TOF/QTOF Users Meeting February 2014 Jim Lau ... - … Users Meeting September 17th, 2008...

TOF/QTOF Users

Meeting February 2014

Jim Lau, Agilent

[email protected]

Technologies.

Agilent Users Meeting

September 17th, 2008

Agenda

• Goal – Share Practices, Assistance to Network of Agilent Users

Today’s Topics:

• Care and Feeding Agilent LCMS Systems

• Data Acquisition: Optimize TOF/QTOF, Methods Development

• MassHunter Qual: Flexible Data Mining

• Overview MassProfiler Software

• Quant/Qual (HRAMS Quant): Setup and Use

• MassHunter Bioconfirm Software

• Questions and Answer Period



Care and Feeding of QTOF/TOF

Current Versions of Software

• MassHunter Acquisition (B.05)

• MassHunter Quant (B.06)

• MassHunter Qual (B.06)

• MH Mass Profiler (B.03)

• MassHunter BioConfirm (B.06)



Data Acquisition Overview

How to Improve Performance

• Optimize Source Parameters:

– Drying Gas Temperature, Gas Flow, and Neb - LC Condition and

Compound Dependent

– Fragmenter Voltage - Compound Dependent

• Optimize Ion Transmission in over the Mass Range

– QTOF For Low Mass Analytes < 200 Change Quad Amu Gain to 100

– TOF/QTOF Fragmentor

• Review How to Calibrate/Tune Q(TOF)

• Optimize Acquisition – Scan the Proper Mass Range

– Saturation and Acquisition Mode

– Set the Proper Acquisition Rate for Chromatographic Peaks



Optimization of ESI Source Parameters non-Ion

Funnel TOF/QTOF

VCap – Entrance of the Capillary, 2000 to 4500V / 5000VMAb

(3500V) Pos, (3000V) Neg

Dependent on compound and MW Concentrated samples (µg/µL) lower VCap

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

Gas Temp - Start at high temperature work down-usually 275-325C

Skimmer - Voltage on skimmer and lens after capillary 65V

Fragmentor- Not Same as QQQ/Quad, Compound Dependent

Vary fragmentor/scan (30V steps) 100V to 240V

FIA with Injection Program

Sheath Gas- Dependent on LC flow rate 10-12 L/min

Shields Up!

Dual ESI Source Schematic

Vcap

Fragmentor

Nebulizer Pressure

Drying Gas Temperature and Flow

Drying Gas Flow high water needs higher flow if too low, spikes in spectra from droplets, dirty cap Drying Gas Temperature higher for low vapor pressure solvents Usually 275 - 350C Vcap optimize with FIA (2000-6000) start with 3000 V in negative mode, look for high chamber current or

blue glow (indicates corona): reduce Vcap if this happens

Electrospray Spray Chamber Settings

HPLC

Flow Rate

(µ/min)

Nebulizer

Pressure

(psi)

Drying Gas

Flow

(l/min)

Drying Gas

Temp

(C)

1 – 10 10 – 15 7 150 - 250

10 – 50 15 – 20 8 - 10 150 - 250

50 – 200 20 – 40 10 - 12 200 - 325

200 – 500 25 – 45 12 325

500 – 1000 50 – 60 12 - 13 350

Vcap

Corona

current

Nebulizer

Pressure

Fragmentor

Drying gas

Temperature

and Flow

Heater

APCI Spray Chamber Settings

HPLC

Flow Rate

(µl/min)

Nebulizer

Pressure

(psi)

Drying Gas

Flow

(l/min)

Drying Gas

Temp

(oC)

200 – 500 30-40 5 - 8 300-350

500 - 1000 40-60 8*-13* 350

APCI

Vap Temp

(oC)

Corona Current

(nA)

Capillary

Voltage

(V)

300 – 500 4000 (pos) & 20,000 (neg) 3500

* Use enough dry gas, but don’t remove reagent

Overview of the Multimode Source

Capillary

HPLC inlet

Nebulizer

Drying gas

Corona needle

ESI Zone

APCI Zone

Thermal container

To convert a method developed with any other source, all that is needed is to go to method and run control and change the Method spray chamber to MM-ES+APCI. Spray chamber values will be the same as for APCI

IR

Multimode Spray Chamber Settings

Mode Nebulizer

Pressure

(psi)

Drying Gas

Flow

(l/min)

Drying Gas

Temp

(oC)

APCI 30-60 5 - 8 300-350

ESI 30-60 8-13 325

APCI/ESI

Vap Temp

(oC)

Corona Current

APCI

(nA)

Capillary

Voltage

(V)

150(ESI)

250(APCI)

4000 (pos) & 20,000 (neg) 3500

Capillary

HPLC inlet

Nebulizer

Drying gas

Corona needle

ESI Zone

APCI Zone

Thermal container

Agilent Jetstream Technology (AJT) source

Non-ion funnel small molecule

The super-heated sheath gas collimates the nebulizer spray and presents more ions to the MS inlet.

