peptide quant workshop santa clara site€¢ ultimate sub-attomole sensitivity for synthetic peptide...

Peptide Quant Workshop

Santa Clara Site

Christine Miller

Omics Market Manager

December 2015

Topics

6495 QQQ

Standard flow vs Nanoflow

Software tools

Automation

Your thoughts

December 2, 2015


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Proven iFunnel Technology

December 2, 2015


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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 noise

Agilent Jet Stream Ion Generation

Nozzle voltage

MS inlet

Resistive samplingcapillary

Nebulizing gas

Heated drying gas

LC sample inlet

Super-heated sheath gas

The collimated thermal containment zone creates a dramatically “brighter source”

ESI

AJS

Jet Stream Proteomics

Agilent’s unique solution for proteomics that uses the 1290+Jet Stream source with our ion funnel MS systems

Jet Stream proteomics is• Used for both discovery and targeted experiments• 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

December 2, 2015

Jet Stream Proteomics Kickoff

5

Why is Agilent Jet Stream Technology an Advantage?

Agilent Jet Stream shows mass-flow dependent behavior • Sensitivity depends on absolute

amount (mass) injected• Using smaller i.d. columns with

standard sources does NOT increase sensitivity!

Jet Stream source provides 3-5x signal increase compared to ESI

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

December 2, 2015


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2x10

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

Counts vs. Acquisition Time (min)

1 2 3 4 5 6 7 8 9

2.1 mm column

0.5 mm column

575.5937.5

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

AJS normalized response

ESI relative response

Journal Article Demonstrates Agilent Jet Stream Performance

December 2, 2015


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Buckenmaier S, Miller CA, van de Goor T, Dittmann MM. J Chromatogr. A 2015, 1377:64-74.

Continual HPLC Innovation

December 2, 2015


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Infinity II series

A-line fittings

AdvanceBio columns

6495 QQQ TechnologiesContinued Development

December 2, 2015


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12

3

• New Detector with High Energy Conversion Dynode

• Improved NEG ion detection with low noise 3

• New Tapered Hexapole Collision Cell• Effective ion collection and transmission2

• New Enhanced Q1 Ion Optics• Improved ion transmission1

Proven iFunnel Technology• Agilent Jet Stream• Hexabore Capillary• Dual Ion Funnel• Increased ion generation• Enhanced ion sampling

Quantitative Applications• Enhanced peak area response• Improved peak area %RSD• More sensitive and precise• Lower Limits of Detection (IDL)and

Quantitation (LLOQ)• More reliable and robust

Improved Sensitivity and Precision – Peak Area %RSDReserpine (+) and Chloramphenicol (-) at low levels, 5 fg on-column

12/2/2015

6495 Webinar

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6495 QQQ Avg. Area = 157Area %RSD = 6.1

6490 QQQAvg. Area = 47Area %RSD = 10.4



5 fg reserpine (+) (n=10) 5 fg chloramphenicol (-) (n=10)

3 x higher area responseLower area %RSD

3 x higher area responseLower area %RSD

• The improved sensitivity of the 6495 QQQ LC/MS system results in enhanced peak area response and improved area precision (%RSD), especially at the low levels

• This ultimately leads to lower instrument detection limits (IDLs) compared to previous designs

2x10

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

1.1

1.15

1.2

1.25

1.3

1.35

Counts vs. Acquisition Time (min)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 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

2x10

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

1.1

1.15 1

Counts vs. Acquisition Time (min)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 3.4 3.6

Nanoflow LC vs. Standard Flow

NanoflowProvides the ultimate sensitivity

Less sample means less trypsin and SIS peptides are required

Lower solvent consumption

Standard flowEasy to learn and use

Fast run times

Flexibility in columns

Robustness

Large loading capacity

Label-free quant possible


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5 µg of protein digest requires only 80 nL of plasma!!!!100 µg digest is 1.7 µL plasma

December 2, 2015

December 2, 2015


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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

1.111.741 5833

12.053 839


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

4.428 5092


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

Superior Standard Flow Chromatography with the AdvanceBio Peptide Mapping ColumnPacking has 120Å pore size with superficially porous 2.7 μmparticles

Specially tested with a challenging peptides mix to ensure reliable peptide mapping performance

Exceptional resolution and speed for UHPLC, and excellent results for conventional HPLC too

Available in a range of column lengths including 250 mm!

