Making Sense of the LCMS Data Differences -David Weil
description
Transcript of Making Sense of the LCMS Data Differences -David Weil
-
MassHunter Overview
March 2008Page 1
Making Sense of Differences in LCMS Data: Integrated Tools David A. Weil
Agilent Technologies
-
Mass Hunter Review Pitcon 2008
March, 2008Page 2
How Clean is our Water?....
-
Mass Hunter Review Pitcon 2008
March, 2008Page 3
Chemical Residue Analysis.. .. From the headlines..
PFOA/PFOS Persistent Chemical
PPCP Steroids and Antibiotics
Fungicides/Pesticides
Residuals in Beverages
-
Mass Hunter Review Pitcon 2008
March, 2008Page 4
Challenges in Differential Analysis
Sample Preparation: Extraction Efficiency Separation Methods: Sharper the Better Ionization Efficiency Effects
ESI preferential Ionization of polar/acid/base compounds APCI for non-polar compounds Polarity Effects
Ionization Suppression: Matrix Effects
Mass Spectrometry Sensitivity: Detection vs Relevant
Data Analysis: Show me the Data
-
Mass Hunter Review Pitcon 2008
March, 2008Page 5
Agilents Solution for Differential Analysis
Data AnalysisLC/MS
Analysis
Sample Preparation
GC/MS Analysis
Separations
Quant QQQ
Volatile Residues
Non-Volatile Residues
TOF/QTOFImpurity IDProfiling
Targeted
Non-TargetedAgilents New SPE Products
Data Analysis
Objective: Detection - ID low-level Impurities in Drugs, Chemical Residues in Water, Food and Carbonated Beverages
-
Mass Hunter Review Pitcon 2008
March, 2008Page 6
The Match Game.Choosing The Mass Spectrometer for the Task
Sensitivity, Selectivity, Identification..
Unknown IdentificationImpurity Profiling by Accurate Mass
Target AnalysisEnhanced Sensitivity by MS/MS
Unknown IdentificationAccurate Mass MS, MSMS
MS/MS Sensitivity
-
Mass Hunter Review Pitcon 2008
March, 2008Page 7
Agilent Suite of MassHunter Products
Qual Quant
Profiler MetID
-
Mass Hunter Review Pitcon 2008
March, 2008Page 8
MassHunter QUAL MSD, QQQ, TOF, QTOF
Method Parameters
Method Editor
Data Navigator
-
Mass Hunter Review Pitcon 2008
March, 2008Page 9
Qualitative Analysis: Compound Centric Data Mining and Navigation
Navigation Tree
Chromatogram
Spectrum
Several automated algorithms to find compounds or sample components from LC/MS and LC/MS/MS datasets
Linking of panes for easier browsing and navigation
-
Mass Hunter Review Pitcon 2008
March, 2008Page 10
Qualitative Analysis: Molecular Feature Extraction
List of Compounds
Compound Spectrum
Compound Chromatogram
-
Mass Hunter Review Pitcon 2008
March, 2008Page 11
How To Determine Complexity of Sample/Matrix
6x10
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
+ BPC(150.00000-800.00000) Scan CoffeeSpiked12.d
1 1
6x10
0.2
0.4
0.6
0.8
1
1.2
1.4
+ BPC(150.00000-800.00000 [-1]) Scan CoffeeSpiked12.d
1 1
Counts vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
Base Peak Chromatogram with Exclude m/z 195.09145 +/- 100 ppm
Acquire MS or AutoMSMS Modes Base Peak Chromatogram Exclude Major Component Find Compounds by Molecular Feature Extraction
-
Mass Hunter Review Pitcon 2008
March, 2008Page 12
3D Plot Before3D Plot Before
Molecular Feature Extraction (MFE)Automated Data Reduction Software
Original TICProcessed TIC
3D Plot No Background
Data Reduced sum intensities of isotopes, adducts, clusters and multiply charges ions together.
