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Introduction to
Protein/Peptide Quantitation Using Normal Flow LC/MS
High Sensitivity Protein/Peptide
Quantitation enabling Biomarker
Translational Research
Overview
Introduction
• Mass Spectrometry and Proteomics
Increasing Sensitivity
• Low Flow and Normal Flow
• Agilent JetStream and Agilent iFunnel technology
Increasing Robustness
• Reproducible analysis with complex samples
Conclusion
For Research Use Only. Not for use in diagnostic
procedures.
Mass Spectrometry and Proteomics
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles
Proteomics is the study of the structure and function of proteins and how they interact within a complex biological system
Since the emergence of many OMICS discovery technologies, thousands of putative biomarkers have been identified and published harkening the increase in translational opportunities for developing new therapeutics destined for the clinics
For Research Use Only. Not for use in
diagnostic procedures.
For Research Use Only. Not for use in
diagnostic procedures.
Agilent 1290-6490 System
Attomole MS Sensitivity
Robustness and Reproducibility
For Research Use Only. Not for use in
diagnostic procedures.
Low-Flow LC/MS Standard-Flow LC/MS
Increasing Sensitivity for Protein Quantitation
For Research Use Only. Not for use in diagnostic
procedures.
• Cap- and Nano-Flow LC provide
excellent sensitivity
• Requires more skill to maintain
and use
• On complex samples, may hit
limit of column capacity (75 µm
id columns)
• Sub-2 micron columns provide
outstanding chromatographic
performance (speed, resolution,
loading capacity)
• Rapid, robust analysis
Enhancing Sensitivity for Higher Flow LC Using More Efficient Ionization of Peptides
2 x10
0.2
0.4
0.6
0.8
1.0
Acquisition Time (min) 3 3.5 4 4.5 5 5.5 6 6.5 7
1 2 3 4 5 6 7 AJS normalized response
ESI relative response
MS Inlet
Microflow LC/MS
Nebulizer Heated Sheath Gas
Thermal Gradient Focusing Region
Heat Sink with Active Cooling
Agilent JetStream interface:
• Thermal gradient focusing electrospray
• Usable with flow rates from 10 µL/min and up
• Yields 3-5x increase in sensitivity for peptides
For Research Use Only. Not for use in diagnostic
procedures.
JetStream Interface Allows Robust LC/MS with High Sensitivity
1 fmol on-column
2.1 mm
column
0.5 mm
column 575.5937.5
2.1 mm column
300 µL/min
100 amol – 10 pmol
0.5 mm column
17 µL/min
100 amol – 1 pmol
For Research Use Only. Not for use in diagnostic
procedures.
•SIX bores
•HALF as long
Six bores, Half the restriction
means…
• 6 times the amount of
atmospheric gas sampled
AND
• 10X the number of ions
sampled over wide mass
range.
But how do we handle all the extra gas molecules?
Compare:
Standard
Capillary is
180mm
6 Bore
Capillary
Enhancing Sensitivity by Increasing Ion Sampling
For Research Use Only. Not for use in diagnostic
procedures.
Enhancing Sensitivity by Transmitting More Ions
Samples
more ions
Removes
neutrals
Gives
higher ion
yield
For Research Use Only. Not for use in diagnostic
procedures.
Impact of Ion Funnel on Sensitivity
Observed a 5-10x increase in sensitivity
Linear dynamic range on 6460 LC/MS QQQ (std flow):
200 fmol to 25 pmol on-column
Linear dynamic range on 6490 LC/MS QQQ (std flow):
20 amol to 25 pmol on-column 6460 QQQ
6490 QQQ with iFunnel technology
1 fmol on-column
LVNEVTEFAK
575.5 937.5
For Research Use Only. Not for use in diagnostic
procedures.
Increasing Robustness: Reproducibility for 110 Injections (10 fmol SIS Peptides and 2.5 µg Plasma Digest On-column)
Protein Response
%RSD
Ret. Time
%RSD
Adiponectin:
IFYNQQNHYDGSTGK 9.8 0.13
Antithrombin-III :
DDLYVSDAFHK 4.7 0.16
Apolipoprotein A-II precursor:
SPELQAEAK 6.7 0.12
Apolipoprotein C-III:
GWVTDGFSSLK 2.3 0.08
Ceruloplasmin :
EYTDASFTNR 9.6 0.14
Heparin cofactor II:
TLEAQLTPR 6.1 0.15
Histidine-rich glycoprotein:
DGYLFQLLR 3.4 0.02
Kininogen-1:
TVGSDTFYSFK 3.3 0.13
L-selectin:
AEIEYLEK 9.5 0.15
Plasminogen:
LFLEPTR 2.2 0.13
Vitamin D-binding protein:
THLPEVFLSK 3.0 0.12
von Willebrand Factor:
ILAGPAGDSNVVK 9.5 0.15
The samples were provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre
2.2% RSD
n=110
4.7% RSD, n=4
7.9% RSD, n=4
12.3% RSD, n=4
Plasminogen LFLEPTR
For Research Use Only. Not for use in diagnostic
procedures.
