Targeted mass spectrometry

Marina Zajec Dept. of Neurology and Clinical Chemistry Lab. of Neuro-Oncology/Clinical&Cancer Proteomics

Outline

Introduction to targeted mass spectrometry • When to use targeted mass spectrometry?

• What is required for a targeted mass spectrometry experiment?

• Selected Reaction Monitoring vs. Parallel Reaction Monitoring

Examples with real data 1. Quantitation of low levels HSP90α by Parallel Reaction Monitoring

2. Development of targeted mass spectrometry assay to detect M-protein in multiple myeloma patient serum

LC-MS/MS proteomics strategies

Shotgun proteomics - discovery

Proteins in the mixture are digested and the resulting peptides are separated by liquid chromatography and analyzed by mass spectrometry

Spectra are generated from all detectable proteins in a sample

Results are interpreted by database searching

Semi-quantitative analysis

Targeted proteomics

Mass spectrometer is analyzing a preselected group of proteins

By use of internal standard quantitative values of proteins can be acquired stable isotope labelled (SIL) reference spiked into the sample

When to use targeted approach?

when predetermined sets of proteins need to be measured across multiple samples in a consistent, reproducible and quantitatively precise manner

Examples:

Picotti P., Aebersold R. Nature, 2012.

What is required for a targeted proteomics experiment?

1. Protein(s) of interest, based on: Previous experiments (e.g. Shotgun proteomics)

Scientific literature

Prior knowledge

2. Selection of the target peptides Optimally represent the protein set –

proteotypic peptides

Proteins of interest

Target peptides

Targeted analysis

Target peptides

Measured as surrogates for proteins

Need to fulfill certain criteria:

• Unique to the target protein – proteotypic peptides

• No variable modifications (e.g. methionine present in the amino acid sequence)

• No ragged ends in the sequence (KK, KR, RK, RR)

• Optimal length: 7-15 amino acids

Target peptides - examples

1. KRNGGGR

2. RNGGGKK

3. LEPADFAVYYCQR

4. YGSSPLIFGGGTR

5. ASTLESGVPSR

6. FLIYK

7. FSGSGSGTAFTLTISSLQPDDFATYYCQQYDSPPYTFGQGTK

OK

Ragged ends

Too short/long

PRM

SRM

Selected Reaction Monitoring (SRM) vs. Parallel Reaction Monitoring (PRM)

Targeted proteomics

Coon et al. MCP, 2012.

Orbitrap (Fusion and Lumos)

Triple quadrupole

Method optimization - SRM

1. Selection of optimal transitions

select the fragment ions for each precursor-ion charge state that provide the highest signal intensity and lowest level of interfering signals

2. Retention time assignment – scheduling

3. Collision energy optimization

maximize the SRM signal response for specific peptides or fragments

PRM compared to SRM

High specificity In PRM all product ions are monitored providing high confidence of peptide identification. The high resolution mass analyzer increases specificity (narrower mass window) compared to a SRM.

Reduced interference Compared to SRM, PRM provides data with high mass accuracy, which allows the removal of noise of interfering signals.

Reduced assay development time PRM-based targeted proteomics requires less effort in assay development compared to SRM as fragment ions can be selected post acquisition.

Examples with real data

Quantitation of low levels HSP90α by Parallel Reaction Monitoring

• Comparing selectivity, sensitivity and repeatability of SRM, PRM, and immunoassay

Development of targeted mass spectrometry assay to detect M-protein in multiple myeloma patient serum

• Clinical application of targeted mass spectrometry

• Personalized proteomics

Example 1

HSP90 (low ng/mL level) quantification

43 control sera

SRM (2D-LC)

ELISA (2 microtiter plates)

SRM by Xevo TQs

PRM (2D-LC)