December 2, 2015


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Comparison of Standard Flow and Nanoflow Chromatography: Sharper Peaks with UHPLC

December 2, 2015


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4x10

0

1

2

3

4

5

6

7

8

39.969 0.932

45.177 0.573

5x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.544.966 1.870

41.594 1.864


38 40 42 44 46 48

4x10

1

2

3

4

5

6

49.719 0.473

58.367 0.337

5x10

1

2

3

4

5

58.100 2.217


48 50 52 54 56 58 60

4x10

0

1

2

3

4

5

6

7

70.047 0.686

72.143 0.451 84.349 0.56680.251 0.608

5x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

82.836 2.650

78.518 1.553


68 70 72 74 76 78 80 82 84 86

Jet StreamProteomics

NanoLC

Stability of Standard Flow LC/MS

December 2, 2015


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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

December 2, 2015


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LVNEVTEFAK - 11 Levels, 11 Levels Used, 110 Points, 110 Points Used, 0 QCs

Concentration (amol on-co

0 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 5000000

Re

spo

nse

s

7x10

-0.2

0

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

y = 5.884906 * x - 2.453268 R^2 = 0.99761971 Type:Linear, Origin:Ignore, Weight:1/x

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

Concentration (amol on-co

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

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

Excellent Precision and Low Attomole Level IDL

December 2, 2015


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Amount measured Replicates %RSD t (99%) IDL5.0 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

Injection # 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

1x10

62.312 29

1x10

62.308 30

1x10

62.312 18

1x10

62.321 24

1x10

62.315 26


1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

1x10

6

2.314 29

1x10

6

2.319 26

1x10

6

2.309 29

1x10

6

2.308 32

1x10

6

2.309 32


1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

5 amol

5 amol

5 amol

5 amol

5 amol

5 amol

5 amol

5 amol

5 amol

5 amol

• Excellent peak area precision (%RSD) are observed at the lowest levels (LLOQs)

Ultimate Sensitivity with Nanoflow LC

December 2, 2015


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• Ultimate sub-attomole sensitivity for synthetic peptide spiked in tryptic digest using the 6495 with nanoflow chromatography

• Wide (over 5 orders) linear dynamic range• Excellent precision and accuracy are observed at all levels including the LLOQ

level

LVNEVTEFAK0.5 amol – 0.1 pmol> 5 OrdersR2 = 0.99996

Levels (amol) %RSD %Accuracy

0.5 4.52 103.21 8.50 80.210 5.75 84.6100 4.71 88.91000 1.23 85.6

10,000 1.09 97.6100,000 1.69 100.4

Robustness Test Strategy

12/2/2015

6495 Webinar

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• Used MRM Proteomics kits to establish initial performance and monitor performance (10 µg load, 40 min method)

• Injected large amounts of plasma digest using short chromatography to accelerate “dirtying” of the system (40 µg load, 15 min method)

• No cleanup of plasma digest and no flow diversion –maximized dirtying of system

Proteomics MRM Kit• To establish baseline

response

40µg plasma digest• To dirty the instrument

(10 min runs)

Proteomics MRM Kit• Every 50 injections• To monitor MRM area

response

Robust Standard Flow Chromatography: 40 SIS Peptides in Plasma

6x10

0.1

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0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.73.032 10.725

7.706

4.834

16.779

8.3845.898 15.13912.224 13.703 19.6723.813 6.6405.443 16.0809.811 21.63318.250

1 1


2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Overlay of 238 transitions from 40 peptides in QC kit from MRM Proteomics

Proven System Robustness in Complex Matrix: Protein Quantitation in Plasma

12/2/2015

6495 Webinar

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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

Post-Robustness Test Cleaning of Source

12/2/2015

6495 Webinar

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After rinsing with water, a biofilm could be seen which was peeled off from the spray shield and endplate.

Increased Signal (60-120%) with Higher HED

ASMS 2014

24

15 kV

20 kV

0102030405060708090

HED

Volta

ge

Resp

onse

Incr

ease

(Rel

ativ

e to

10

kV)

Product Ion (m/z)

Enolase Digest

15 kV

18 kV

20 kV

-15 kV

-20 kV-50

0

50

100

150

204.

0859

366.

1385

528.

1903

1199

.597

651

1253

.600

822

1352

.669

236

1392

.583

314

51.7

3765

1465

.634

248

1538

.639

515

94.6

7684

116

11.7

6829

916

67.7

5812

816

93.7

4525

517

12.8

1597

717

64.7

8236

818

77.8

6643

218

93.8

5348

519

92.8

9337

619

92.9

2189

921

76.9

7816

822

27.8

787

HED

Vol

tage

Res

pons

e In

crea

se (R

elat

ive

to -1

0 kV

)

Product Ion (m/z)

Anti-HER2/neu mAb Peptides

-15 kV-18 kV-20 kV

Increased Mass Range Useful for Peptides

ASMS 2014

25

2x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

3

3.25

3.5

3.75

4

4.25

4.5

4.75

5

5.25

5.5

5.75

204.

