Utilizing Automated Sample Preparation for Sensitive and ......Utilizing Automated Sample...
Transcript of Utilizing Automated Sample Preparation for Sensitive and ......Utilizing Automated Sample...
Utilizing Automated Sample Preparation for Sensitive and Reproducible Protein Quantitation with LC/MS
Maryann Shen, Ph.D.
Automation Marketing Manager
Agilent Technologies, Inc.
Topics
Challenges in peptide quantitation
Automation
Standard flow vs Nanoflow
Software tools
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For Research Use Only. Not For Use in Diagnodtic Procedures.
Challenges in Peptide Quantitation
Efficiently discover the protein of interest
Sample preparation focused on target protein• Specificity• Recovery• Reproducibility
MRM method development that are unique for the protein of interest
Sensitivity of LC/MS detection• Nanoflow vs. regular flow• Mass range
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AssayMAPAutomated Sample Prep
Quanco
Simplede
R b t
Excellent Reproducibility
Ultra High Sensitivitywide dynamic range
For Research Use Only. Not For Use in Diagnostic Procedures.
AssayMAP Technology Components
Microchromatography CartridgesQuantitative binding & elution
Protein purification• PA-W (protein A)• PG-W (protein G)• SA-W (streptavidin)Reversed-phase cleanup: • C18 (peptide)• RP-S (peptide)• RP-W (denatured mAbs)Peptide Fractionation:• SCX• RP-S• C18Phosphopeptide enrichment:• TiO2• Fe(III)-NTAPositive Displacement Pipetting
Syringes interface directly with cartridges and enable precise, controlled liquid flow through cartridges with no air bubbles to disrupt binding
Simple User InterfaceUses customer language - not automation language• Affinity (protein) Purification• In-Solution Digestion• Peptide Cleanup (desalting)• Protein Cleanup (desalting)• Phosphopeptide Enrichment• IMAC Cartridge Customization• Fractionation• Sample Normalization• Liquid handling utilities
Target Customer
Automated workflows designed for analytical chemists
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For Research Use Only. Not For Use in Diagnostic Procedures.
AssayMAP Technology
Each AssayMAP cartridge is slurry packed, back-pressure tested, and inspected for voids
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For Research Use Only. Not For Use in Diagnostic Procedures.
Workflow Library
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For Research Use Only. Not For Use in Diagnostic Procedures.
Workflows
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For Research Use Only. Not For Use in Diagnostic Procedures.
Current AssayMAP Application Portfolio
Protein Digestion
Protein/Peptide Clean Up
Fractionation
Antibody Purification
ImmunoaffinityPurification
PhosphopeptideEnrichment
N-glycan Sample Prep
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For Research Use Only. Not For Use in Diagnostic Procedures.
AssayMAP Application Portfolio-Affinity Purification
Antibody Purification
ImmunoaffinityPurification
Protein A (PA-W) CartridgeProtein G (PG-W) Cartridge
Protein A (PA-W) CartridgeProtein G (PG-W) CartridgeStreptavidin (SA-W) Cartridge
Streptavidin (SA-W) CartridgeImmunoaffinityPurification
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For Research Use Only. Not For Use in Diagnostic Procedures.
AssayMAP Automation Portfolio-Protein Analysis
Protein Digestion
Peptide Cleanup
Peptide Fractionation
PhosphopeptideEnrichment
No Cartridge used
Reversed-Phase (RP-S) CartridgeReversed-Phase (C18) Cartridge
Strong Cation Exchange (SCX) CartridgeReversed-Phase (RP-S) Cartridge
TiO2 CartridgeFe(III)-NTA Cartridge
Protein Cleanup Reversed-Phase (RP-W) Cartridge
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For Research Use Only. Not For Use in Diagnostic Procedures.
Purification Reproducibility and Recovery Using PG-W Cartridges
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For Research Use Only. Not For Use in Diagnostic Procedures.
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Consistent and Robust
Across 96 sample replicates
EIC overlays of mAb peptides from a single row of samples (n = 12)
• Affinity purification
• Denaturing• Reduction• Alkylation• Digestion
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For Research Use Only. Not For Use in Diagnostic Procedures.
All 25 peptides
C18 and RP-W Cartridge: Peptide Recovery
RH = relative hydrophobicity
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For Research Use Only. Not For Use in Diagnostic Procedures.
Low %CVs for both intra- and interdaydigestion and cleanup for samples prepared using urea or guanidine-based denaturation
Day 1 cleanup = C18Day 2 cleanup = RP-S
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C18 and RP-W Cartridge: Reproducibility
For Research Use Only. Not For Use in Diagnostic Procedures.