PRM by Orbitrap Fusion anti-HSP90

Triple quadrupole High resolution MS

Performed by C. Guzel

Selection of stable isotope labeled peptides for quantification

MPEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNKEIFLRELISNSSDALDKIRYESLTDPSKLDSGKELHINLIPNKQDRTLTIVDTGIGMTKADLINNLGTIAKSGTKAFMEALQAGADISMIGQFGVGFYSAYLVAEKVTVITKHNDDEQYAWESSAGGSFTVRTDTGEPMGRGTKVILHLKEDQTEYLEERRIKEIVKKHSQFIGYPITLFVEKERDKEVSDDEAEEKEDKEEEKEKEEKESEDKPEIEDVGSDEEEEKKDGDKKKKKKIKEKYIDQEELNKTKPIWTRNPDDITNEEYGEFYKSLTNDWEDHLAVKHFSVEGQLEFRALLFVPRRAPFDLFENRKKKNNIKLYVRRVFIMDNCEELIPEYLNFIRGVVDSEDLPLNISREMLQQSKILKVIRKNLVKKCLELFTELAEDKENYKKFYEQFSKNIKLGIHEDSQNRKKLSELLRYYTSASGDEMVSLKDYCTRMKENQKHIYYITGETKDQVANSAFVERLRKHGLEVIYMIEPIDEYCVQQLKEFEGKTLVSVTKEGLELPEDEEEKKKQEEKKTKFENLCKIMKDILEKKVEKVVVSNRLVTSPCCIVTSTYGWTANMERIMKAQALRDNSTMGYMAAKKHLEINPDHSIIETLRQKAEADKNDKSVKDLVILLYETALLSSGFSLEDPQTHANRIYRMIKLGLGIDEDDPTADDTSAAVTEEMPPLEGDDDTSRMEEVD

Primary structure of HSP90α

No known modifications or problematic cleavage sites

High quality stable isotope labeled peptides are required for correct quantitation

Performed by C. Guzel

SRM/PRM assay 2D-LC approach (SCX prefractionation)

+ stable isotopes (references)

mRP C18

digestion

peptides proteins

13 C/ 15 N label

SCX prefractionation

0 2 4 6 0

5

10

concentration

i n t e

n s i t y

SpeedVac

heavy light

Calculation concentration

(ratio heavy/light)

n = 43 control sera SRM/PRM

Based on 2 HSP90α peptides:

YIDQEELNK

DQVANSAFVER

RP-LC SCX-LC

Performed by C. Guzel

Targeted method

4 target HSP90 peptides (time scheduled):

light heavy light heavy

Performed by C. Guzel

SRM SRM

heavy peptide light peptide

PRM

heavy peptide light peptide

PRM

SRM vs PRM on serum digest ∼ 60 ng/mL HSP90

Interfering peak

• High resolution data obtained by PRM • No or less interfering peak detected by PRM

Performed by C. Guzel

Distribution fragments obtained by SRM and PRM

1 11 21 31 410

50

100

s a m p le n o .

pe

rce

nta

ge

(%

)

Y ID Q E E L N K y 7 e n d o g e n o u s

Y ID Q E E L N K y 6 e n d o g e n o u s

Y ID Q E E L N K y 5 e n d o g e n o u s

1 11 21 31 410

50

100

sample no.

perc

enta

ge (%

)

YIDQEELNK y7 endogenous

YIDQEELNK y6 endogenous

YIDQEELNK y5 endogenous

peptide y5 y6 y7 YIDQEELNK

(endogenous) 632.33 760.38 875.41

mean %CV 31.8 47.7 3.9

peptide y5 y6 y7 YIDQEELNK

(endogenous) 632.33 760.38 875.41

mean %CV 13.3 9.9 1.1

Pure compound (reference)

Pure compound (reference)

SRM

PRM

Performed by C. Guzel

Distribution ratio’s of fragments obtained by SRM and PRM

1 1 1 2 1 3 1 4 10

5 0

1 0 0

s a m p le n o .

pe

rce

nta

ge

(%

)

D Q V A N S A F V E R y 9 e n d o g e n o u s

D Q V A N S A F V E R y 8 e n d o g e n o u s

D Q V A N S A F V E R y 7 e n d o g e n o u s

1 11 21 31 410

50

100

pe

rce

nta

ge

(%

)

D Q V A N S A F V E R y 9 e n d o g e n o u s

D Q V A N S A F V E R y 8 e n d o g e n o u s

D Q V A N S A F V E R y 7 e n d o g e n o u s

s a m p le n o .

peptide y7 y8 y9 DQVANSAFVER (endogenous)

