Neutral OptiMS Cartridge
Instruction Guide

RUO-IDV-05-2782-D

May 2024

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Contents

1 Operational Precautions and Limitations...............................................................................5
2 Neutral OptiMS Cartridge......................................................................................................... 6
3 Prepare the Reagents............................................................................................................... 9
Initial Sample Concentrations................................................................................................. 10
Prepare the 50 mM AmAc Buffer, pH 3.0................................................................................10
Prepare the 100 mM AmAc Buffer, pH 3.0.............................................................................. 11
Prepare the 10 mM AmAc Buffer, pH 3.0................................................................................ 11
Prepare the 10% HAc Solution................................................................................................11
Prepare the 20% HAc Solution............................................................................................... 12
Prepare the 200 mM AmAc LE Buffer, pH 4.0........................................................................ 12
Reconstitute the Protein Test Mix............................................................................................12
Prepare the Protein Test Mix Sample......................................................................................13
Prepare the Peptide Test Sample........................................................................................... 13
Prepare the PepCalMix Test Sample...................................................................................... 13
4 Prepare the Trays....................................................................................................................14
About Vials.............................................................................................................................. 14
Fill a CESI Vial................................................................................................................... 15
Fill a Microvial.................................................................................................................... 16
Fill a nanoVial.....................................................................................................................17
Prepare the Buffer Trays......................................................................................................... 18
5 Prepare the System................................................................................................................ 20
Condition the Capillaries......................................................................................................... 20
Establish a Stable Spray.........................................................................................................22
Optimize the ESI Voltage........................................................................................................ 25
Fine-Tune the Position of the Sprayer Tip...............................................................................29
Manual Calibration.................................................................................................................. 30
Prepare for Manual Calibration.......................................................................................... 31
Create a Reference Table.................................................................................................. 32
Manually Calibrate in TOF MS Mode................................................................................. 35
Manually Calibrate in Product Ion Mode............................................................................ 40
6 Methods for the Neutral OptiMS Cartridge........................................................................... 45
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Contents
Initial Conditions Tab...............................................................................................................45
Time Program Tab...................................................................................................................46
7 Do a Cartridge Performance Test.......................................................................................... 50
Analyze the Data (Protein Test Mix Sample)...........................................................................50
8 Separation of Peptide Mixtures............................................................................................. 53
9 About Autocalibration............................................................................................................ 55
Create a Reference Table....................................................................................................... 56
Create the MS Acquisition Method for Autocalibration............................................................59
Add Autocalibration to the Sequence......................................................................................63
Analyze the Autocalibration Data............................................................................................ 64
10 Shut Down and Disconnect................................................................................................. 70
Remove the Cartridge............................................................................................................. 70
Stow the Cartridge.................................................................................................................. 74
11 Add Capillary Cartridge Coolant..........................................................................................75
12 Troubleshooting....................................................................................................................77
Troubleshoot an Autocalibration Failure................................................................................. 82
Conductive Liquid Capillary Contamination............................................................................ 82
Condition the Conductive Liquid Capillary......................................................................... 83
Clean Blockage from the Sprayer Tip..................................................................................... 84
Contact Us.................................................................................................................................. 86
Customer Training...................................................................................................................86
Online Learning Center...........................................................................................................86
Purchase Supplies and Reagents...........................................................................................86
SCIEX Support........................................................................................................................86
Cybersecurity.......................................................................................................................... 86
Documentation........................................................................................................................86

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Operational Precautions and Limitations

