDA and Skyline workflow

Ultimate Sensitivity and Robustness with Agilent 6495 QQQ

- Low attomole LOQ for peptides

- Six orders of linearity

- Great robustness

- Excellent precision at the lowest levels

- Extended mass range benefits glycopeptides

SUMS_Peptide_Quant_Skyline

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October 13, 2014

Skyline Targeted Proteomics Environment


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• Freely available Windows

client application

• Multi-vendor software

• Initially funded with the

CPTAC project

• Rapidly evolved using

feedback from top

proteomics labs

• Widely used and highly

regarded

October 13, 2014

Skyline and MassHunter Quant Why Does Agilent Support Skyline?

MassHunter

Excellent full-featured quant package

Provides many metrics and navigational

aids such as Compound Browser

MH Acquisition software includes

Peptide Optimizer (pre-dates Skyline)

But it is NOT protein-centric!!

Skyline

Widely used and highly regarded by

leaders in targeted proteomics

Protein-centric organization of

information

More sophisticated transitions prediction

Excellent visualization of optimization

results

Excellent evolution of tools including

SSRCalc, spectral library support etc.


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October 13, 2014

MRM Method Development

• Selection of target proteins

• Selection of target peptides that represent the protein list

• Selection of best charge states and MRM transitions for each

targeted peptide

• Optimization of collision energy for each MRM transition

• Optimization of LC and source conditions

• Application of the assays to the detection and quantitation of

the proteins


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October 13, 2014

Important Criteria

For Peptide:

- Unique to the target protein

- 6-25 amino acids

- Avoid peptides prone to artifactual modifications (oxidation; deamidation)

- Avoid ragged end (2×R;RK;2×K-miss cleavages)

For MRM transition:

- Highest signal with lowest level of interference

- Select product ions with m/z higher than precursors (less interference)


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October 13, 2014

Agilent’s QQQ + Skyline: Collision Energy Optimization


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October 13, 2014

Agilent’s QQQ + Skyline: Workflow Manuals Standard With Installation


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October 13, 2014

Skyline Support for tMRM and MassHunter Method Export


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October 13, 2014

New Automation Link in Skyline


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Automation tool automatically

creates QQQ methods and

acquire data for CE

optimization, reloads and

analyzes the results, export

the final optimized QQQ

method (MRM, dMRM, tMRM)

and use it to runs any real

samples queued up in the

worklist

October 13, 2014

New Agilent Automation Tool For Skyline and the QQQ Acquisition Software


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MRMRT dMRM CE opt dMRM/tMRMQuant

October 13, 2014

Agilent’s QTOF and Skyline

• Data Dependent Acquisition (DDA): MS1 Filtering

• Data Independent Acquisition (DIA)

All ions MS/MS

No isolation in quadrupole

2 or more collision energies applied

Simplify the MRM method development

Wide band isolation

Isolate 10 Da

Typically use same formula as DDA


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Collision Cell

Target Peptides Library or Standard

Skyline

Quadrupole Collision

Cell

Target Peptides Library or Standard

Skyline

October 13, 2014

All Ions MS/MS Skyline QQQ MRM Workflow

• All Ions acquisition on a Q-TOF

- Two channels: 0 V and 30 V

• Load All Ions data into Skyline

- Create new Skyline project and import FASTA

- Select target peptides for each protein of interest

- Select charge state for each peptide precursor

- Select multiple transitions for each peptide (>6)

• Export MRM list or method with CE opt to run on a QQQ

• Load CE opt results into Skyline and export final QQQ

method (MRM, dMRM or tMRM) with optimized CE

• Quantitation using the optimized method on a QQQ


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October 13, 2014

All Ions MS/MS on Agilent QTOF

y3+

y5+ y12++

y14++

y8+ y9+

y10+ y11+ y7+

CE=0

CE=25 V

mAb Tryptic Digest

y15++


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October 13, 2014

All Ions Settings in Skyline SW

MS1 Filtering setting extracts

information from low energy

channel

MS/MS Filtering setting extracts

information from high energy

channel


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October 13, 2014

All Ions Skyline Workflow Step 1: Show p & y Ions for Peptide Confirmation

Precursors (p ions) from low energy channel and products (y ions) from high

energy channel are automatically extracted and plotted together to easily

confirm the identity of the peptides.

