TOF/QTOF Users Meeting Jim Lau, Agilent …September 17th, 2008 Optimization of ESI Source Parameters VCap – Entrance of the Capillary, 2000 to 4500V (3500V) Dependent on compound

TOF/QTOF Users Meeting

Jim Lau, Agilent Technologies.

Agilent Users Meeting

September 17th, 2008

Agenda • Goal – Share Best Practices, Develop Network of Agilent Users

Today’s Topics:

• Care and Feeding Agilent LCMS Systems

• Data Acquisition: Optimize TOF/QTOF for Best Mass Accuracy

• MassHunter Qual: MFG and MFE for Small and Large Molecule Applications

• Overview MassProfiler Software

• MassHunter Bioconfirm Software

• Questions and Answer Period



Care and Feeding of QTOF/TOF (Cont.)

• Defragment Hard Drives (Weekly) – Use Diskeeper to Defragment Drives – If Errors – Run Check Disk for Errors Program

• Check LC Components Firmware – If older version update LC Firmware



Data Acquisition Overview How to Improve Mass Accuracy

• Optimize Source Parameters: – Drying Gas Temperature and Flow; Fragmenter Voltage

• Optimize Ion Transmission in Quads – For Low Mass Analytes < 300 Change Quad Amu Gain to 100

• Review How to Calibrate/Tune Q(TOF) • Optimize Points Defining a Peak

– Reduce maximum mass from m/z 3200 to 1700 – Increase Acquisition Rate MS (2-4 spectra/s)

• Causes of High Mass Error (> 3ppm) – Saturated Ions – Unresolved Ions



Optimization of ESI Source Parameters

VCap – Entrance of the Capillary, 2000 to 4500V (3500V) Dependent on compound and MW Concentrated samples (µg/µL) lower VCap

Drying Gas - Dependent on LC flow rate 10-13 L/sec

Gas Temp - Start at high temperature work down 275-325oC

Skimmer - Voltage on skimmer and lens after capillary 65V

Fragmentor- Not Same as QQQ/Quad, Compound Dependent

Vary fragmentor/scan (30V steps) 120V to 240V FIA with Injection Program or MultiFragmentor acquis.



Vary Fragmentor with FIA Injection Program Multiple Methods or Scan Functions Auto Sampler Injection Program • REMOTE Startpulse • REPEAT 5 times • VALVE bypass • DRAW Def Amount from Sample, def.

speed, def offset • VALVE mainpass • WAIT 0.75 min • END REPEAT

Vary Fragmentor Voltage • Right Click in Expt Area and select “Add

Experiment” • Vary Fragmentor Voltage by 20-40V per

Experiment • Acquire Data using Injector Program • Determine Optimum Voltage from EIC as a

Function of Fragmentor Voltage



Extracted Ion Chromatograms: Advanced Tab “Fragmentor Setting”

Review How to Tune and Calibrate the Q(TOF)



Entering Calibration and Tuning

In Mass Hunter Workstation Data Acquisition, select the Tune Context.



Set Instrument State for Calibration

• Mass Range: Low (1700 m/z), Standard (3200 m/z) or

High (20000)

• Instrument Modes: • High Resolution (4GHz) • 4GHz (High Resolution disabled) • Extended Dynamic Range (2GHz) • Minimum Storage Size (1 GHz)

Advanced showing

Agilent 6520



Calibration

• Calibrate weekly. • Generates fifth order polynomial to accurately assign masses over entire mass range. •Takes 10 –15 seconds.

1. Turn on MS. 2. Select Instrument State. 3. Select Bottle B. 4. Press Calibrate. 5. Apply



Calibration Result - Mass Calibration Curve

• The Mass Calibration Curve is generated by selecting the Calibrate button.

• The calibration curve shows the time-to-mass fit of the polynomial correction across the full mass range.

• Apply accepts new TOF Mass Coefficients to polynomial mass correction function.

• Residual Error display often exhibits polynomial “wave” pattern.



Calibration Result - Detailed Residual Plot

Detailed Residual Plot shows: • uncorrected mass error in ppm (blue diamonds). • corrected mass error in ppm (red squares) after applying fifth-order polynomial mass correction. • actual calibration curve – green line.