Operational Precautions andLimitations

1

WARNING! Electrical Shock Hazard. To prevent a shock, do not touch anadapter on a mass spectrometer that is connected to the CESI 8000 Plus
system when the separation voltage is on.
CAUTION: Potential System Damage. Use the lowest ESI voltage possible. If the ion
source is exposed to high temperatures, then blockage in the emitter and damage canoccur.
CAUTION: Potential System Damage. To prevent capillary blockage, use volatile buffers
such as phosphate and borate buffers.
CAUTION: Potential System Damage. Do not expose the cartridge to temperatures thatare < 2 °C. For storage, keep the cartridge at 2 °C to 8 °C.
CAUTION: Potential System Damage. Do not rinse the separation capillary with solutions
that have a pH < 2 or > 9. The capillary is stable in the pH range from 2 to 9. Exposure
to solutions with a pH that is < 2 and > 9 can cause damage to the neutral coating. The
result of damage to the neutral coating can be poor separation and a shorter run life.
CAUTION: Potential System Damage. Do not use buffers that let the separation current
become > 5 μA. A separation current that is > 5 μA can cause permanent damage to thecoating of the separation capillary.
For instructions about how to use the CESI 8000 Plus system safely, refer to the document:
CESI 8000 Plus High Performance Separation-ESI Module User Guide.

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Neutral OptiMS Cartridge

Neutral OptiMS Cartridge

2

The cartridge assembly has a conductive liquid capillary and a separation capillary that ends
in an ESI sprayer tip. The separation capillary and the conductive liquid capillary are different
capillaries. The contents of the separation capillary never touch or mix with the contents of the
conductive liquid capillary. Each capillary is inside a liquid-cooling tube.
The internal surface of the separation capillary is covalently coated with a neutral polymer toprevent the interaction between protein samples and the capillary surface, and to minimize
electroosmotic flow. The separation of sample components (analytes) occurs in the separation
capillary.
The conductive liquid capillary is filled with a conductive solution. The distal end of the capillary
is porous, to let ion flow and electric contact occur.
Protective sleeves prevent damage to the inlet tips of the capillaries. The protective sleeves
retract when the cartridge is installed in the system. When the cartridge is not installed in thesystem, locking mechanisms prevent the retraction of the protective sleeves.
Figure 2-1 OptiMS Cartridge

Item

Description

1

Cartridge body

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Neutral OptiMS Cartridge

Item

Description

2

Separation capillary in liquid-cooling tube

3

Conductive liquid capillary in liquid-cooling tube

4

Protective sleeves that retract when the cartridge is installed

5

Sprayer housing

6

Sprayer tip

7

Protective sleeve shown retracted

8

Exposed capillary when protective sleeve is retracted

9

Inlet end of separation capillary

10

Outlet end of cartridge body

The sprayer tip is made of polyether ether ketone (PEEK), and holds the ESI needle.
Figure 2-2 Conceptual View of Inside the Sprayer Tip

Item

Description

1

ESI needle

2

Conductive liquid3

Separation capillary

4

Etched segment of separation capillary

5

Plume

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Neutral OptiMS Cartridge
The sprayer locks into an adapter that is made for the NanoSpray ion source on the mass
spectrometer. The ESI needle in the sprayer tip closes the circuit between the CESI 8000 Plus
system and the mass spectrometer to cause electrospray ionization. During the separation step,
the spray of analytes goes into the mass spectrometer.
Figure 2-3 Adapter for the NanoSpray Ion Source

Item

Description

1

High-voltage input cable2

High-voltage output cable

Note: Adapters for other mass spectrometers are available from SCIEX. For more information,
go to store.sciex.com.

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Prepare the Reagents

3

Prepare the ReagentsThe following reagents are used with the neutral OptiMS cartridge.