Fe(III)-NTA and TiO2 are the two most common methods for untargeted (discovery) phosphopeptide enrichmentPA-W, PG-W, or SA-W in combination with anti-phosphopeptide antibodies allow more targeted phosphopeptide enrichment
High selectivity• Routinely enrich phosphopeptides (>90%) from complex matrices compared to
starting material having ~1-2% phosphopeptides
Unparalleled reproducibility• %CV of <10%
High Binding Capacity• Capture >95% of phosphopeptides from complex matrices
Phosphopeptide Enrichment Portfolio
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For Research Use Only. Not For Use in Diagnostic Procedures.
Elution with 1% aqueous NH3 (~ pH 11)
Fe(III)-NTA Cartridge: Small Elution Volume
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For Research Use Only. Not For Use in Diagnostic Procedures.
150 μg load mass of digested α-casein
Fe(III)-NTA Cartridge: Phosphopeptide Enrichment
92% of identified peptides are phosphopeptides
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For Research Use Only. Not For Use in Diagnostic Procedures.
Fe(III)-NTA Cartridge: Phosphopeptide Enrichment from Yeast Digests
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For Research Use Only. Not For Use in Diagnostic Procedures.
Sensitivity: NanoLC vs. Regular Flow LC
Nano LC
Pros: • Ultimate sensitivity
Cons:• Requires more skill to maintain and
use• Column capacity is limited for complex
samples (75 um i.d.)
Regular Flow LC
Pros: • Sub-2 micron columns provide
outstanding chromatographic performance (speed, resolution, loading capacity)
• Rapid, robust analysis
Cons:• Less sensitive than nanoLC
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For Research Use Only. Not For Use in Diagnostic Procedures.
Enhancing Sensitivity for Higher Flow LC Using More Efficient Ionization of Peptides
2x10
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 34 5 6 7AJS normalized response
ESI relative response
MS Inlet
Microflow LC/MS
NebulizerHeated 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
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For Research Use Only. Not For Use in Diagnostic Procedures.
Proven iFunnel Technology
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Agilent Jet Stream Hexabore Capillary Dual Ion Funnel
• Thermal gradient focusing• Efficient desolvation • Creates an ion rich zone
• Six capillary inlets• Samples x10 times more ion
rich gas
• Removes the gas but captures the ions
• Removes neutral noiseFor Research Use Only. Not For Use in Diagnostic Procedures.
Comparison of Standard (0.3 mL/min), Capillary (17 µL/min) and Nanospray (600 nL/min)
100 amol on-column2x10
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1.4
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1.8
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2.2
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2.6
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Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11
2.1 mm column 0.5 mm
column
75 µm column
3x10
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Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11
2.1 mm column 0.5 mm
column
75 µm column
1 fmol on-column
575.5937.5 575.5937.5
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For Research Use Only. Not For Use in Diagnostic Procedures.
Jet Stream Proteomics
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Agilent’s unique solution for proteomics that uses the 1290+Jet Stream source with ion funnel MS systems Jet Stream proteomics is• Enables near nanoflow sensitivity for proteomics• Intended for scientists who are not sample limited (plasma, cell
cultures, plants, food, etc.)• Offers the ease-of-use, robustness and reproducibility associated
with standard flow separations
Combined with sensitivity of ion funnel systems, makes standard flow feasible!
For Research Use Only. Not For Use in Diagnostic Procedures.
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For Research Use Only. Not For Use in Diagnostic Procedures.
MRM Analysis of INDISHTQSVSSK in Mouse Plasma
3x10
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1.1 11.7415833
12.053839
Counts vs. Acquisition Time (min)11 11.2 11.4 11.6 11.8 12 12.2
3x10
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Counts vs. Acquisition Time (min)3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9
10x more injected on Agilent Jet Stream system
Jet Stream Proteomics Nanoflow
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For Research Use Only. Not For Use in Diagnostic Procedures.
Stability of Standard Flow LC/MS
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Retention time stability
Peak area stability
High retention time stability
Narrow RT windows in DMRM
Fewer overlapping transitions
Longer dwell times
Better data
For Research Use Only. Not For Use in Diagnostic Procedures.
Outstanding Sensitivity with Standard Flow LC
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Levels %RSD(n = 10)
%Accuracy
RT %RSD(n = 100)
5 amol 14.0 109.8
0.12
7.5 amol 16.0 108.715 amol 9.4 105.030 amol 9.0 87.1300 fmol 1.6 85.23 fmol 1.2 81.430 fmol 0.6 86.4300 fmol 0.7 87.43 pmol 2.1 105.65 pmol 10. 97.5
Zoom-in 5 – 30 amol
LVNEVTEFAK 5 amol – 5 pmol6 OrdersR2 = 0.998
• Low attomole sensitivity for synthetic peptide spiked in tryptic digest using the 6495 with standard flow chromatography
• Six orders of linear dynamic range• Excellent precision and accuracy are observed at all levels including the LLOQ
level
Blank
3 amol
5 amolArea %RSD = 14.0, n = 10%Accuracy = 109.8
7.5 amol
LOD
LLOQ
Injection volume = 1 µL
For Research Use Only. Not For Use in Diagnostic Procedures.
