1

Evaluation of SRM-, PRM- and DIA-based Targeted Quantification

Ehwang Song1, Tujin Shi1, Hui Wang1, Anil K. Shukla1, Thomas L. Fillmore1, Yuqian Gao1, Song Nie1, Ronald J. Moore1, Matthew J. Gaffrey1, Athena A. Schepmoes1, Karin D. Rodland1,

Richard D. Smith1, Wei-Jun Qian1, and Tao Liu1

1Pacific Northwest National Laboratory, Richland, WA, USA

Biological Sciences Facility (BSF) & Computational Sciences Facility (CSF)

Environmental Molecular Sciences Laboratory (EMSL)

Outline

Introduction

Initial Evaluation of Targeted Proteomics Performance

Large Scale Demonstration- Quantification of EGFR Pathway Proteins With/Without Fractionation

Summary

2

Targeted Proteomics Workflow

3

Peptides

…

Transition Selection &

Optimization

SRM

PRM & DIA

K^R^

Isotopically labeled peptides

Sample

46.0 46.2 46.4Retention Time

0

200

Inte

nsi

t y(1

0^3)

46.1

46.0 46.2 46.4

Retention Time

0

1

Inte

nsi

t y(1

0^6)

46.1

Light (or endogenous)

Heavy

47.4 47.6 47.8 48.00

200

400

Inte

nsity

(10^

3 )

47.7+0.3 ppm

47.4 47.6 47.8 48.0

Retention Time

0

0.5

1

Inte

nsity

(10^

6 )

47.7+0.8 ppm

47.4 47.6 47.8 48.00

200

400

Inte

ns i

ty(1

0^3 )

47.7+0.3 ppm

47.4 47.6 47.8 48.0

Retention Time

0

0.5

1

Inte

ns i

ty(1

0^6 )

47.7+0.8 ppm

Method including Precursor (Isolation

Window) m/z & Retention Time

Post-data Refinement

Method including Transition m/z,

Collision Energy, and Retention Time

SRM: Selected Reaction MonitoringPRM: Parallel Reaction MonitoringDIA: Data Independent Acquisition

Light (or endogenous)

Heavy

Aim: Evaluation of Targeted Quantification Performance

4

1. SRM and PRM: A.C. Peterson et al., Mol Cell Proteomics, 2012, 11, 1475.2. SRM and PRM: S. Gallien et al., J Proteomics, 2013, 81, 148.3. SRM and DIA: T. Schmidlin et al., Proteomics, 2016, 16, 2193.4. SRM, PRM and DIA: T. Kockmann et al., Proteomics, 2016, 16, 2183.5. IS-PRM: S. Gallien et al., Proteomics, 2015, 14, 1630.6. TOMAHAQ: B.K. Erickson et al., Mol Cell, 2017, 65, 361.

PRISM Fractionation

Same LC System

Sensitivity

Selectivity

AccuracyReproducibility

Throughput

Experimental Design

5

1. Reverse Response Curve Matrix: Shewanella oneidensis (bacteria)

2. Large Scale DemonstrationTargets: 133 EGFR Peptides

23 Heavy Stock Concentration

(fmol/uL)

0

0.0025

0.0125

0.025

0.125

0.25

2.5 Throughput Sensitivity

Sensitivity Selectivity Accuracy

Reproducibility

SRM: TSQ Vantage; PRM&DIA: Q Exactive-HFTechnical triplicate

PRISM Fractionation

No Fractionation

vs.

PRISM: Shi et al., Proc Natl Acad Sci, 2012, 109, 15395.

Linear Dynamic Range of Response: SRM ≥ PRM > DIA

6

y = 1.0184x - 0.0043R² = 0.9979

y = 1.0125x + 0.0128R² = 0.9902

y = 0.9709x - 0.0136R² = 0.9841

9.77E-04

3.91E-03

1.56E-02

6.25E-02

2.50E-01

1.00E+00

4.00E+00

9.77E-04 3.91E-03 1.56E-02 6.25E-02 2.50E-01 1.00E+00 4.00E+00

Log2

[H/L

]

Averaged Log2[H/L]

SRM

PRM

DIA

SRM PRM DIA

1.00x2-9

1.00x2-7

1.00x2-5

1.00x2-3

1.00x2-1

1.00x21

1.00x23

1.00x25

Log2

[H/L

]

3 Orders

LOQ Values: SRM ≤ PRM < DIA

7

1.00E+00

4.00E+00

1.60E+01

6.40E+01

2.56E+02

1.02E+03

LOQ

(log

2[pg

/mL]

)

SRMPRMDIA

SRM, PRM <(=) DIA

LOQ Values: SRM ≤ PRM < DIA

8

1.00E+00

4.00E+00

1.60E+01

6.40E+01

2.56E+02

1.02E+03

LOQ

(log

2[pg

/mL]

)

SRMPRMDIA

SRM=PRM :10 SRM < PRM: 7 PRM < SRM : 5

PRM: Selectivity Sensitivity

9

y = 1292.0x - 169.33R² = 0.9935

y = 1305.4x - 135.91R² = 0.9926

y = 1326.2x - 138.32R² = 0.9926

3.91E-03

3.13E-02

2.50E-01

2.00E+00

1.60E+01

1.28E+02

1.02E+03

8.19E+03

1.95E-03 1.17E-02 7.03E-02 4.22E-01 2.53E+00

Mea

sure

d H

eavy

pep

tide

(fg/m

L)

Spiked Heavy Peptide (fmol/uL)