Note: The preparation of every reagent is not required. Different experiments require different
reagents. Prepare the reagents that are applicable to the experiment.
Table 3-1 Required Reagents for the Neutral OptiMS Cartridge
Reagent

Purpose50 mM ammonium acetate (AmAc) buffer,
pH 3.0Used as the background electrolyte (BGE) andconductive liquid

100 mM AmAc buffer, pH 3.0

Used to make the 50 mM AmAc buffer, pH 3.0

10 mM AmAc buffer, pH 3.0

Used to make the Protein Test Mix sample

10% acetic acid, glacial (HAc)

Used as the BGE separation solution andconductive liquid

20% HAc

Used to adjust the pH for the 200-mM AmAc
leading electrolyte (LE) buffer

200 mM AmAC LE buffer, pH 4.0

Used to make the sample become aconcentrate during separation

Reconstituted Protein Test Mix

Used to make the Protein Test Mix sample

Protein Test Mix sample

Used to make sure that the cartridge operates
correctly

Peptide test sample

Used to optimize the position of the capillary
tip in front of the mass spectrometer to get thebest signal intensity

PepCalMix test sample

Used to prepare for manual calibration andautocalibration

Table 3-2 Required Supplies from SCIEX
Material

Part Number

CE Grade water

C48034cIEF Peptide Marker Kit

A58481

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Prepare the Reagents

Table 3-2 Required Supplies from SCIEX (continued)
Material

Part Number

CESI Vials (100 Pack)

B11648CESI Vial caps (100 Pack)

B24699MS Synthetic Peptide Calibration Kit

5045759

nanoVials (100 Pack)

5043467

Neutral OptiMS Cartridge

B07368PCR Microvials (100 Pack)

144709Protein Test Mix

477436

Table 3-3 Other Required Reagents or Supplies
Reagent

VendorPart Number

Acetic acid, glacial (HAc)

Sigma-Aldrich

A6283

7.5 M Ammonium acetate (AmAc)

Sigma-Aldrich

A2706

Ammonium hydroxide (NH4OH) (30%)

Sigma-Aldrich

05002

0.1 M Hydrochloric acid (HCl)Sigma-Aldrich

1.09060

Initial Sample Concentrations
Use the following sample concentrations as initial conditions.
If required, then remove the salt from the sample to optimize the concentration for the sensitivityof the mass spectrometer and injection parameters.
Sample Type

Concentration

Protein Test Mix or intact
proteins in pH 3.0 buffer

0.1 mg/mL in 10 mM AmAc buffer, pH 3.0

Peptide mixtures

3 μg/μL or 1.5 μM in 100 mM LE buffer, pH 4.0

Prepare the 50 mM AmAc Buffer, pH 3.0
1.

In a 50 mL Falcon tube, add 10.0 mL of 100 mM AmAc buffer, pH 3.0, and 10.0 mL of CE
Grade water.

2.

To mix the contents, invert the Falcon tube three times.

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Prepare the Reagents3.

Attach a label with the name 50 mM AmAc buffer, pH 3.0 and the preparation date to
the Falcon tube.

4.

When it is not in use, keep the buffer at 2 °C to 8 °C for as long as 3 months after
preparation.

Prepare the 100 mM AmAc Buffer, pH 3.0
1.

In a 500 mL volumetric flask, add 2.9 mL of HAc, and then add CE Grade water to make
500 mL of 100 mM HAc.

2.

Transfer the solution to a 500 mL Nalgene bottle.

3.

In a 50 mL volumetric flask, add 0.7 mL of 7.5 M AmAc, and then add CE Grade water tomake 50 mL of 100 mM AmAc.

4.

Use a calibrated pH meter to measure the initial pH of the 100 mM HAc solution.

5.

At the same time, use a magnetic stirrer to mix the 100 mM HAc solution and add aliquots of
100 mM AmAc solution until the pH is 3.0 ± 0.1.

6.

Attach a label with the name 100 mM AmAc buffer, pH 3.0 and the preparation date to
the bottle.

7.

When it is not in use, keep the buffer at 2 °C to 8 °C for as long as 1 year after preparation.

Prepare the 10 mM AmAc Buffer, pH 3.0
1.

In a 15 mL Falcon tube, add 1.0 mL of 100 mM AmAc buffer, pH 3.0, and 9.0 mL of CE
Grade water.

2.

To mix the contents, invert the Falcon tube three times.

