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Ultimate Software
Solutions for Improving
Your Productivity
All Ions MS/MS
Multi-omics
Biopharma Solutions
Bernhard Wuest
Christine Miller
Ning Tang
ASMS 2013
September 2013
ASMS eSeminar
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All Ions MS/MS LC/MS Software that
Differentiates Agilent
TOF and Q-TOF Instruments
Liquid Phase Division, LC/MS
Bernhard Wuest LC/MS Director of Applications
Applied Markets
Agilent Confidential
June 9, 2013
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Topics
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What is All Ions MS/MS?
Compound identification with All Ions MS/MS
Quantitation with All Ions MS/MS
All Ions MS/MS-What is it?
Step 1: Fragmentation without isolation on TOF or Q-TOF
a) First Scan: Low fragmentation energy to analyze precursors
b) Second Scan: High fragmentation energy to analyze fragment ions
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Low energy spectrum
High energy spectrum
Shows
Precursor
Ions
Shows
Fragment
Ions
All Ions MS/MS-What is it?
Step 2: Software Extracts, Correlates, and Confirms
a) Find by Formula uses library to extract precursors from low energy MS
channel
b) All Ions MS/MS uses library to extract fragments from high energy
channel, gives coelution score
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Software automatically
matches precursors with
fragment ions:
Coelution Plot with
Coelution Score
The Toolbox for Accurate Mass Screening The complete solution from Agilent
6 September 2013
ASMS eSeminar
Accurate Mass LC/MS Application Kits Untargeted Screening with TOF or Q-TOF
LC/MS Application
Kits
Method
Standards
On Site Training
LC Column
Accurate Mass
MS/MS Library
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Pesticides
-Test Mix: 253 compounds
-DB: 1600+ compounds
-Library: 700+ compounds
Veterinary Drugs
-Test Mix: 146 compounds
-DB: 1000+ compounds
-Library: 600+ compounds
Forensic Toxicology
-Test Mix: 139 compounds
-DB: 9000+ compounds
-Library: 3000+ compounds
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Topics
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What is All Ions MS/MS?
Compound identification with All Ions MS/MS
Quantitation with All Ions MS/MS
Untargeted Drug Screening in Postmortem Blood Compound Identification with All Ions MS/MS
• Higher Productivity in confirmation work
• Easier methods and processes
• Reanalyze without reinjection
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5991-2191EN 5991-2319EN
5991-2465EN
Untargeted Drug Screening in Postmortem Blood All Ions MS/MS Automated Workflow
Step 1: Find by Formula Extracts Precursors using PCDL database
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Qualify Precursor Match with Accurate Mass and Isotopic Distribution
Untargeted Drug Screening in Postmortem Blood All Ions MS/MS Automated Workflow
Step 2: Extract Corresponding Fragment Ions
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Fragments from PCDL
Accurate Mass MS/MS Library for Thousands of Forensic Compounds
Untargeted Drug Screening in Postmortem Blood All Ions MS/MS Automated Workflow
Step 3: Precursor and Fragment Correlation/Coelution
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Overlaid Precursor and
Fragment Ion
Chromatograms
Coelution Plot
Agilent’s UNIQUE Coelution Score Enables Confident Identification
Untargeted Drug Screening in Postmortem Blood Differentiation and Identification of Isomers
Coeluting with ~50x
Concentration Difference
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Alprazolam
Methadone
Worst Case Scenario: Coeluting Isomers at Different Concentrations
Untargeted Drug Screening in Postmortem Blood Differentiation and Identification of Isomers
Similar precursor
m/z values and
overlapping isotopic
clusters
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Worst Case Scenario: Overlapping Isotopic Distribution
Untargeted Drug Screening in Postmortem Blood Compound Confirmation-Methadone
Good Isotopic pattern
and abundance match
Confirmation with
seven qualified
fragments
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Confident ID for High Concentration Coeluting Isomer
Untargeted Drug Screening in Postmortem Blood Compound Confirmation-Alprazolam
Good isotopic pattern
match-despite low
concentration
Alprazolam confirmed
with two qualified
fragments
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AND Confident ID for Low Concentration Coeluting Isomer
High Productivity: Compound Details View
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Co-elution Plot
Overlaid
Chromatograms
Compound List Compound ID Results
Low Energy (MS) Spectrum
High Energy Fragment Spectrum
Topics
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What is All Ions MS/MS?