Optimization for Low Mass Operation

1. Calibrate using the entire mass range (High 3200 m/z). 2. Perform an AutoTune. 3. Instrument State – change mass range from 3200 to

1700. 4. Deselect higher masses not in new range from mass list. 5. Re-calibrate.



Mass Calibration Error Reduced

•The errors have been reduced. •Must have 6 points. •No gain with fewer points.



Automatic and Manual Tuning

Automated tuning for most users

Manual tuning for diagnostics and special applications



Order of Tuning Choices

(Check TOF Tune) • If necessary, do… (Quick TOF Tune) • If necessary, do… (TOF Tune) • If necessary, do…FIRST every few months Initial TOF Tune

(Check Quad Tune) • If necessary, do… (Quad Tune) • If necessary, do…AT PM time (Yearly) Initial Quad Tune



Autotunes – TOF and Quad Autotune

TOF Autotune steps are displayed while the system tunes.

Autotune Report



Save Tune File When Changing Context

Make certain to specify correct tune file in acquisition method.

Or, save and load tune files in the Instrument State tab.







New Makeup Procedure for Reference Solution

Changing from Acetonitrile to Methanol Solution • 2 mL of HP121 into 98 mL of 95/5 MeOH/Water

• 0.8 mL of HP921 into 99.2 mL of 95/5 MeOH/Water

• Using a 500 mL volumetric flask, mix 25 mL of HP121 and 921 into 450 mL of 95/5 MeOH/Water with 0.2% Acetic Acid.

• Internal Reference Mass Correction – Two Ions (1 < m/z 350, second 500 amu higher) – (m/z 121.050873, 149.02332, 322.048121, 922.009798, 2421.91339) – Neb Pressure at 5 psi

Better for Negative ion performance and for use with APCI and Multimode sources without changing reference solution.

Addition of Negative Ion Reference Ion

• From Acquisition • MS TOF Tab • General Tab

– Ion Polarity – Negative

• Ref Mass Tab – Right Click “Add” – Input Mass 966.000725 – Input Mass 980.016375

Reference Solution can also be introduced using a Binary or Isocratic with a 100:1 splitter designed by Agilent. Flow rate of LC set at between 0.5-1.0 mL/min and gives very stable flow.



My Mass Error is Above 3 ppm What should I do?

• Check for Interference from Chemical Background – Change Chromatography – Create Narrow (+/- 10ppm) Extracted Ion Chromatogram with subtraction

• Check Resolution of Peak in Spectrum Peak List • Check for Saturated Peak – Peak List or “*” in Mass Spectrum









Poor Mass Accuracy Example – Unresolved Peaks “Spectrum Peak List 1 Table – Resolution Column”

EIC m/z 337.09847

EIC m/z 338.17521

BPC

1.59 ppm error



Compare Manual versus Automated Analysis “Find Compounds by Feature Extraction”

Manual Spectral Subtraction

Automated Spectral Extraction



QUALITATIVE ANALYSIS

Find Compounds by Formula

Find Compounds in analysis by inputting: •Formulas separate by “,” •A database with Formulas •Worklist Formulas

Find Compounds by Formula - Options Input Comma Separated Formulas

Find Compounds by Formula: Accurate Mass Database Search

Modify default.csv file adding formula, RT and exact mass of compound

Results “Compound Found” in Table

Find Compounds by Formula

Compounds Found

Identify Compounds

Generate Formulas after a Find Compounds

Search a Database after Find Compounds

Example: Characterization of Synthetic Urine

Mixture of 72 Compounds MW 100-625 Daltons, Mixed Classes.

Acquire ESI Positive and Negative ion MS and MSMS .

First Step: Find Compounds by Molecular Feature.

Second Step: Search Compound Table by Metlin & Exact Mass Database.

Compare Accurate Mass, Coverage, Dynamic Range.

Database Searching

Search Mode

Automated Compound Search Metlin Database

Creating a Database

Modify existing database file and save to unique name.

MassHunter\Databases\default.csv.