3.

Attach a label with the name 10 mM AmAc buffer, pH 3.0 and the preparation date to
the Falcon tube.

4.

When it is not in use, keep the buffer at 2 °C to 8 °C for as long as 3 months after
preparation.

Prepare the 10% HAc Solution
Use this solution as the background electrolyte (BGE). Prepare new 10% HAc solution everyday.
1.

In a clean 20 mL glass vial, add 18 mL of CE Grade water.

2.

Inside a fume hood, add 2 mL of HAc to the vial.

3.

Invert the vial three times to mix the contents.

4.

Keep the 10% HAc solution at room temperature.

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Prepare the Reagents5.

At the end of the day, discard the solution that was not used.

Prepare the 20% HAc Solution
Only prepare new 20% HAc solution when the 200 mM AmAC LE buffer, pH 4.0 is prepared.
1.

In a 100 mL glass bottle, add 80 mL of CE Grade water.

2.

Inside a fume hood, add 20 mL of HAc to the bottle.

3.

Invert the bottle three times to mix the contents.

4.

Attach a label with the name 20% HAc solution and the preparation date to the bottle.

5.

Keep the 20% HAc solution at room temperature.

6.

At the end of the day, discard the solution that was not used.

Prepare the 200 mM AmAc LE Buffer, pH 4.0
1.

To prepare 50 mL of 400 mM AmAc LE buffer, pH 4.0, do this:a.

In a 50 mL glass volumetric flask, add 20 mL of CE Grade water.

b.

Inside a fume hood, add 2.7 mL of 7.5 M AmAc to the flask.

c.

To increase the volume to 50 mL, add CE Grade water.

d.

Invert the flask three times to mix the contents.

2.

Pour the 50 mL of 400 mM AmAc solution into a 100 mL beaker.

3.

Use a calibrated pH meter to measure the initial pH of the solution.

4.

Add aliquots of newly prepared 20% HAc solution until the pH of the solution is 4.0.

5.

Transfer the solution to a 100 mL volumetric flask.

6.

To increase the volume to 100 mL, add CE Grade water.

7.

Invert the flask three times to mix the contents.

8.

Attach a label with the name 200 mM AmAC LE buffer, pH 4.0 and the preparation
date to the flask.

9.

When it is not in use, keep the buffer at 2 °C to 8 °C for as long as 2 years after preparation.

Reconstitute the Protein Test Mix
1.

Add 2.0 mL of CE Grade water to a vial of Protein Test Mix.

2.

Use a vortex mixer to mix the solution until all of the solid material is dissolved.

3.

Divide the Protein Test Mix into aliquots of 20 μL.

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Prepare the Reagents
4.

When it is not in use, keep the reconstituted Protein Test Mix frozen at –35 °C to –15 °C for
up to 1 year after preparation.

Prepare the Protein Test Mix Sample
1.

If required, then let the vial of reconstituted Protein Test Mix thaw to ambient temperature.

2.

In a 0.5 mL centrifuge tube, add 20 μL of reconstituted Protein Test Mix and 80 μL of 10 mM
AmAc buffer, pH 3.0.

3.

Attach a cap to the tube.

4.

Use a vortex mixer to mix the sample for 3 seconds.

5.

When it is not in use, keep the sample at 2 °C to 8 °C for as long as 1 year after preparation.

Prepare the Peptide Test Sample
1.

In a centrifuge tube, add 100 μL of BGE and 5 μL of a peptide marker, such as pI 9.5.

2.

Attach a cap to the tube.

3.

Use a vortex mixer to mix the sample for 3 seconds.

4.

Keep the sample at 2 °C to 8 °C.

5.

At the end of the day, discard the solution that was not used.

Prepare the PepCalMix Test Sample
1.

In a 0.5 mL centrifuge tube, add 10 µL of PepCalMix and 90 µL of 200 mM AmAC LE buffer,
pH 4.0.