Compound identification with All Ions MS/MS
Quantitation with All Ions MS/MS
Automatic Creation of Quantitation Method
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Compound results
- Major adducts
- Accurate mass EICs for
quantifier and qualifier
- Relative responses
- Retention times
Quantitation method
-Automatic selection of quantifier
and qualifier(s)
-Reporting of qualifier ratios
- Isotope pattern comparison and
accurate mass metrics as QC
• 161 Pesticides Quantified
• Great Sensitivity
• Accurate Identification
Pesticides Screening with Accurate Mass Validation data for 3 different matrices
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5991-2295EN
5991-1995EN
Blank 0.005 0.01 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
Acetamiprid
Aldicarb
Azoxystrobin
Bifenazate (D 2341)
Buprofezin
Carbaryl
Carbendazim (Azole)
Chlorfenvinphos(I)
Chloroxuron
Chlorpyrifos
Chlorpyrifos-methyl
Cyprodinil
Difenconazole(I)
Dimethoate
Dimethomorph(E)
Dimoxystrobin
Dinotefuran
Dioxacarb
Ethoxyquin
Fenamiphos
Fenhexamid
Fluquinconazole(I)
Flutriafol
Imazalil(II) (Enilconazole)
Imidacloprid
Metalaxyl
Methidathion
Myclobutanil
Penconazole(I)
Pendimethalin (Penoxalin)
Phosmet (Imidan)
Pirimicarb
Propamocarb
Propiconazole(II)
Pyraclostrobin
Pyridaben
Quinalphos (Diethquinalphione)
Spinosyn A
Spiroxamine
Sulfentrazone
Tebuconazole(II) (Terbuconazole)
Tebufenpyrad
Thiabendazole
Thiacloprid
Thiamethoxam
Triadimefon
Triazophos
Trifloxystrobin
Uniconazole-P(I)
Vamidothion
Zoxamide
Tomato Lemon AvocadoCompound
5991-2465EN
Quantitation of Pesticides Using Accurate Mass Batch review process including accurate mass metrics
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ASMS eSeminar
Qualitative and Quantitative results in a single view
Quantitation of Pesticides
• Single Qual/Quan SW Platform
• Greater efficiency
• Faster data review
• Faster, simpler method setup
• Powered by application kits
and accurate mass libraries
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800
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Without All Ions With Allons
min
ute
s
Processing time
Quant
Qual
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All Ions MS/MS is a great Productivity Tool
Setup takes
minutes instead
of hours
So How Do We Do All Ions MS/MS for Peptides?
Skyline and Agilent’s Q-TOF
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MS1 Filtering
Data independent acquisition
• All Ions (currently limited to 2 CE steps)
• Wide band isolation (SWATH-like)
Spectral Libraries
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Spectral Library Exported From Spectrum Mill
Results
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E. Coli Digest With All Ions MS/MS CE = 0 and 30
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Transition Settings for Wide Isolation MS/MS on
the Agilent Q-TOF
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Wide Isolation Target List With 6550 Q-TOF
ASMS eSeminar
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Biology-directed Workflows to Targeted
Proteomics
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Targeted Proteomics
Transforming target proteins to target peptides
Spectrum Mill and/or Skyline
List of target proteins
Pathway Architect
MassProfiler Professional
Metabolomics
Spectrum Mill
Discovery Proteomics
GeneSpring
Genomics Literature
September 2013
Spectrum Mill: Unique Protein Abundance Feature Works Remarkably Well!
During protein database search in
Spectrum Mill:
• Precursor area is extracted and
stored
• MS/MS spectrum is matched to a
peptide
• Peptide precursor area is
assigned to that peptide
• Protein area = total of peptides
matched to protein
ASMS eSeminar
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Unique Agilent Workflow: Protein Summary to
Pathway Analysis
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Spectrum Mill protein-protein
comparison mode groups
proteins across the entire
data set
Export results to MPP
Pathway visualization in Pathway Architect
September 2013
Exporting Protein Areas From Skyline to MPP
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Review and process QQQ
results in Skyline
Export results to MPP
Pathway visualization in Pathway Architect
September 2013
Multi-omics Pathway Visualization of Metabolomic
and Proteomics Results
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metabolites proteins
September 2013
Integrated Biology: Enabling Pathway-based Multi-
omic Discovery-to-Validation
Pathway
Architect
Skyline
Spectrum
Mill
Pathway to
PCDL
MPP
Q-TOF
Acquisition
QQQ
Acquisition
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BIOCONFIRM Designed for biopharmaceutical characterization
Intact protein
Peptide mapping
Glycan analysis
Synthetic peptide
Oligo-nucleotide
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BioConfirm
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BioConfirm
Intact protein
analysis
Peptide mapping
Glycan analysis
Synthetic peptide
Oligo-nucleotide
Acquire data Integrate and extract MS
Deconvolute Match protein
sequences and predict PTMs
Report generation
Intact Protein Workflow
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A New Deconvolution Algorithm -- pMod Resolves Overlapped Peaks
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IgG 1
IgG2
IgG1 and IgG2
mix
These two peaks are 39 Da apart
Not resolved by Maximum Entropy
Resolved by pMod IgG1 and IgG2
mix
pMod
Acquire data Extract
compounds
Match protein sequences and identify PTMs
Sequence coverage
Report generation
Peptide Mapping Workflow
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Acquire data Extract
compounds
Database search and identify
glycans
Calculate percentage and
report generation
mAb Glycan Workflow
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mAb-Glyco Chip Kit – A Comprehensive Workflow
Solution
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mAb-Glyco chip Reagent Pack
Structure Viewer
Total
workflow
solution
Glycan Database
Reporting Templates
ASMS eSeminar
Intact Protein
Peptide Mapping
Analysis Comparison
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AGILENT MASSHUNTER
EASY-ACCESS SOFTWARE
Walk-up solution for the biochemist with no LC/MS experience
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Walkup LC/MS for Biologists
Three steps to get the results
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1. Identify User
2. Enter Protein Information
3. Place Samples Where Directed
Receive confirmation report by e-mail
Behind the Scene - Administration
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All system setup, user
group set up, method
setup, queue
management and error
clearing is done from
here
Automatic calibration of
TOF on Wakeup or
through Events
Scheduler
E-mail Notification of System Problems
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