Absolute quantification
of serum apolipoprotein A-I and B
by SID-MRM-MS
in clinical research
Christa Cobbaert, PhD, clinical chemist
Department of Clinical Chemistry and Laboratory Medicine,
Leiden University Medical Center, Leiden, The Netherlands
SID-MRM-MS approach for assay development
of high abundant serum apo A-I and B
For Research Use Only. Not for use in diagnostic
procedures.
Serum Desirable specification
CVw CVg I (%) B (%) TE (%)
Apo A-I 6.5 13.4 3.3 3.7 9.1
Apo B 6.9 22.8 3.5 6.0 11.6
CVw; within-subject biological variation, CVg; between-subject biological variation
I; desirable specification for imprecision, B; desirable specification for inaccuracy
TE; desirable specification for allowable total error
Biological variation
Reproducibility of the SID-MRM-MS method
for serum apo A-I and B
CV (%) normal poolserum (NPS 1) CV (%) normal poolserum (NPS 2)
Apo A-I Instrumental Intrarun Interrun Instrumental Intrarun Interrun
AKPAL 0.9 2.9 4.2 1.6 3.3 4.9
VQP 1.7 2.7 4.9 1.5 4.6 7.2
QGLL 5.1 3.2 8.6 3.2 5.6 7.1
VSFL 3.5 4.3 7.8 3.8 5.6 8.2
Apo B
SVSL 3.8 3.1 6.4 5.4 1.0 4.5
FPEV 2.3 0.8 5.2 1.0 3.3 5.6
TEVIP 3.0 1.8 4.2 0.1 1.4 4.6
TGISP 1.0 1.6 4.3 0.7 0.2 5.1
Instrumental = 5 repeated measurements; intrarun = 5 sample work-ups;
interrun = triplicate work-ups on 5 days (N = 15)
For Research Use Only. Not for use in diagnostic
procedures.
R² = 0,9594
0,7
1,2
1,7
2,2
0,7 1,2 1,7 2,2
LC
-MS
g/L
ITA g/L
Apo B SVSL
R² = 0,9821
0,7
1,2
1,7
2,2
0,7 1,2 1,7 2,2
LC
-MS
g/L
ITA g/L
Apo B FPEV
R² = 0,9804
0,7
1,2
1,7
2,2
0,7 1,2 1,7 2,2
LC
-MS
g/L
ITA g/L
Apo B TEVIP
R² = 0,9512
0,7
1,2
1,7
2,2
0,7 1,2 1,7 2,2
LC
-MS
g/L
ITA g/L
Apo B TGISP
R² = 0,9621
0,4
0,8
1,2
0,4 0,8 1,2
LC
-MS
g/L
ITA g/L
Apo B SVSL
R² = 0,9814
0,4
0,8
1,2
0,4 0,8 1,2
LC
-MS
g/L
ITA g/L
Apo B FPEV
R² = 0,988
0,4
0,8
1,2
0,4 0,8 1,2
LC
-MS
g/L
ITA g/L
Apo B TEVIP
R² = 0,9846
0,4
0,8
1,2
0,4 0,8 1,2
LC
-MS
g/L
ITA g/L
Apo B TGISP
Method comparison of SID-MRM-LCMS with a clinical
immunoturbidimetric assay for apo B
serum triglycerides:
0.6 - 2.6 mmol/L
serum triglycerides:
4.1 - 17.8 mmol/L
Y = X
For Research Use Only. Not for use in diagnostic
procedures.
Conclusions
• LC/MS is a powerful tool in the targeted proteomics workflow
with its superior sensitivity and specificity
• Agilent JetStream and Agilent iFunnel technologies allow you
to achieve highly sensitive analyses using standard-flow
LC/MS
• Standard-flow LC/MS provides excellent robustness and
reproducibility for reliable protein/peptide biomarker
quantitation
For Research Use Only. Not for use in diagnostic
procedures.
