From Biomarker Jaume C.

Morales
Discovery to Validation: LC/MS Product Specialist
Routine Nano-LC/QQQ Agilent Technologies
for Quantitation of
Peptides

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Agilent Proteomics Biomarker Workflow
SAMPLE1 DATA
SAMPLE2 Candidate
Extraction Biomarker
Identification

6520
Depletion QTOF Candidates

Fractionation Proteolytic
Digest
Extraction

6410
QQQ
Identification
Validation

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 Biomarker validation workflow
• Perform statistical
analysis for Step 3
identification of
Spectrum Mill
biomarker candidates
• Run samples on Q- • Search QTOF data
TOF for protein ID in using Spectrum Mill
data-dependent
MS/MS mode • Use Spectrum Mill
MRM Builder to
create a list of MRM
Step 1 transitions with RT

Q-TOF

Step 2 and 5
Mass Profiler Pro
• Run samples on
• Integrate the MRM QQQ in Dynamic
chromatograms MRM mode
• Import quantitation
results into MPP to
perform statistical
analysis Step 4
QQQ

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 Workflow for quantitative peptide analysis using MRM
1. Creation of a (D)MRM acquisiton method from
discovery data
MRM
optimization

QTOF MRM
SpectrumMill MRM Builder
acquisition acquisition

DMRM
Export MRM transitions and optional retention time information acquisition
for optimization or direct (D)MRM acquisition analysis

1. Export transitions from SpectrumMill.wmv

2. Import SpectrumMill results into Optimizer for Peptides.wmv
Workflow for quantitative peptide analysis using MRM
2. In-silico prediction of MRM transitions

SpectrumMill MRM MRM

Peptide 1. + 2. optimization 3.
acquisition
Selector

Predict proteotypic peptides (BLAST search) and import optimized

transitions into MRM method

1. Peptide selector import into MassHunter Qual.wmv

2. Import transitions from MassHunter Qual.wmv
3. Import transitions into MRM method.wmv
Workflow for quantitative peptide analysis using MRM
3. Generation of a (D)MRM acquisition method

MRM MRM DMRM

1. + 2. acquisiton 3.
optimization acquisition

Verify results from Optimizer for peptides and perform (D)MRM analysis

1. Optimizer for peptides data.wmv

2. Import transitions into MRM method.wmv
3. Update RT information for DMRM method.wmv
 Prerequisites for successful profiling and
quantitation of biomarkers
• Sensitivity
• Linearity
Signal Response • Reproducibility

• Separation Speed
• Peak Capacity
• Reproducibility

Mass Spectrum
• Selectivity
• Acquisition Rate

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HPLC-Chip/QQQ LCMS Technology
Nanospray chip configuration brings new era in high sensitivity
quantitation

HPLC Chip Cube system

NanoLC system for
analytical chromatography

CapLC pump for sample

loading on enrichment column

QQQ LCMS

Sensitivity: low -mid amol

Dynamic range: 103 -105

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Triple Quadrupole Mass Spectrometer
Extending Outstanding Performance

6400 Series – Triple Quad – NEW Functionality

No Cross Talk collision cell – commonality with QTOF

Peptide Optimizer

Dynamic MRM – 4000 MRMs

High Sensitivity – 10attomols on peptides

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HPLC-Chip/MS Interface:
Fluid Connections to the HPLC-Chip
Autosampler
Waste
Nanopump
Stator

Side View
Rotor

inner rotor

outer rotor
Rotor

Microvalve
Stator

HPLC-Chip

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Retention Time Reproducibility

RT SD %RSD
EIC 487.8 3.618 0.014 0.40
EIC 752 3.788 0.011 0.29
EIC 740.6 5.018 0.010 0.20
EIC 874.4 3.968 0.012 0.31
EIC 653.6 4.289 0.012 0.28
EIC 511.7 3.681 0.012 0.31
EIC 722.7 3.547 0.012 0.35
Extracted ion chromatograms for 17 peaks from a EIC 778 4.143 0.010 0.23
BSA tryptic digest (50 fmol on-column) EIC 526.3 4.399 0.015 0.34
EIC 547.5 4.472 0.011 0.25
EIC 746.7 5.196 0.011 0.20
EIC 519.1 4.142 0.011 0.26