Mass Hunter Qual…Molecular Formula Generation



Molecular Formula Generator



Molecular Formula Generation Parameters

• Pull Down Menu.. – Compounds or Spectrum Peaks

• Adduct Selection

• Electron State: Even Electron

– Allows “none”

• Add Natural Isotopes with “[ ]”

• Limits for Matches using MS, MSMS Mass Error, DBE.

• DBE can be Negative for Sulfate and Phosphate Compounds.



Molecular Formula Generator

MS Formula Results

MS/MS Formula Results



Molecular Formula Generation



Allowed Species, Limits and Charge State User Inputs



Charge State and Isotope Distribution (B.02.00)



New in B.02.00

Isotope Model:

Common Organic Molecules Peptides Unbiased (unique isotope patterns)

Charge State: 1 or 2 for small molecule 10-15 for large molecule

Isotope Distribution (Experimental/Theoretical)



Cross Score (using MFG with MFE)



MFG Overview





Molecular Feature Extractor (MFE) – How it works

• Map signals in the 3-dim. space in time and mass at the MS level. IMPORTANT: High resolution in mass and time!

• Remove areas which only contain noise, and NO signals. • Identify all mass signals with a common RT (narrow time window) • Combine mass signals with common RT and chemical relation (isotope,

adduct, dimer, higher charge state) => Molecular feature or Compound. • Create Extracted Compound Chromatograms (ECC) and Compound Mass

Spectra from ions associated to a molecular feature (compound)

RT m/z Abund … … …

… … … 2.11 195.1745 21000 2.11 257.2566 33550

2.34 224.2134 11784

… … …

… … …

Compound list or Molecular feature list

Find by Molecular Feature



Method Editor – MFE (cont.)

Extraction Parameters •Target Data Type Small Molecule Large Molecule Infusion

•Peak Filters S/N Profile only Peak Height both















Executed



Find Compounds by Molecular Feature 6x10

0

0

0

0

1

1

1

1

1

2

2

2

2

2

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



Compound List

Use found compounds for further analysis: •Use Molecular Formula Generator on list. •Search a Database and match findings •Upload the MFE results to Mass Profiler or GeneSpring for analysis.



Large Molecule Applications Proteins, Oligonucleotides, Peptides



API-Electrospray: Multiply-Charged Data

Multiply charged compounds acquire a range of charge states yielding a series of mass peaks for a single compound.

Each mass peak represents a different charge state.

Compounds in excess of 100,000 Da may be analyzed.

Picomole to femtomole full scan spectra.

Useful for obtaining molecular weights

Find Molecular Weight of Intact Protein

1000 1500

100000

120000

20000

40000

60000

80000

706

.85

726

.25

746

.15

767

.55

789

.95

839

.45

866

.45

895

.45

926

.15

959

.15

994

.85

103

2.95

1

074.

15

111

8.85

1

167.

55

122

0.45

127

8.75

134

2.65

141

3.25

157

9.65

Green Fluorescent Protein MW 27 kDa - Multiple Charged Envelope

Determine Charge State: • Assume that a pair of ions at m1 and m2 are two charge

states of the same ion (n1 and n2), where n2 = n1 - 1 • Assume an adduct ion weight, x.

(H+ in this example, mass = 1) • Calculate charge state for the second ion n2. If these peaks

are related, it must be an integer. n2 = (m1-x)/(m2-m1) = (694.0-1)/(867.3-694) = 4 n2 = 4

550 600 650 700 750 800 850 900

578.5

694.0 100

50

0

867.3

m1

m2

Determining Charge State of a Multiply Charged Ion

Calculate Molecular Weight, M • Use X = hydrogen = 1.0 • M = n2(m2-x) 4(867.3-1) = 3465.2

Repeat for next pair of peaks in the series • n2 = (578.5-1)/(694.0-578.5) = 5 • M = 5(694.0-1) = 3465.0

The next pair(s) form a coherent set if:

• They calculate out to an integer charge state in the proper series with earlier pairs

• The calculated MW is close enough to the MW derived from earlier pairs

550 600 650 700 750 800 850 900

578.5

694.0 100

50

0

867.3

5+ +4

6+

Determining Molecular Weight of a Multiply Charged Ion

Deconvolute Myoglobin

•Open the data file. •Integrate the data. •Obtain background subtracted peak spectrum. •Fill in Deconvolute Method parameters.