2.

Mix the solution in a vortex mixer for 10 seconds.

3.

To remove any precipitant, use a centrifuge to spin the solution at 12,000 × g for 5 minutes.

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Prepare the Trays

4

Prepare the Trays
Use only CESI vials and caps in the CESI 8000 Plus system buffer and sample trays.

Make sure that each buffer and sample vial is in the location identified in the tray layout. The
locations of the buffer and sample vials are related to the method in the 32 Karat software. If
a vial is put in a different position, then the position must be identified in the method in the
32 Karat software or manually in the sequence table.

About Vials
CAUTION: Potential System Damage. Do not overfill the vials. If the vials are too full, thenliquid can go into the pressure system and cause damage.
CAUTION: Potential System Damage. Do not underfill the vials or let the liquid level get
too low. If the liquid level in the vials is too low, then the separation capillary can fill with
air and cause the vials to break if voltage is applied.
Three types of vials are used with the CESI 8000 Plus system:• CESI vials are used for buffer and as holders for microvials and nanoVials.
• Microvials are used for sample volumes from 50 µL to 100 µL. These vials must be put in a
CESI vial and then put in the sample tray.
• nanoVials are used for sample volumes from 5 µL to 50 µL. These vials are put in the sample
tray.
Note: Always use a CESI vial cap.
Note: Do not use any vial or cap more than once.

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Prepare the Trays

Fill a CESI Vial
Note: To prevent splashing, put the empty vials in the tray, and then add liquid and attach the
caps.
1.

Fill the CESI vial to the maximum fill line.
Note: Do not put more than 1.5 mL in a CESI vial.
Figure 4-1 CESI Vial

2.

Item

Description

1

CESI Cap

2

Maximum fill line

3

CESI Vial

Attach a CESI cap.
Note: When filling the vials, do not make air bubbles. If there are air bubbles in the vial, thenspin the vial in a centrifuge for a few seconds to remove the bubbles.

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Fill a Microvial
Note: To prevent splashing, put the empty vials in the tray, and then add liquid and attach the
caps.
1.

Fill the microvial with at least 50 μL of sample.
Figure 4-2 Microvial

Item

Description

1

CESI Cap

2

Microvial

3

CESI Vial

4

Microvial inside CESI vial

2.

Put the microvial inside the CESI vial.

3.

Attach a CESI cap.
Note: When filling the vials, do not make air bubbles. If there are air bubbles in the vial, thenspin the vial in a centrifuge for a few seconds to remove the bubbles.

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Prepare the Trays

Fill a nanoVial
For sample volumes between 5 μL and 50 μL, use a nanoVial.
Note: To prevent splashing, put the empty vials in the tray, and then add liquid and attach the
caps.
1.

Use a thin pipette tip to transfer the sample to the deeper well of a nanoVial.
When the tab points to the user, the deeper well is on the left side of the nanoVial.
Figure 4-3 nanoVial

Item

Description

1

nanoVial inside the CESI vial with the thin pipette tip transferring sample

2nanoVial inside the CESI vial with the cap attached2.

Attach a CESI cap.

3.

Put the sample vial in the applicable position in the inlet sample tray.
Note: Make sure that the tab on the outside of the nanoVial points to the front of the sample
tray.

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Prepare the Trays
Figure 4-4 Orientation of the nanoVial Tab in the Sample Tray

Item

Description

1

Electrode at the left side of the nanoVial

2

Capillary at the right side of the nanoVial (for use in loading sample)3

Tab outside of the nanoVial

4

Sample tray

Note: If the number of sample vials will not fill the tray, then keep every second column inthe sample tray empty. This will make it easier to remove the nanoVials from the tray.
Note: When filling the vials, do not make air bubbles. If there are air bubbles in the vial, thenspin the vial in a centrifuge for a few seconds to remove the bubbles.