*Nozzle voltage

Resistive sampling capillary exit: FragV

Nebulizing gas: pressure

*Sheath gas: flow and temperature

Drying gas:

flow and temperature

Resistive sampling capillary entrance: Capillary V

12

*New parameters unique to AJT source

Parameter

Starting Conditions

Agilent Jetstream

Pos/Neg

Nebulizer pressure 35-45 psi

Drying gas flow 10-12 LPM

Drying gas temp 275°C/325°C

Sheath gas flow 10-12 LPM

Capillary voltage 3500V/3000V

Nozzle voltage (AJT) 0V/1500V

Pos - low/Neg - higher

Fragmentor voltage

(cpd-dependent)

100-175V QTOF/TOF

Drying Gas Experiment on Agilent Jet Stream

Source 5l/min


Source 8l/min


Source 10l/min


Source at 12l/min

Drying Gas Experiment on Agilent Jet Stream Source

12l/min Note: a Higher Vcap increases High m/z response


Non-ion funnel intact protein


*Nozzle voltage

Resistive sampling capillary exit: FragV



Drying gas:



18


Parameter

Starting Conditions

Agilent Jetstream

Pos/Neg

Nebulizer pressure 55 psi

Drying gas flow 10 LPM

Drying gas temp 350°C


Capillary voltage 4500-5500V

Nozzle voltage (AJT) 2000V

Fragmentor voltage

(cpd-dependent)

225-400V QTOF, TOF

Note that “quad amu” will need to be increased 100-250amu with old quad drive in quad tuning parameters for QTOF


Ion funnel intact protein


*Nozzle voltage

Resistive sampling capillary exit:



Drying gas:



19


Parameter

Starting Conditions

Agilent Jetstream

Pos/Neg

Nebulizer pressure 55 psi

Drying gas flow 15LPM

Drying gas temp 250°C

Sheath gas flow 12 LPM

Sheath gas temp 250-300

Capillary voltage 4500-5500V

Nozzle voltage (AJT) 2000V

Fragmentor voltage

(cpd-dependent)

NA

Note that “quad amu” will need to be increased 100-250amu with old quad drive

in quad tuning parameters for QTOF


Ion funnel small molecule


*Nozzle voltage

Resistive sampling capillary exit:



Drying gas:



20


Parameter

Starting Conditions

Agilent Jetstream

Pos/Neg


Drying gas flow 15-17LPM

Drying gas temp 200-250°C


Sheath gas temp 200-400°C


Nozzle voltage (AJT) 0-500V/1500-2000V

Fragmentor voltage

(cpd-dependent)

NA

APCI source

Ion funnel small molecule

21

Parameter

Starting Conditions

Agilent Jetstream

Pos/Neg


Drying gas flow 14-15LPM

Drying gas temp 150-200°C

Vap Temp 350-400°C

Sheath gas temp NA


Chamber current 35 µa

Fragmentor voltage

(cpd-dependent)

NA

Vcap

Corona

current

Nebulizer

Pressure

Fragmentor

Drying gas

Temperature

and Flow

Heater

Ion Funnel Settings for Different Applications

6550 Tune (default)

6550 Tune

Peptide/Protein

6550Tune very

small molecule

(m/z <120) and

fragile adduct

6550 Generic

small molecule

6490 Tune

(default)

6490 Tune

generic small

molecule

6490 Tune Fragile

Compound and

Adduct

Funnel DC 50 50 30 50 15 15 15

HP Funnel Voltage Drop 150 150 100 150 180 180 130

HP Rf Voltage 200 200 90 150 200 200 150

LP Funnel Voltage Drop 100 100 50 100 100 100 90

LP Rf Voltage 100 100 40 60 110 110 40

Fragmenter to 175 Fragmentor to 325

Quad amu 100 Quad amu 120

These are suggested starting funnel parameters and will need optimization. Settings can be very analyte specific. They can be used “as is” should a user need to find out very quickly if it is worth the time to optimize the parameters further. These parameters may not be the same as listed from other sources that have been optimized for a particular analyte or class of analytes.