Deconvolution Parameters

Mass Range – Expected Mass Range of the Protein. Sets the x-axis of the deconvolution result display. Mass Step – Resolution of the deconvoluted protein mass range. Use limited m/z range – mass range examined in algorithm. Adduct – expected adduct, necessary for proper calculation. Proton,ammonium, potassium, sodium, proton loss. Average mass %peak – The % of the peak used for the calculation. Isotope width – Automatic, old automatic, specified

Highlight spectrum then Execute

Deconvolution Result

Mass intact protein if it were singly charged.

5x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

Abundance vs. Mass-to-Charge (m/z)

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900

+ Scan (7.949-8.052 min, 6 scans) 4000-200myo.d

893.1872

1211.8108

1304.9478

1413.6178

1696.1247

MS Spectrum and Deconvolved Spectrum of Horse Myoglobin 6x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

Abundance vs. Mass (amu)

16750 16800 16850 16900 16950 17000 17050 17100 17150 17200 17250 17300 17350

+ Scan (474.4-483.1 sec, 8 scans) 4000-200myo.d Deconvoluted (1)

16951.6584

17050.3704

17218 125116781 0598

Theoretical: 16951.44 Observed: 16951.66 Error: -0.22 Da -12.9 ppm

MS

Deconvolved Spectrum

Export Data

Resolved Charge State Deconvolution



Converts Isotopically Resolved Charged States to Singly Charged Spectrum

Applications:

Surfactants Peptides Oligonucleotide HRES Mode Resolves to +13

Large Molecule Molecular Feature Extraction





Mass Profiling – A Powerful Workflow

Molecular Feature: • a discrete molecular entity defined by the combination of

RT, mass and response in an LC/MS or GC/MS analysis • typically represents a known or unknown compound

LC/MS or GC/MS Analysis

Identification Find molecular features

Compare sample sets

Validation

Mass Profiling: • is used to find differences between sample sets • originally developed for proteomics/metabolomics • can be applied to many analytical problems where finding

differences between samples is important • is done using statistical analysis

Mass Profiler GeneSpring MS

MFE (LC/MS) AMDIS (GC/MS)

Spectrum Mill Molecular Formulas DB Search (METLIN) Library Search

GC/MS LC/MS

QQQ MRM GeneSpring MS



MassProfiler Dependent on MFE Feature Extraction

Don’t extract all the compounds only Important ones

How to Mine for Gold… Generated a Base Peak Chromatogram Input Mass Range of Compounds Exclude Reference Ions Input m/z to 4 decimal places Set window to 20 ppm Run Standards to Determine RT Information



Good Quality *.MHD Gives Good Quality Profiler

1. Centroid versus Profile data 2. S/N versus Total Counts Typically use 100 counts Too high may not detect C13 peak Profile S/N 10 First Pass

3. Focused MHD generation Limit Retention Time Limit m/z Range

4. Ion Species (NH4 If buffer) 5. Check Box if High Salt 6. Check if Free Radicals/Quats



Good Quality *.MHD Gives Good Quality Profiler

7. Charge State = 1 to detect only singly charged species

8. Last Tabs only for Display in MassHunter QUAL

9. *.ProPos from Profile

10. *.CentPos from Centroid



Find Compounds by Molecular Feature

Large Molecule Applications

• Protein Characterization Software

– Resolved Charge State Deconvolution – Maximum Entropy Software – Large Molecule Molecular Feature Extraction – Bioconfirm B.02.00 – Peptides, Intact Proteins, Oligonucleotides





Mass Profiler B.01.00 – Highlights

• Impurity Profiling/Differential Analysis Software • Graphical Displays

– Mass vs Retention Time – Log2 Ratio (Group1/Group2) vs Retention Time – Log2 Abundance Group 2 vs Log2 Abundance Group 1 – Unique to Group 1 vs RT and Unique to Group 2 vs RT (New)

