Targeted mass spectrometry Marina Zajec Dept. of Neurology and - - PowerPoint PPT Presentation

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

MPEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNKEIFLRELISNSSDALDKIRYESLTDPSKLDSGKELHINLI PNKQDRTLTIVDTGIGMTKADLINNLGTIAKSGTKAFMEALQAGADISMIGQFGVGFYSAYLVAEKVTVITKHNDDE QYAWESSAGGSFTVRTDTGEPMGRGTKVILHLKEDQTEYLEERRIKEIVKKHSQFIGYPITLFVEKERDKEVSDDEAEE KEDKEEEKEKEEKESEDKPEIEDVGSDEEEEKKDGDKKKKKKIKEKYIDQEELNKTKPIWTRNPDDITNEEYGEFYKSLT NDWEDHLAVKHFSVEGQLEFRALLFVPRRAPFDLFENRKKKNNIKLYVRRVFIMDNCEELIPEYLNFIRGVVDSEDLP LNISREMLQQSKILKVIRKNLVKKCLELFTELAEDKENYKKFYEQFSKNIKLGIHEDSQNRKKLSELLRYYTSASGDEMV SLKDYCTRMKENQKHIYYITGETKDQVANSAFVERLRKHGLEVIYMIEPIDEYCVQQLKEFEGKTLVSVTKEGLELPED EEEKKKQEEKKTKFENLCKIMKDILEKKVEKVVVSNRLVTSPCCIVTSTYGWTANMERIMKAQALRDNSTMGYMAA KKHLEINPDHSIIETLRQKAEADKNDKSVKDLVILLYETALLSSGFSLEDPQTHANRIYRMIKLGLGIDEDDPTADDTSA AVTEEMPPLEGDDDTSRMEEVD

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

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 41 50 100 s a m p le n o . p e rc e n ta g e (% ) 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 41 50 100 sample no. percentage (%) 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 1 5 0 1 0 0 s a m p le n o . p e rc e n ta g e (% ) 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 41 50 100

p e rc e n ta g e (% ) 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

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

s a m p le n o . H S P 9 0 (n g /m L )

L O Q P R M /EL ISA L O Q S R M

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

sam p le no H S P 9 0 (n g /m L )

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

Performed by C. Guzel

Correlation SRM vs ELISA

YIDQEELNK DQVANSAFVER

1 0 0 2 0 0 3 0 0 4 0 0 1 0 0 2 0 0 3 0 0 4 0 0 con cen tratio n H S P 90 Y ID Q E E LN K (ng /m L) c o n c e n tra tio n H S P 9 0 E L IS A (n g /m L )

0.764

R

C

Y = 1.6*X - 3.8

1 0 0 2 0 0 3 0 0 4 0 0 1 0 0 2 0 0 3 0 0 4 0 0 co n ce n tra tion H S P 9 0 D Q V A N S A F V E R (n g /m L) c o n c e n tra tio n H S P 9 0 E L IS A (n g /m L )

0.652

R

D

Y = 0.799*X + 27.1

Correlation PRM vs ELISA

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 con cen tratio n H S P 90 Y ID Q E E LN K (ng /m L) c o n c e n tra tio n H S P 9 0 E L IS A (n g /m L )

0.878

E R

Y = 0.845*X + 13.3

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 co n ce n tra tion H S P 9 0 D Q V A N S A F V E R (n g /m L) c o n c e n tra tio n H S P 9 0 E L IS A (n g /m L )

0.811

R

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

5 0 1 0 0

A v e ra g e % D iffe re n c e + 9 5 %

• 9 5 %

B ia s 2 5 0 5 0 0

• 1 0 0
• 5 0

5 0 1 0 0 A v e ra g e % D iffe re n c e + 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