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 Mode for Acquisition and

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)

• High Mass Range (2 GHz)

Advanced showing

Agilent 6520



Ultra High Speed Acquisition From Agilent’s Leadership in GHz Speed Electronics (Integration and active sampling

to TOF detection)

Picture of 4GHz board

Goes here

FPGAs

Dual Input Agilent

pre-amplifiers

4 GHz Agilent ADC

• 4 GHz Acquisition for Maximum

Resolving Power and <1ppm

Mass Accuracy

• 5 Decades of in-Spectrum

Dynamic Range from 2-Channel x

2 GHz Dual Gain Mode

• Allows changing from Highest

Resolution to Highest Dynamic

Range without changing

Sensitivity

Brings Research Grade HRAMS Performance in a routine format

Best Performance Obtained by not Overscanning

the Chromatographic Peaks: 4-peaks elute 0.6 to

0.9min and 10 scans/sec gives 24scans/peak

Calibration

• Calibrate daily to weekly.

• Generates fifth order polynomial to

accurately assign masses over entire

mass range.

•Takes 10 –15 seconds.

•6550 B.05 should use Extended

Calibration

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)


(TOF Tune)

• If necessary, do…every few months

Initial TOF Tune

(Check Quad Tune)


(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.







**First, try reducing Nebulizer pressure

**



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), One ion OK

– (m/z 121.050873, 149.02332, 322.048121, 922.009798, 2421.91339)

– Do not use any ion with an interference

– Neb Pressure at 5 psi, B.05 not revealed

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

New Ref Mass Solutions AJT

Improved recipes for ref mass abundances above sample background (>100k) Adjust quantities as necessary to ensure both ref masses in both pos and neg modes are greater than 75,000 counts throughout the gradient, with both UNX and plasma extracts.

uL per 100mL of 95/5 MeOH/water, reagents from Agilent Ref Mass kit:

AJST sources

Ammonium TFA 40 Purine 720 HP-0921 300

dual-nebulizer AJST source; roughly half the concentration needed vs UIRM introduction.

Ammonium TFA 20 Purine 300 HP-0921 75 (reduce to 50uL if 922 still >150k at both ends of gradient)

Behavior of the these compounds in the ref mass systems is not linear with concentration; some empirical adjustment might be required for each instrument, hence dedicated ref mass bottles with good labeling are required for each instrument.



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



10000 transients/sec = 10000@1scan/sec*255 =2.6 x 10^6, 5000@2scan/sec, 3333@3scan/sec 10000 transients/sec - 10000*255=2.5x10^6 @ 1scan/sec (no worries 400k)

- 5000*255=1.2x10^6 @2scan/sec (no worries 200k)








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

Manual Spectral Subtraction

Automated Spectral Extraction

AutoMS/MS Features In Acquisition B.04 and

B.05 Acquisition

July 2011 52

Topics

The Decision Engine (“DE”)

Variable number of transients

– TIC target (MS/MS TIC abundance)

– Use MS/MS abundance limit

– Reject precursors that cannot reach target TIC

Purity

Isotope model

Multiple collision energies

Directed MS/MS

July 2011 53

Decision Engine

586.33420

464.79648

561.27494418.91250 1221.99064650.79033

726.36857363.25277

Counts vs. Mass-to-Charge (m/z)300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300

575 580 585 590

3+ charge state

580.60775

719.3670786.09825

645.83725302.09533

783.89034462.21703525.27282

935.42894187.072131178.58929

Counts vs. Mass-to-Charge (m/z)250 500 750 1000 1250 1500

681.34779586.33438

86.09667 863.43679

314.17494499.24340940.89651

728.30139157.13583

1048.493531184.64735

Counts vs. Mass-to-Charge (m/z)250 500 750 1000 1250

675 680 685 690

2+ charge state

MS/MS

MS/MS

July 2011 54

Quad Isolation Behavior Medium isolation (4 m/z) and wide isolation (9 m/z) modes

– 400.0000 m/z, 1+, medium isolation: window centered at 401.7000 m/z, isolates

approximately 399.7 to 403.7 m/z

– 400.0000 m/z, 2+, medium isolation: window centered at 401.4000 m/z, isolates

approximately 399.4 to 403.4 m/z

– Charge states >3 treated like 3; charge state ‘unknown’ (0) treated like 2

Narrrow (1.3 m/z) mode

– Isolation centered at requested m/z value

Values stored in .xml file (can be changed, use care!)