• Feature Identification (NEW) – Molecular Formula Generation and AMRT database – Single Feature or Batch Processing (summary report) – Web Internet Database Searching



Mass Profiling Software

Processed TIC Processed TIC Processed TIC Group A (Impure)

Processed TIC Processed TIC Processed TIC Group B (Control)

Aligns Data RT, Mass,

Abundance

All Data RT/Mass

What’s Changed? Mass/RT >2 Fold Change

What’s Unique!! Impurity Control



Graphical Displays



Mass Profiler – Reproducibility of Results

Spread in ChromatographyRT

Reproducible Retention Time Stability

Spread of Peak in Time



Mass Profiler – Reproducibility of Results

Outlier Sample or Feature



Blue Unique Group 2 and Red Unique Group 1



Mass Profiler – The Basics…

Double Click Mass Profiler Icon

“1” for Single Group Comparison “2” for Differential Analysis



Mass Profiler – Load MHD Files

*.MHD Data File Location

Select “Add” then add in the *.MHD files in Groups

Holding Down ctrl key select all *.MHD to add

Then Select “OK”



Set Up Mass Profiler “Method”

*.mpm is Extension of Mass Profiler Project



Mass Profiler – Edit Method

1. Limit Mass Range and RT

2. For Small Molecule Apps Multiply Charge Forbidden

3. For Biomolecules Apps Multiply Charged Required

4. Abundance to 0.1 to 0.01%

5. Number of Ions >=2

6. Isotope Pattern Matching



Mass Profiler – How to Import Exclusion List

Sort Table By Height/Volume

Right Click on Table Select “EXPORT”

From EXCEL Copy Masses From Table



Mass Profiler – Exclusion List of Masses

Paste List of Masses From Blank Into Exclude Masses



Mass Profiler – Alignment



Mass Profiler – Results Filters



Mass Profiler – Special Case – No Replicates

For Example without Replicates Min Differential Score MUST be set to ZERO before loading *.MHD Files



Feature Identification – Set Parameters for MFG



Identify Composition of Features

Right Click any feature in Graph

Right Click any feature in Table



Individual Feature Results # Compositions and Database Hits



Identification Summary Report



Feature Details



Exact Mass/Formula Database Searching



Export Feature Information



Questions and Answers



METLIN Personal Metabolite Database Software



METLIN Personal Database G6825AA

Metabolite-specific database for metabolomics research

• Database installed on a PC • Contains ~15000 compounds • Manual and batch searches

– Query based on monoisotopic mass

• Customizable – Add compounds – Assign chromatographic retention

times to metabolites – Create subset databases

• Works with other Agilent software • Cost: $3759



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



METLIN Personal Database Initial View

User search mode



METLIN Manual Single Mass Search

Metabolite search results

Active metabolite structure Single mass search settings

Hyperlinks to Metabolite websites



METLIN Batch Search

Load file for batch search

Active mass

Active metabolite structure

Active mass search results



METLIN Batch Summary

Active metabolite structure

Batch summary – best metabolite match

Exportable list of best metabolite search results



Creating a New METLIN Database

Create a new personal database from METLIN

METLIN is read only and can not be changed



Edit Metabolite Database – Update Entry

Add retention time to existing metabolite



METLIN Mass Search With Retention Time Adds Specificity

Adding retention time eliminates isobaric metabolites

Only 1 metabolite instead of 3 !!!!



Edit Metabolite Database – New Entry

Select add new then type new metabolite information



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



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



Summary

• Care and Feeding of QTOF/TOF – Remember to Defrag Hard Drives Weekly – Software updates available

• Mass Profiler B.01.00 $3,150 – Differential Analysis Software – Batch Molecular Formula Generation and Database Searching

• METLIN Personal Metabolite Database B.01.00 $3,759 – Accurate Mass Retention Time Database (AMRT) – Convert *.CSV to MTL Formats – User Generated Databases (Plant Extract, Pesticides, AA’s)



Questions and Answers Thank You

Documents

TOF/QTOF Users Meeting Jim Lau, Agilent …September 17th, 2008 Optimization of ESI Source Parameters VCap – Entrance of the Capillary, 2000 to 4500V (3500V) Dependent on compound