Proven System Robustness in Complex Matrix: Protein Quantitation in Plasma
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• No significant signal degradation observed after 853 injections of 40 µg plasma digest per injection and 3.5 weeks of continuous operation
• Response %RSD: 6 - 15
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
120.00%
140.00%
01/06/14 01/11/14 01/16/14 01/21/14 01/26/14 01/31/14 02/05/14
Apolipoprotein E
L-selectin
Plasminogen
Albumin_serum
Kininogen
Transthyretin
Hemopexin
Response Normalized to Day 1 Response
For Research Use Only. Not For Use in Diagnostic Procedures.
Software Tools
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For Research Use Only. Not For Use in Diagnostic Procedures.
MRM Method Development: Manual Chromatographic Process
• Create Skyline document
• Add proteins/peptides• Export MRM method• Run MRM analyses
Determine RT
• Import MRM results (determine RT)
• Export CE optimization method
• Run CE optimization
Optimize CE• Import CE optimization
results• Export final RT-
scheduled method• Run final method
Create final method
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For Research Use Only. Not For Use in Diagnostic Procedures.
Skyline Software: Agilent Automation Tool for Automatic CE Optimization
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For Research Use Only. Not For Use in Diagnostic Procedures.
MassHunter Quant Software
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Quantifier/Qualifier ratiosFlag outliersCalculate absolute concReporting
For Research Use Only. Not For Use in Diagnostic Procedures.
SIS Peptides Analysis in Human Plasma12 SIS peptides standard mixture
Biomarker information Peptide Label Compound nameAdiponectin IFYNQQNHYDGSTGK K+6 Adiponectin_IFY_hAntithrombin‐III DDLYVSDAFHK K+6 Antithrombin‐III_DDL_hApolipoprotein A‐II precursor SPELQAEAK K+6 Apolipoprotein A‐II_SPE_hApolipoprotein C‐III GWVTDGFSSLK K+6 Apolipoprotein C‐III_GWV_hCeruloplasmin EYTDASFTNR R+6 Ceruloplasmin_EYT_hHeparin cofactor II TLEAQLTPR R+6 Heparin cofactor II_TLE_hHistidine‐rich glycoprotein DGYLFQLLR R+10 Histidine‐r‐g_DGY_h Kininogen‐1 TVGSDTFYSFK R+6 Kininogen‐1_TVG_hL‐selectin AEIEYLEK K+6 L‐selectin_AEI_hPlasminogen LFLEPTR R+6 Plasminogen_LFL_hVitamin D‐binding protein THLPEVFLSK K+6 Vitamin D‐binding_THL_hvon Willebrand Factor ILAGPAGDSNVVK K+6 von Willebrand_ILA_h
Sample preparation
• SIS peptides are spiked into 2.5 ug/uL non-depleted human plasma tryptic digest solution with final peptide concentrations as 10, 5, 1, 0.75, 0.5, 0.1, 0.075, 0.05, 0.025, 0.01 and 0.005 fmol/uL.
The samples were provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre
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For Research Use Only. Not For Use in Diagnostic Procedures.
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
Plasminogen_LFL_h - 6 Levels, 6 Levels Used, 15 Points, 15 Points Used, 110 QCs
Relative Concentration5 10 50 100 500 1000 5000 10000
Rel
ativ
e R
espo
nses
0.001
0.0025
0.005
0.00750.01
0.025
0.05
0.0750.1
0.25
0.5
0.751
2.5
5 y = 3.602471E-004 * x - 4.854952E-004R^2 = 0.99909469
2.2% RSD n=110
4.7% RSD, n=4
7.9% RSD, n=4
12.3% RSD, n=4
Plasminogen LFLEPTR
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For Research Use Only. Not For Use in Diagnostic Procedures.
Summary
Agilent peptide quantitation workflow enables • Automated and reproducible
sample enrichment, digestion and clean up
• Software tools to easily develop protein specific quantitation methods
• Sensitive LC/MS instrument to deliver accurate quantitation at ultra low levels
AssayMAPAutomated Sample Prep
Quanco
Simplede
R b t
Excellent Reproducibility
Ultra High Sensitivitywide dynamic range
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For Research Use Only. Not For Use in Diagnostic Procedures.