Finds Features in TOF/QTOF Data Coeluting Features
-
Mass Hunter Review Pitcon 2008
March, 2008Page 13
Processing large amount of data with MFEDisplay filtering:
- S/N > 2
- m/z 150 to 800
- [M+Na] and [M+NH4] Adducts
2 4 6 8 10 12 14
0
5
10
15
I
n
t
e
n
s
i
t
y
(
1
0
^
6
)
Processed TIC
min
2 4 6 8 10 12 14
0
5
10
15
I
n
t
e
n
s
i
t
y
(
1
0
^
6
)
Original TIC
min
- At least 2 ions
- Relative intensity > 0.01%
5131 features
Original TIC Processed TIC
-
Mass Hunter Review Pitcon 2008
March, 2008Page 14
In MassHunter QualSelect Find by Molecular Feature
-
Mass Hunter Review Pitcon 2008
March, 2008Page 15
Method Editor: Parameters for Molecular Feature
Display Filters
-
Mass Hunter Review Pitcon 2008
March, 2008Page 16
Mass Defect Filtering
-
Mass Hunter Review Pitcon 2008
March, 2008Page 17
Executed
Compound Table
-
Mass Hunter Review Pitcon 2008
March, 2008Page 18
Find Compounds by Molecular Feature6x10
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
Cpd 279:+ ECC Scan CoffeeSpiked12.d
Counts vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
Over 280 Compounds Found in Coffee Spiked with Pesticide
-
Mass Hunter Review Pitcon 2008
March, 2008Page 19
Prednisolone: C21H28O5 360.193674
OHOH
OO
O
O
CH3
CH3
CH3
2 year old Suspension
-
Mass Hunter Review Pitcon 2008
March, 2008Page 20
Sequential Neutral Water Losses Detected5x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
+ ECC Scan Prednisolone0002.d
13.42123081.8
Counts vs. Acquisition Time (min)8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14
-
Mass Hunter Review Pitcon 2008
March, 2008Page 21
12 Potential Degradation Products in Prenisolone
-H2, -H2O, -CO2H2, +H2, +CH4+H2-O,
CH2O Loss
-
Mass Hunter Review Pitcon 2008
March, 2008Page 22
Molecular Formula Generation..
Technical Overview 5989-7409EN
-
Mass Hunter Review Pitcon 2008
March, 2008Page 23
Generate Formulas Parameters
-
Mass Hunter Review Pitcon 2008
March, 2008Page 24
Theoretical Isotope Pattern
-
Mass Hunter Review Pitcon 2008
March, 2008Page 25
Accurate Mass of Compounds
-
Mass Hunter Review Pitcon 2008
March, 2008Page 26
Identify Compounds by Database Searching
Search database by mass or by molecular formula
Select either exact mass or Personal Metlin database
-
Mass Hunter Review Pitcon 2008
March, 2008Page 27
Automated Compound Search Metlin Database
-
Mass Hunter Review Pitcon 2008
March, 2008Page 28
Mass Profiling Version 2.0
Processed TICProcessed TICProcessed TICGroup A (Impure)
Processed TICProcessed TICProcessed TICGroup B (Control)
Aligns Data RT, Mass,
Abundance
All Data RT/Mass
Whats Changed? Mass/RT >2 Fold Change
Whats Unique!! Impurity Control
-
Mass Hunter Review Pitcon 2008
March, 2008Page 29
How to Create a ProjectSelect 2 Groups for DifferentialSelect 1 Group for Concentration Change
Need at least 3 replicates
-
Mass Hunter Review Pitcon 2008
March, 2008Page 30
Develop a Method
Select Retention Time and Mass Range
For Small Molecules Exclude Multiply Charged Species
Set Relative Intensity Limit Mass Defect Option Special Masses:
Exclude ions present in blank Display only Targeted Ions
-
Mass Hunter Review Pitcon 2008
March, 2008Page 31
Results Filters
Application of Results Filters will reduce the number of features displayed in the graph
To Display only Features with Greater than 2 fold change set Min Log2 value to 1.0
Relative Frequency filter by presence in samples.
Exclude Saturated Features
-
Mass Hunter Review Pitcon 2008
March, 2008Page 32
Multiple Display OptionsMass vs RT (Average of Features)
-
Mass Hunter Review Pitcon 2008
March, 2008Page 33
Differential Display by Retention Time
-
Mass Hunter Review Pitcon 2008
March, 2008Page 34
Unique Features..
-
Mass Hunter Review Pitcon 2008
March, 2008Page 35
Single Group Example:Compounds Spiked into Plant Extract
-
Mass Hunter Review Pitcon 2008
March, 2008Page 36
Feature Table Single Group Example
For Single Group: Large SD of abundance indicates compounds that have concentrations that vary over the samples
-
Mass Hunter Review Pitcon 2008
March, 2008Page 37
Graphical Display of Concentration Change of Feature
Change Plot Style to Colored by Feature
-
Mass Hunter Review Pitcon 2008
March, 2008Page 38
Abundance and Feature Details
-
Mass Hunter Review Pitcon 2008
March, 2008Page 39
Batch Chemical Composition and Database Searchfrom Mass Profiler
-
Mass Hunter Review Pitcon 2008
March, 2008Page 40
Batch Molecular Formula Generation and Database Searching from Profiler
-
Mass Hunter Review Pitcon 2008
March, 2008Page 41
Web Searching of CompoundsNIST, ChemIDPlus, PubChem,
-
Mass Hunter Review Pitcon 2008
March, 2008Page 42
MetID Software For Impurity Profiling
-
Mass Hunter Review Pitcon 2008
March, 2008Page 43
MassHunter Workstation - Metabolite IDQuickly Identify More Metabolites with Greater Confidence