Center of window = monoisotopic m/z + (charge state * -0.3) + (window width / 2)

399 399.5 400 400.5 401 401.5 402 402.5 403 403.5 404

1+

399 399.5 400 400.5 401 401.5 402 402.5 403 403.5 404

2+

Decision Engine Flowchart

Centroid

spectrum

Eliminate peaks

below storage

threshold

Find isotope

groups by

applying isotope

model, “de-ring”,

determine

charge state

Rank by charge

state and/or

abundance

Profile

spectrum

Eliminate

candidates with

undesired charge

states and below

precursor

threshold

Calculate purity,

apply stringency,

rerank

Apply variable

number of

transients cutoff

Schedule

top N

Apply active

exclusion criteria

Match with

preferred list,

rerank

Limit to

directed list

typical

directed

preferred

LCMS Default Conditions for “Average Analysis”

0.4-0.6ml/min and 5-8min Gradient Time

LCMS Default Conditions for “Walkup Self-Serve

MS” 0.4-0.6ml/min and 5-8min Gradient Time

LCMS All Ions MSMS Experiment Low energy

parent ion scan

LCMS All Ions MSMS Experiment High energy

fragmentation scan

LCMS Setting up AutoMSMS experiment

Precursor Threshold Selection for MSMS

TIC Target: Summary

63 July 2011

Lower TIC target

Higher acquisition rate

Higher TIC target

Higher MS/MS spectral quality

•Trying to strike the compromise between too many poor quality MS/MS spectra, and

too few high quality spectra

•‘Just enough quality’

•Optimum value differs by experimental goals

Variable Number Of Transients

Power Ramp Correlation Plot—6530 Q-TOF M

S/M

S to

tal io

n c

urr

en

t/tr

an

sie

nt (lo

g s

ca

le)

MS precursor abundance/transient (log scale)

July 2011 64

Use MS/MS Accumulation Time Limit

This is the maximum number of transients

Unchecked: the maximum number of transients is the DE limit (16383, ~1.5 seconds on a 6530)

July 2011 65

**

Reject Precursors That Cannot Reach Target TIC

•From precursor abundance, calculate number of transients needed to

achieve TIC Target

•If number of MS/MS transients in Spectral Parameters tab is exceeded, it is

likely to yield a poorer quality product ion spectrum, so don’t use this

precursor

July 2011 66

Purity Feature and Isotope Model

Avoid isolating multiple precursors / minor precursors which produce

mixed (“chimeric”) product ion spectra

Purity: minimum acceptable percentage of target precursor cluster

Stringency: weighting factor for the ‘contaminants’

Purity re-orders abundance sorting (higher purity precursors move ahead

of higher abundance precursors with lower purity)

Disable purity: 0 stringency, 0 cutoff

600 601 602 603 604

Isolation window

July 2011 67

Collision Energy Tab Examples

3 CE fixed (e.g., small molecule libraries)

Suggested default

value for peptides

More aggressive fragmentation

for 2+ charge state peptides

July 2011 69

Watch for Acquisition Centroid Threshold Set Too

Low: periods of no automsms

Cause:

Too many mass peaks in the MS1 spectrum, exceeding the maximum number of isotope

groups permitted for the DE to handle. All isotope groups are then assigned as “unknown

charge state”, and the unknown charge state is excluded from sampling for this analysis.

Therefore, no autoMS/MS occurs until the number of isotope groups decreases below the

limit.

Solution: increase the centroid storage threshold so fewer isotope groups are being detected

and processed

July 2011 70

Directed MS/MS, A Special Mode of AutoMS/MS

•Box is checked: Directed MS/MS “Do autoMS/MS, but only if the precursor

candidate is in this table”.

•Box is unchecked: Preferred MS/MS “Do autoMS/MS, and if the precursor

candidate is in this table, move it to the top of the precursor candidate list.”

•Alternative concept—targeted MS/MS but using autoMS/MS selection criteria

NOTE: The preferred/directed table is executed in the order listed. No sorting is

applied to items in this table.

July 2011 71

MFE Results

12 February 2014

AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis

72

Compounds in a Compound List Exported for

Acquisition

Compounds showing H+ and Na+ limited to just H+

(For H+ and NH4+ may have 2 precursors/cmpnd)

Setup Precursor Charge States (Calculated from

Neutral Mass)

Choose Ion Species (Added to Charge States)

Imported into Acquisition



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

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TOF/QTOF Users Meeting February 2014 Jim Lau ... - … Users Meeting September 17th, 2008...

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