1-3%RSD Interchip RT EIC 508.2 4.972 0.011 0.23

EIC 582.4 4.679 0.011 0.23
differences – Applied EIC 461.9 3.905 0.012 0.30

Proteomics reference EIC 474 4.759 0.011 0.22

EIC 628 4.584 0.010 0.22

RT reproducibility evaluated using 69 repeat injections

 Ion Intensity Reproducibility
Scatter plot of intensities: Run 1 vs, Run 10
1 Scatter plot of ions intensities
5,5

5
log individual intensity

4,5

4 Replicate 1
Replicate 2
3,5 Replicate 3
Replicate 4
3 Replicate 5

2,5 N =234
2,5 3 3,5 4 4,5 5 5,5

log average intensity 2

 234 ions were monitored over 5 replicate runs

 ~ 94 % of all ions show less than 20% variation in intensity
 5000 glycopetides monitored over 10
across all 5 replicates.
replicate runs.
 HPLC-Chip/TOF
 HPLC-Chip/TOF
1 Data courtesy of Dr. Pierre Thibault University of 2 Data courtesy of Dr. H. Zhang, X.J. Li and Dr. R.
Montréal Immunology and Cancer Research Institute Aebersold, Institut für Molekulare Systembiologie,
Switzerland
Rapid Nanoflow LC/MS – 3 Minute Analysis
21 MRM Transitions From 7 Peptides
MRM Optimizer for QQQ
Programa que permite encontrar los méjores valores de
Fragmentor y/o Energía de colisión
para aquellas transiciones que nos interesan en base a su abundancia.

Es necesario especificar la m/z de precursor y product ion.

Peptide Optimizer for QQQ

Caso particular del MRM optimizer.
Las transiciones se especifican tan sólo detallando el péptido.
El péptido escogido puede provenir de SM Peptide Selector
o bien de MH Qual
¿Que es diferente en Peptide Optimizer?

Secuencia de péptidos en lugar de fórmulas químicas

Precursor generalmente +2 y/o +3 de estado de carga
El espectro MS/MS muestra varios product ions
Algunos product ions tienen m/z > precursor m/z
Es predecible la fragmentación de los deseados iones “b” y “y”
aunque algunos product ions pueden presentar también
multicarga
Típicamente se buscan 2 -3 péptidos por proteína y 2-3
transiciones por péptido.
Desarrollo y Optimización de métodos SRM para
Péptidos
Selección de transiciones basada en
• Observaciones de MS/MS durante el descubrimiento ó
predicciones de iones “b” y “y”.
• Predicción de exclusividad de la secuencia peptídica en la base
de datos.
Optimización de las transiciones
• El parámetro más importante en QQQ es la energía de colisión.
• Debe seleccionar las transiciones que ofrezcan una mayor
relación S/N y menor interferencias en la matriz.
MRM Method Optimizer for Peptides
Ejecutar Optimizer: Importar las secuencias de péptidos, luego ver las
predicciones de fragmentos b/y y seleccionar iones para la optimización.

Run 1 (MRM): Optimiza la CE en modo MRM utilizando la fórmula del Q-TOF

como punto de partida, en un sólo análisis.
Ventajas:
• Reduce considerablemente la cantidad de muestra utilizada.
• Puede optimizar en más de una carga para el mismo péptido.
• Pre-selección de iones b/y (incluyendo multicarga)
• El único límite está en el numero total de transiciones por inyección de
optimización. El propio software nos da información basado en la anchura
de los picos y Cycle Time. Conviene adquirir ≥10 puntos por pico.
Enter Sequences and Select b/y Ions For
Optimization
Results of Peptide Optimization

Peptide
sequence
Precursor

Abundance of each transition

allows customer to choose
Optimized
best transitions for the final
product ions
method

19
Dynamic MRM
 Comparison of MRM and Dynamic MRM
Time Segment 1 Time Segment 2 Time Segment 3 Time Segment 4
Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

MRM

Compounds (10/block) 50 80 100 70

Cycle Time (sec) 0.5 0.8 1 0.7

Dynamic MRM

Max Coincident 20 40 40 30
Cycle Time (sec) 0.4 0.4 0.4 0.4

• 2 x shorter cycle times supports narrow chromatographic peaks, more analytes or longer dwell per
analyte.