P ions including M, M+1, M+2,

extracted from low energy

channel

y ions extracted from high energy

channel

Contaminant


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October 13, 2014

Retention time and peptide sequence confirmation

All Ions Skyline workflow Step 2: Show only p Ions to Select Best Charge State

Best charge state for each peptide is easily determined by comparing p ions only

+2

+3


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October 13, 2014

All Ions Skyline workflow Step 3: Show only p ions to Compare Peptide Abundance

Peptides with the highest signal are easily determined by comparing p ions from the best charge state of all peptides.

Skyline peptide rank refine tool picks top n peptides


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October 13, 2014

All Ions Skyline workflow Step 4: Show Only y Ions to Select Top MRMs

Skyline transition rank refine tool picks top n MRMs


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October 13, 2014

All Ions Skyline workflow Step 5: CE Optimization

CE optimization process can be automated using new Skyline Automation Tool

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


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October 13, 2014

6495 QQQ LC/MS - Premium Performance


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Improved sensitivity (IDL / MDL) – Average 3x in S/N specifications and applications

Improved precision and excellent accuracy at the lowest levels – 4 to 5x in IDL spec

Proven 6 orders of linear dynamic range

Proven robustness in complex matrix – Food matrix and biological matrix (plasma)

Improved mass range, fast scan speed and MRM acquisition rate

October 13, 2014

LVNEVTEFAK: Excellent Precision and Low Amol IDL


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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

October 13, 2014

Peptide Quantitation: Outstanding Sensitivity with Standard Flow Chromatography


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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

October 13, 2014

Peptide Quantitation – 6 Orders of Linear Dynamic Range


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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

October 13, 2014

Peptide Quantitation: Ultimate Sensitivity with Nanoflow Chromatography


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blank

0.25 amol

on-column

0.5 amol

on-column

1 amol

on-column

Instrumentation: 1260 HPLC-Chip/MS + 6495 QQQ

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

Injection volume: 1 µL

LLOD

LLOQ

October 13, 2014

Peptide Quantitation With NanoLC/MS – Over 5 Orders of Linear Dynamic Range


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0.5 amol – 100 fmol

R2 = 0.99996

5.5 orders of linear dynamic range

LVNEVTEFAK Calibration Standards (amol/µL; 1 µL injected)

0.5 1 10 100 1000 10,000 100,000

%Accuracy 103.2 80.2 84.58 88.9 85.6 97.6 100.4

Reproducibility (%RSD, n=5) 4.52 8.50 5.75 4.71 1.23 1.09 1.69

RT (%RSD, n=35) 1.4

Zoom-in 0.5 – 10 amol

October 13, 2014

Proven System Robustness in Complex Matrix: Protein Quantitation in Plasma


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• 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

October 13, 2014

Increased Signal (60-120%) with Higher HED


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-15 kV

-20 kV-50

0

50

100

150

204

.08

59

366

.13

85

528

.19

03

119

9.5

976

51

125

3.6

008

22

135

2.6

692

36

139

2.5

833

145

1.7

376

5

146

5.6

342

48

153

8.6

395

159

4.6

768

41

161

1.7

682

99

166

7.7

581

28

169

3.7

452

55

171

2.8

159

77

176

4.7

823

68

187

7.8

664

32

189

3.8

534

85

199

2.8

933

76

199

2.9

218

99

217

6.9

781

68

222

7.8

787

HE

D V

olt

ag

e

Resp

on

se I

ncre

ase (

Rela

tive t

o -

10 k

V)

Product Ion (m/z)

Anti-HER2/neu mAb Peptides

-15 kV

-18 kV

-20 kV

October 13, 2014

Increased Mass Range Useful for Peptides


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

October 13, 2014

Increased Mass Range Useful for Peptides


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MRM Spectrum Skyline Overlay of MRM Chromatograms

MRM Spectrum for IgG Peptide: GFYPSDIAVEWESNGQPENNYK

October 13, 2014

Summary

• Agilent LCMS coupled with Skyline software enables a simple

automated method development workflow for targeted proteomics

and Biopharm research.

• The Agilent 6495 Triple Quadrupole LC/MS achieves low attomole

sensitivity for peptides. The calibration curves show excellent

linearity with six orders of dynamic range with great accuracy and

reproducibility.

• With a complex plasma digest matrix, the 6495 QQQ showed

excellent response and retention time reproducibility over an

extended period (Total of 34 mg of plasma digest injected over 3.5

weeks!)

• Improved performance in peptide quantitation can be achieved by

the increased precursor ion transmission, enhanced detection

efficiency (HED voltage up to -20kV) and extended m/z range of the

6495 QQQ

October 13, 2014


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THANK YOU !

October 13, 2014


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targeted protein and peptide quantitation using agilent lc ......but it is not protein-centric!!...

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