822.41 893.45 992.52

mean %CV 26.9 15.0 22.3

peptide y7 y8 y9 DQVANSAFVER (endogenous)

822.41 893.45 992.52

mean %CV 3.7 2.5 2.9

Pure compound (reference)

Pure compound (reference)

SRM

PRM

Performed by C. Guzel

Comparison of HSP90 levels by SRM, PRM and ELISA

SRM PRM ELISA YIDQEELNK DQVANSAFVER

Peptide LOD (ng/mL) LLOQ (ng/mL) SRM 5.6 17.4 PRM 1.0 2.9 ELISA 0.4 1.2

Peptide LOD (ng/mL) LLOQ (ng/mL) SRM 6.7 20.4 PRM 1.3 3.8 ELISA 0.4 1.2

0 1 0 2 0 3 0 4 00

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

s a m p le n o .

HS

P9

0 (

ng

/mL

)

L O Q P R M /E L IS AL O Q S R M

0 1 0 2 0 3 0 4 00

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

s a m p le n o

HS

P9

0 (

ng

/mL

)

L L O Q S R ML L O Q P R M /E L IS A

Performed by C. Guzel

Correlation SRM vs ELISA

YIDQEELNK DQVANSAFVER

0 1 0 0 2 0 0 3 0 0 4 0 00

1 0 0

2 0 0

3 0 0

4 0 0

c o n c e n tra tio n H S P 9 0 Y ID Q E E L N K (n g /m L )

co

nc

en

tra

tio

n H

SP

90

EL

ISA

(n

g/m

L) 0.764R 2 =

C

Y = 1.6*X - 3.8

0 1 0 0 2 0 0 3 0 0 4 0 00

1 0 0

2 0 0

3 0 0

4 0 0

c o n c e n tra tio n H S P 9 0 D Q V A N S A F V E R (n g /m L )

co

nc

en

tra

tio

n H

SP

90

EL

ISA

(n

g/m

L)

0.652 R 2 =

D

Y = 0.799*X + 27.1

Correlation PRM vs ELISA

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 00

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

c o n c e n tra tio n H S P 9 0 Y ID Q E E L N K (n g /m L )

co

nc

en

tra

tio

n H

SP

90

EL

ISA

(n

g/m

L)

0.878

E

R 2 =

Y = 0.845*X + 13.3

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 00

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

c o n c e n tra tio n H S P 9 0 D Q V A N S A F V E R (n g /m L )

co

nc

en

tra

tio

n H

SP

90

EL

ISA

(n

g/m

L)

0.811R 2 =

F

Y = 0.726*X + 20

Performed by C. Guzel

Bland-Altman plots (method comparison)

2 0 0 4 0 0

-1 0 0

-5 0

0

5 0

1 0 0

A v e ra g e

%D

iffe

ren

ce

+ 9 5 %

- 9 5 %

B ia s

2 5 0 5 0 0

-1 0 0

-5 0

0

5 0

1 0 0

A v e ra g e

%D

iffe

ren

ce

+ 9 5 %

- 9 5 %

B ia s

SRM vs ELISA

PRM vs ELISA

significant different: YES (p < 0.0001)

significant different: NO (p = 0.1581)

YIDQEELNK

YIDQEELNK

Performed by C. Guzel

Summary

PRM is highly reproducible compared to SRM assay to determine HSP90 concentrations in SCX fractionated sera at relative low ng/mL level

PRM could be used as an attractive alternative for ELISA to quantify multiple proteins

highly reproducible in biological samples including sera

Targeted mass spectrometry could be used for personalized cancer diagnostics and follow-up (e.g. in multiple myeloma)

ACKNOWLEDGEMENTS

Radboud University Medical Center Hans Jacobs Patricia Groenen Irma Joosten Alain van Gool

Dept. of Clinical Chemistry, Erasmus MC Yolanda de Rijke Henk Russcher

Dept. of Neurology, Erasmus MC Christoph Stingl Lennard Dekker Coskun Guzel Martijn van Duijn Theo Luider

Dept. of analytical biochemistry, Groningen Natalia Govorukhina Alexander Boichenko Rainer Bischoff