Acknowledgements
University of Victoria
Derek Smith
Christoph H. Borchers
Leiden University Medical
Centre
Christa Cobbaert
Agilent Technologies
Yanan Yang
Craig Love
Martin Haex
Christine Miller
For Research Use Only. Not for use in diagnostic
procedures.
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
19
AssayMAP Automated Protein Sample Preparation
Rachel Bolger, Product Manager
April 2014
The LC/MS Influence
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
20
Light Chain
Fc
Fab
Glycosylation
site
Disulfide
shuffling
Pyroglutamate
Deamidation/oxidation
Heavy Chain
21
Peptide Mapping Intact molecular
weight Glycan Analysis
A complete menu of automated methods for protein analysis • with method-specific cartridges
• on a single instrument
• with simplified user-interface
The Vision
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
AssayMAP Technology Components
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
22
Robust &
Reproducible quantitative
chromatographic
separation method
provides highly
reproducible results
Easy to Use Standardized user interfaces enable control
over key assay parameters, are easy to use
and make methods simple to transfer
Precision & Increased
Throughput AssayMAP Bravo Liquid Handling
platform provides precision pipetting
and parallel processing
AssayMAP Technology - Components
Why Chromatography? AssayMAP is like microscale HPLC columns, run 96 at a time, yielding chromatographic-like performance
AssayMAP Cartridge Traditional Pipet Tip
Column
• Single-pass contact (chromatography!)
• Precise, controlled flow rate
• Quantitative binding & elution
• Efficient removal of contaminants
• Recover 100% of capture
• Minimal volume (10 µL elution)
• Multi-pass contact (equilibrium adsorption)
• No flow rate control
• Non-quantitative extraction
• Less efficient removal of contaminants
• Recover < 100 % of capture
• Diluted >10 X
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
24
VS
Bat
h
Shower
AssayMAP Cartridge Portfolio
Affinity
Purification Protein A and G
Cartridges
Protein Digestion (protocol only - no
cartridge)
Peptide Clean
Up C18 & RP-S
Cartridge
Fractionation SCX, C18 & RP-
S Cartridge
Workflows: combine individual Apps into powerful workflows
Purify Digest
Cleanup
Peptide Sample Prep
for mass spec analysis
N-glycan Sample Prep
for HPLC, CE or mass spec
analysis
Release and Label
Glycans
Applications (Apps)
Page 25
Software Designed for Scientists Flexible and intuitive software, out of the box
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
26
Workflow-specific
control parameters
Standardized
deck layout
“Pushbutton”
run controls
Complete Spectrum of Scale
Plate
Hub
PlateLoc Centrifuge
AssayMAP Bravo BenchBot Robot
Multidrop Combi
INHECO
Incubator MP
XPeel
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
27
AssayMAP Automated Protein Sample Prep Platform
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
28
Powerful enrichment due to small volume
Total workflow reproducibility
Dramatically reduces human variability
Standardized interfaces are easy to use
Out-of-the box automation
Integrated workflows
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
29
AssayMAP Automated Peptide Sample Preparation Workflow
Rachel Bolger, Product Manager
4/11/2014
Peptide Sample Prep Workflow
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
30
Protein Purification App Protein A or Protein G cartridges
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
31
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
32
Quantitative recovery >95%
Less than 2% CVs
Protein Purification App Quantitative Recovery
30 min/96 samples
Integrates with other workflows
Very reproducible, CVs = <5%
Minimal hands on time
Elution in < 25µl
In-Solution Digestion App Dramatically reduces hands on time
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
33
Peptide Cleanup App
Up to 25X concentration factor!
C18 or RPS Reversed Phase cartridges
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
34
BSA digestion and cleanup
Person to person reproducibility
Low intra- and interday CVs
Low elution volume = powerful enrichment
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
35
Peptide Fractionation App
Simplify peptide samples by stepwise ionic strength or pH elution.
Strong Cation Exchange cartridges
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
36
Fractionation – Optimized
May 4, 2014
Confidentiality Label
37
peptides elute mostly in one fraction
Fractionation – Needs More Optimization
May 4, 2014
Confidentiality Label
38
peptides elute across multiple fractions
E. coli fractionation by SCX
Elution by
increasing ionic
strength (KCl)
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
39
AssayMAP Automated Peptide Sample Prep
May 4, 2014
For Research Use Only. Not for use in diagnostic
procedures.
40
Consistent recovery across mass loads
Powerful enrichment due to:
small elution volume
fractionation
Total workflow reproducibility
Dramatically reduces human variability
Standardized interfaces are easy to use
Out-of-the box automation
Integrated workflows
Recommended