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Dynamic MRM – what happens?

Increases
Sensitivity

Improves cycle
time

Provides better
chromatographic
definition

4 ions – not 11 7 ions - not 11

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Dynamic MRM: Retention Time Based Scheduling
of MRM for Optimal Sensitivity
 Absolute Protein UCD Conway Institute
Quantification in the University college Dublin
Context of Non-clinical And
Drug Safety Evaluation Agilent Technologies

Vehicle Low Dose High Dose

Day 2,4,15 Day 2,4,15 Day 2,4,15 Day -3/-4, 1/2, 3/4, 12/13

Histopathology Histopathology Transcriptomics Metabonomics

Transcriptomics Transcriptomics Proteomics Clinical Biochemistry
Proteomics Proteomics Metabonomics
Clinical Biochemistry

Collins B. C. et al. ASMS 2008 MPQ 477

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Experimental Design

Rat liver lysate were prepared from

Catalase was selected based on
rats treated with troglitazone or
previous 2D-DIGE data
vehicle control

Peptides and MRM transitions were

1 mg of soluble protein extract was
selected using Peptide Selector in
reduced, alkylated, acetone
Spectrum Mill and 13C, 15N labeled
precipitated and trypsin digested
peptides were synthesized

The liver digest were spiked with the isotope-labeled peptides

and analyzed by Agilent 6410 QQQ system

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Using Spectrum Mill Peptide Selector for
Optimising MRM Transitions
Chemically reactive residues
(Cys = C, Met = M, Trp = W)

Peptides adjacent to multiple cleavage

site

Chemically unstable residues (Asp-Gly

= D-G; Asn-Gly = N-G; N-term Glu =
E; N-term Asn = N)

Eliminate “LC-incompatible” peptides

Uniqueness

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Spectrum Mill – Peptide Selector

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Peptide Selector – Catalase Results

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Catalase Peptide LAQ – Peptide Selector

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Catalase Peptide EAE – Peptide Selector

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External Calibration on Catalase Peptides
Linearity : five order of magnitude
780 fMol

78aMol External quantitation curve of

catalase peptide L*AQEDPDYGLR
78fMol
from 78 amol to 7800 fmol

0.9965

RSD < 6%

Page 30
Catalase Quantitation Results

Page 31
 Quantitation of protein Erk1 protein was quantified
phosphorylation using from depleted human serum.
MRM

+3 PO4
Erk1
+2 PO4 intact
+4 PO4 protein

Peptide MRM
Lorne
Page 32
Selection of MRM transitions

b14 Precursor ion Product ions

TY: IADPEHDHTGFLTEYVATR
545.3 615.3 782.5
y5 TY
y16 P [M+3H] 3+ y5 b14 2+

t202: IADPEHDHTGFLTEYVATR 753.3 615.3 979.9

y5 t202
[M+2H] 2+ y5 y162+
y16 P
753.3 979.9 695.3
y204: IADPEHDHTGFLTEYVATR
y204
y5 [M+2H] 2+ y162+ y5
y16 P P
780.0 647.6 695.3
t202y204: IADPEHDHTGFLTEYVATR t202y204
y5 [M+2H] 2+ y163+-H3PO4 y5

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Chromatographic Separation of the Four Peptide Standards
allowed the selection of the same Q1 and Q3 transitions for
two different peptides

x10 4 + MRM (545.29999 -> 615.29688) mix500f-r001.d

6 TY
5.5

4.5

4 t202
3.5
t202y204
3

2.5

2 y204
1.5

0.5

0
9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5
Counts vs. Acquisition Time (min)

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 TY13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs
Relative Responses T432tY434y13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs
x10 2 y = 0.3630 * x + 0.0337

Relative Responses
x10 1 y = 0.0764 * x + 0.2516
R^2 = 0.99816859 4 R^2 = 0.99673108
1.9
3.8