SRM

PRM

DIA

ERRFI1_NSPSLFPCAPLCERSRM PRM DIA

Retention Time

30 310

0.51

1.530.5

+1.8 ppm

30 31Retention Time

0500

10001500 30.5

-1.1 ppm ND

y9+y8+y5+

Inte

nsity

(10^

6)In

tens

ity

30.5

0

530.5

+0.8 ppm

30.5

0

1030.5

+0.5 ppm

y9+y8+y5+

Inte

nsity

(10^

6)In

tens

ity (1

0^3)

29.0 29.50

100

200

30029.1

29.0 29.5Retention Time

0

200

400 29.1

Inte

nsity

(10^

3)In

tens

ity

Y9+y8+y12++

S/N:6

30.0 30.50

1

2 30.3+1.7 ppm

30.0 30.5Retention Time

0

4000

800030.3

+0.2 ppm

Inte

nsity

(10^

6)In

tens

ity

30.0 30.50

530.3

+0.8 ppm

30.0 30.5

Retention Time

0

5030.3

+0.7 ppm

Inte

nsity

(10^

6)In

tens

ity (1

0^3)

29.0 29.50

100200300400

29.1

29Retention Time

0100020003000

29.1

Inte

nsity

(10^

3)In

tens

ity

PRM: Selectivity Quantification Accuracy

Potential matrix interference in SRMLow mass resolution and accuracy afforded by QqQ mass spectrometer

High accuracy for PRMHigh resolution and tight mass tolerance applied for data acquisition and extraction 10

in HMEC cell line

1.56E-02

6.25E-02

2.50E-01

1.00E+00

4.00E+00

1.60E+01

Log2

[H/L

]

Heavy Peptide Conc. (ng/mL)

SRMPRMDIA

HRAS_LVVVGAGGVGK

3.91E-03

3.13E-02

2.50E-01

2.00E+00

1.60E+01

Log2

[H/L

]

Heavy Peptide Conc. (ng/mL)

KRAS_SYGIPFIETSAK

Reproducibility among Three Techniques

11

Spiked in Shewanella oneidensis Spiked in HMEC

SRM PRM DIA

40

30

20

10

0

SRM PRM DIA

CV

(%)

40

30

20

10

0

Large Scale Demonstration: Quantification of EGFR Pathway Proteins

12

EGFR pathway One of the most important pathwaysOne of the most well-studied signaling networksUnderlying diseases (dysregulation of signaling networks)Key players (low abundance regulatory proteins) and their PTMs

133 peptides/ 32 proteins (including 6 phosphopeptides)Quantification with and without PRISM fractionationSample

PRISM: 25 ug of peptidesNo PRISM: 0.5 ug of peptides

www.qiagen.com/productsOda et al., Mol Syst Biol 2005, 1:2005.0010.

Shi et al., Sci Signal 2016, 9(436):rs6.

PRISM

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PRISM: high-pressure high-resolution separations with intelligent selection and multiplexing

200 nL/min

2 µL/min

2.2 µL/min

Heavy stock of 133 EGFR pathway peptides spiked in HMEC cell line digests

Time (min)

QQQ MS

Q1 Q2 Q33 µm, C18 column(200 µm i.d. × 50 cm)

1.7 µm, C18 column(75 µm i.d. × 25 cm)

iSelection

96 well plate

On-line monitoring heavy peptides

Capillary LC (high pH)

Nano LC (low pH)

#2 #6 #10

Multiplexing: 12 concatenated fractions

SRM PRM & DIA

Shi et al., Proc Natl Acad Sci, 2012, 109, 15395.

Quantification of EGFR Pathway Proteinsby LC vs. PRISM-S/PRM and -DIA

14

* grey: non-detected

53 10546 9916 64

-14.90

-11.58

-8.260

-4.940

-1.620

1.700

Log2[L/H]

PRIS

M-S

RM

PRIS

M-P

RM

PRIS

M-D

IA

LC-S

RM

LC-P

RM

LC-D

IA20

40

60

80

100

120N

umbe

r of P

eptid

es

Dynamic Range of Quantification: PRM ≥ SRM >DIA

15

LC-S

RM

LC-P

RM

LC-D

IA-18

-16

-14

-12

-10

-8

-6

-4

-2

0

2

4

Log2

[L/H

]

3 Orders 5 Orders

PRIS

M-S

RM

PRIS

M-P

RM

PRIS

M-D

IA

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

2

4

0505 0 ( 02 468 ( 00005 012 ( 3 0000 2 4 0 00 (8

XICs of Very Low Abundant Peptide Quantified by PRISM

16

L/H: 0.0045L/H: 0.0028

PRISM-SRM PRISM-PRM

SPRY4_LQHPLTILPIDQVK (copies/cell: ~900)

N/D

PRISM-DIAy6+y4+y6++

y5+y4+y6++

y6+y5+b7+

Summary

Sensitivity: SRM ≥ PRM > DIASelectivity: PRM > DIA > SRMAccuracy: PRM > DIA > SRMReproducibility: SRM ≥ PRM > DIAThroughput: SRM ≥ PRM > DIA

In combination with the front-end fractionation, overall quantification performance was improved for all three techniques. 17

SRM PRM DIAPost-Data

Refinement

Front-end Assay

Development

MultiplexingThroughput

Reproducibility

Accuracy

SelectivitySensitivity Post-Data

Refinement

Front-end Assay

Development

MultiplexingThroughput

Reproducibility

Accuracy

SelectivitySensitivity Post-Data

Refinement

Front-end Assay

Development

MultiplexingThroughput

Reproducibility

Accuracy

SelectivitySensitivity

Acknowledgements

18

The Integrative Omics Group at PNNL