Targeted mass spectrometry - MolMed...Introduction to targeted mass spectrometry • When to use...
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Targeted mass spectrometry Marina Zajec Dept. of Neurology and Clinical Chemistry Lab. of Neuro-Oncology/Clinical&Cancer Proteomics
Transcript of Targeted mass spectrometry - MolMed...Introduction to targeted mass spectrometry • When to use...
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