Relative Responses
1.8 Relative Responses

1.7
1.6
5fm R2 =0.9981 3.6
3.4
5fm R2 =0.9967
3.2
1.5 2.5fm 3
2.5fm
1.4
2.8
0.5fm
1.3 0.5fm 2.6
1.2 2.4
1.1 2.2
1 2
0.9 1.8
0.8 1.6
0.7 1.4
0.6
0.5
0.4
TY 1.2
1
0.8
t202y204
0.3 0.6
0.2 0.4
0.1 0.2
0 0
-0.1 -0.2

-25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 -25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525
Concentration (fmol/ul) Concentration (fmol/ul)

Y434y13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs T432t13 - 5 Levels, 5 Levels Used, 15 Points, 15 Points Used, 0 QCs
Relative Responses

x10 2 y = 0.3447 * x + 0.1682

Relative Responses
x10 1 y = 0.1069 * x + 0.2840
R^2 = 0.99564716 R^2 = 0.99593214
1.8 5.5

R2 =0.9956 R2 =0.9959
Relative Responses

Relative Responses
1.7
1.6 5
1.5 5f 4.5 5fm
1.4 2.5f m 2.5fm
1.3 0.5f m 4
1.2
m 3.5
0.5fm
1.1
1 3
0.9
0.8 2.5
0.7
2
0.6
0.5
0.4
0.3
y204 1.5

1 t202
0.2
0.5
0.1
0 0
-0.1
-0.5
-25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 -25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525
Concentration (fmol/ul) Concentration (fmol/ul)

Page 35
Quantitation of the degree of phosphorylation at
T202 and Y204 in active Erk1 protein

% Molar
peptide RSD (n=9)
ratio
TY 20% 0.13

t202 25% 0.15

y204 21% 0.12

t202y204 34% 0.08

In this batch of active Erk1 sample, 59% of T202

and 55% of Y204 were phosphorylated

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 From Discovery Mode Peroxidase in Human Plasma
to Validation Step

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 Discovery phase to Validation : MRM Selector

Biomarker validation workflow

Step-2 Step
Spectrum Mill • Import the MRM list Mass Prof
• Run samples on Q- into QQQ Acquisition
TOF for protein ID in • Search QTOF data software • Integrate the MRM
data-dependent using Spectrum Mill • Run samples on QQQ chromatograms
MS/MS mode. • Use Spectrum Mill in Dynamic MRM • Import quantitation
MRM Selector to mode results into MPP to
create a list of MRM perform statistical
transitions with RT analysis
Step-1 Step-3
Q-TOF QQQ

Page 38
Depleted Human Plasma Sample analysis

Replicate LC/MS runs

HPLC – Chip / QTOF
Spiked with 0,0.5 and 5fmol
per 0.5ug plasma

Data Dependent
Protein IDs from
Spectrum Mill

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MRM Selector
Generates MRM method from discovery QTOF data

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Dynamic MRM

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Dynamic MRM
Overlaid 2000 MRM chromatograms acquired in a single run using Dynamic MRM

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Sensitivity :
Peroxidase in plasma matrix (per 0.5ug)

M A B

matrix 500amole 5fmol

RT Cycle Min. Max. # RSD

# %RSD
window time dwell concurrent RT
MRM Area
(min) (ms) (ms) MRM (min)

443 2 1000 16.5 50 2.5 0.038

443 1 1000 29.83 30 3.2 0.016

2000 2 1000 2.75 160 4.5 0.030 Reproducibility

3293 1 1050 2.18 185 4.7 0.025
of MS response
and RT

Page 43
Mass Profiler Professional
Statistical Processing of MRM Data

Four peptides from

peroxidase were
highlighted in green.
The mean of 443 MRM
abundances is
displayed (black) to
show the peptides from
plasma did not vary
from sample to sample.

B1 B2 B3 A1 A2 A3 M1 M2

All Samples

Page 44
Principle Component Analysis
Matrix and 2 different peroxidase levels

Samples at
different
peroxidase
concentrations
were correctly
grouped
together.

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Hierarchical Clustering
comparing different peroxidase concns.

A condition was generated

with peroxidase
concentration color-coded
on the tree branches, along
with the peptide features
labeled on each row. The
heat map is colored from
blue to red, where blue is
low abundance and red is
high abundance. The full
view of all the features is on
the left. The zoom view is M1 M2 A1 A2 A3 B1 B2 B3
on the right.

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