; Powerful Sample-Control Comparison Algorithm based on Molecular Feature Extraction (MFE); Find Metabolites via Mass Defect Filter; Empirical Formula Generation supports MS/MS data;Confirm Metabolites via Noviatia Autoshift of MS/MS data;Applications beyond Metabolites;Impurity and Environment Profiling
-
Mass Hunter Review Pitcon 2008
March, 2008Page 44
Molecular Formula Generation MS and MSMS Data
Metabolite Table w/ Find + Confirm Results
MetaboliteChros + Spectra
-
Mass Hunter Review Pitcon 2008
March, 2008Page 45
Overview MetID1. Parent Compound: MW, Formula and Structure
2. Transformations: List Proposed Degradation Products
3. Identification Criteria: Vary Importance of Tests
4. Find Compounds by MFE: Molecular Feature Extraction
5. Find Compounds by AutoMSMS: For MSMS data
6. Sample Comparison: Mass Profiler What is Different
7. Isotope Pattern Filtering: Best for Halogenated Species
8. Mass Defect Filtering: From Proposed Compounds
9. EIC Generation: Confirms Presence of Compounds
-
Mass Hunter Review Pitcon 2008
March, 2008Page 46
-
Mass Hunter Review Pitcon 2008
March, 2008Page 47
Method Input
Parent Compound Propose Degradation Products
-
Mass Hunter Review Pitcon 2008
March, 2008Page 48
Proposed Degradation ProductsQualified by Mass Defect and Isotope Pattern
Lets Determine Composition of MW 411
-
Mass Hunter Review Pitcon 2008
March, 2008Page 49
Accurate Mass Generates CompositionAcid Hydrolysis (H2O+CO2H-OH)
Accurate Mass of Fragments Ions
-
Mass Hunter Review Pitcon 2008
March, 2008Page 50
Overlay MSMS Spectrum Parent/Product
-
Mass Hunter Review Pitcon 2008
March, 2008Page 51
3D of MSMS Spectra Fragmentation Pattern
-
Mass Hunter Review Pitcon 2008
March, 2008Page 52
METLIN Personal Database Search Capabilities
Manual searches Search on neutral or charged
monoisotopic mass User-specified search tolerance Optionally restrict by retention time Molecular formula
Compound name Retention time (user defined)
Batch and manual mass searches from Agilent products GeneSpring MS MassHunter Qual Mass Profiler
-
Mass Hunter Review Pitcon 2008
March, 2008Page 53
Personal Metlin Database
-
Mass Hunter Review Pitcon 2008
March, 2008Page 54
METLIN Personal Database Initial View
User search mode
-
Mass Hunter Review Pitcon 2008
March, 2008Page 55
METLIN Manual Single Mass Search
Metabolite search results
Active metabolite structureSingle mass search settings
Hyperlinks to Metabolite websites
-
Mass Hunter Review Pitcon 2008
March, 2008Page 56
METLIN Batch Search
Load file for batch search
Active mass
Active metabolite structure
Active mass search results
-
Mass Hunter Review Pitcon 2008
March, 2008Page 57
METLIN Batch Summary
Active metabolite structure
Batch summary best metabolite match
Exportable list of best metabolite search results
-
Mass Hunter Review Pitcon 2008
March, 2008Page 58
Creating a New METLIN Database
Create a new personal database from METLIN
METLIN is read only and can not be changed
-
Mass Hunter Review Pitcon 2008
March, 2008Page 59
Edit Metabolite Database Update Entry
Add retention time to existing metabolite
-
Mass Hunter Review Pitcon 2008
March, 2008Page 60
METLIN Mass Search With Retention Time Adds Specificity
Adding retention time eliminates isobaric metabolites
Only 1 metabolite instead of 3 !!!!
-
Mass Hunter Review Pitcon 2008
March, 2008Page 61
Edit Metabolite Database New Entry
Select add new then type new metabolite information
-
Mass Hunter Review Pitcon 2008
March, 2008Page 62
METLIN Search from Mass Hunter Qual
Identify compounds: search database
Specify the METLIN database Set search parameters
Manual search options
Select ion in spectra Select MFE compound(s)
Batch search
Automate by Find and Identify Find compounds by MFE Search database Generate formula (optional)
Name and CAS number Database and MFG results
Set Parameters
View detailed results
-
Mass Hunter Review Pitcon 2008
March, 2008Page 63
METLIN Searching From Mass Profiler
Mass Profiler METLIN searching is virtually the same as METLIN Mass Profiler can optionally add
MFG
MFG is used to assist METLIN search
METLIN results are exportable
Database customization is done only in METLIN
Retention time changes
New compounds
New database
METLIN search integrated into Mass Profiler
-
Mass Hunter Review Pitcon 2008
March, 2008Page 64
Summary
Accurate Mass and Reproducible Retention Time are Critical for Differential Analysis and Trace Level Impurity Detection
Data Mining Software Tools Provide Unbiased Automated Data Analysis
Feature Extraction: Find Compounds in Complex Matrix MassHunter Profiler: Differential Analysis Software MetID: Metabolite ID and Impurity Profiling Metlin Database: Personalize Destktop Database Searching
-
Mass Hunter Review Pitcon 2008
March, 2008Page 65
THANK YOU..