0859

0

366.

1385

0

1392

.583

30

1538

.639

50

528.

1903

0

2227

.878

70

1920

.771

00

Counts vs. Mass-to-Charge (m/z)

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200

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

Increased Mass Range Useful for Peptides

ASMS 2014

26

MRM Spectrum Skyline Overlay of MRM Chromatograms

2x10

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

1594

.676

84

1465

.634

25

1764

.782

37

1693

.745

26

1877

.866

43

2176

.978

17

1992

.893

38

Counts vs. Mass-to-Charge (m/z)

1400 1500 1600 1700 1800 1900 2000 2100 2200

Predicted5.0

4.4 4.5 4.6 4.7 4.8 4.9 5.0Retention Time

0

1000

2000

3000

4000

5000

Inte

nsity

y19 - 2176.9782+ y17 - 1992.8934+y16 - 1877.8664+ y15 - 1764.7824+y14 - 1693.7453+ y13 - 1594.6768+y12 - 1465.6342+

5.04.5 4.6

4.7

MRM Spectrum for IgG Peptide: GFYPSDIAVEWESNGQPENNYK

Software Tools

December 2, 2015


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December 2, 2015


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December 2, 2015


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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

AFO PS 11/19/2013

30

Skyline Software: Agilent Automation Tool for Automatic CE Optimization

Export directly from Skyline to MPP

December 2, 2015


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December 2, 2015


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Import QQQ data and metadata into MPPNew correlation analysis tools 5991-4984EN

5991-5165EN

MassHunter Quant Software

December 2, 2015


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Quantifier/Qualifier ratiosFlag outliersCalculate absolute concReporting

MassHunter Quant: Compounds at a Glance

December 2, 2015


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Rapid visualisation of large data sets

Absolute vs Relative Quant

Absolute Quant

‘Gold standard’

Normalise variation in LC/MS analysis (but not sample prep)

Allows comparison between studies and techniques

SIS peptides useful for interference testing

Relative Quant (Label-free)

Cheap

No waiting for peptides in the post

50% less transitions (longer dwell times)

Absolute quant not always required

December 2, 2015


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Label-free Quant of Pregnancy Specific Proteins

December 2, 2015


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Raw

pea

k ar

ea

All 20 samples MARS-14 depleted, digested and desalted

QQQ as a Discovery Platform

December 2, 2015


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Map proteins onto pathwaysIdentify pathways and biology

KEGG, BioCyc, WikiPathways

Increase pathway coverage

• Export proteins to Skyline

• Export metabolitesto new method

Find discriminatingProteins

t-test, ANOVA

Automated Sample Preparation

December 2, 2015


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The Bravo platform

96-well liquid handling platform• 96LT head (5-250ul)• 96ST head (x-xul)• AssayMap Head

Shakers, heaters, wash station etc.

Bravo can do pipetting

AssayMAP Bravo can do pipetting and chromatography

December 2, 2015


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AssayMAP Technology

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

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December 2, 2015

AssayMAP Cartridge Portfolio

Affinity PurificationProtein A and G Cartridges

Protein Digestion(protocol only - no cartridge)

Peptide Clean UpC18 & RP-S Cartridge

FractionationSCX, C18 & RP-S Cartridge

Workflows: combine individual Apps into powerful workflows

Purify Digest Cleanup

Peptide Sample Prep for mass spec analysis

N-glycan Sample Prepfor HPLC, CE or mass spec analysis

Release and Label Glycans

Applications

EnrichmentTiO2

Agilent’s Peptide Quant Solutions

Maximum performance, maximum productivity

Highest specification QQQ on the market

Tightest integration with Skyline

Use MRM as a discovery tool as well as validation

Digest, clean-up and LC-MRM analysis on 96 samples in one week.

Use AM Bravo for protein/peptide enrichment or fractionation

December 2, 2015


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Requested feedback

Do you agree with the perceived benefits of standard flow?Is giving up nanoflow too much of a risk?• How can we minimize that risk?

What limits your peptide quant experiments?• Availability of samples?• Time taken for sample prep?• Time taken for LC-QQQ analysis?• Time for data analysis?• Cost per sample?

What are your priorities? What can get funded?• Walk-away time• Reproducibility• Clever chromatography• Flexibility in applications

December 2, 2015


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peptide quant workshop santa clara site€¢ ultimate sub-attomole sensitivity for synthetic peptide...

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