Prepare the Buffer Trays
CAUTION: Potential System Damage. Use low-concentration buffers for separations withpH values > 7. High-concentration buffers can cause blockage in the separation capillary.
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Prepare the Trays
1.

Use the following table to prepare the required reagents in CESI vials. Refer to the section:
Fill a CESI Vial.
Table 4-1 Buffer Vial Contents by Sample Type
Sample Type

2.

Position
BI:A1

BI:B1

BO:A1

Protein Test Mix or
intact proteins in pH
3.0 buffer

50 mM AmAc buffer,
pH 3.050 mM AmAc buffer,
pH 3.050 mM AmAc buffer,
pH 3.0Peptide mixtures

10% HAc solution

10% HAc solution

10% HAc solution

Use the following figure to put each reagent vial in the correct position in the buffer inlet andoutlet trays.
Figure 4-5 Buffer Tray Layout

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Prepare the System

5

Prepare the System
To make sure that the system operates correctly, do the following procedures.

Condition the Capillaries
CAUTION: Potential System Damage. To prevent the sprayer tip from breaking, makesure that the sprayer tip is immersed in CE Grade water when the neutral CESI washingmethod is run. Remove the sprayer from the CE Grade water, and then install the sprayer
in the adapter on the mass spectrometer before the neutral CESI electrical conditioning
method is started.
Condition the capillaries before a new cartridge is used for the first time and before a cartridgethat has been in storage is used again.
Use both the neutral CESI washing method and the neutral CESI electrical conditioning method
to condition the neutral capillaries.
• The neutral CESI washing method rehydrates the neutral coating of the capillaries.
• The neutral CESI electrical conditioning method establishes the electrical connection in thesprayer tip.
Refer to the section: Methods for the Neutral OptiMS Cartridge.
If both methods are not used, then earlier migration times, poor separation performance, loss ofelectrospray, and different electrical currents during separations can occur.
1.

Remove the protective sleeve from the sprayer tip.

2.

To make sure that the flow of liquid through the separation capillary is sufficient, do this:

3.

4.

a.

Do a forward rinse with CE Grade water at 100 psi for 3 minutes.

b.

When a liquid droplet shows at the end of the sprayer tip, stop the application ofpressure.

To make sure that the flow of liquid through the conductive liquid capillary is sufficient, do
this:
a.

Do a reverse rinse with CE Grade water at 100 psi for 5 minutes.

b.

When a liquid droplet shows at the end of the stainless steel needle, stop theapplication of pressure.

Install the protective sleeve on the sprayer.

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Prepare the System5.

Put 5 mL of CE Grade water in a 50 mL Falcon tube, and then put the Falcon tube in theholster on the side of the system.

6.

Carefully immerse the sprayer tip in the Falcon tube.
Figure 5-1 Sprayer Immersed in Liquid

7.

Run the neutral CESI washing method.

8.

When the method is complete, remove the sprayer from the Falcon tube.

9.

Use lint-free wipes to dry the sprayer carefully.
Figure 5-2 Dry the Sprayer

CAUTION: Potential System Damage. To prevent the sprayer tip from breaking, do not
move the retractable protective guard during drying.
10. Make sure that the ESI voltage on the mass spectrometer is off.

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Prepare the System
11. Install the sprayer in the adapter on the mass spectrometer.
12. Run the neutral CESI electrical conditioning method.
When the method is complete, the cartridge is ready to set a stable spray.
13. Remove the Falcon tube from the holster, and discard the contents.

Establish a Stable Spray
1.

Make sure that the sprayer tip is in the correct position for the inlet on the mass
spectrometer.

2.

In the Analyst TF software, make sure that the ESI voltage is zero.

3.

To fill the conductive liquid capillary with BGE, do this:a.

In the 32 Karat software, go to the Direct Control window.

b.

Click the Pressure field.
Figure 5-3 Pressure Settings Dialog to Fill the Conductive Liquid Capillaryc.

In the Pressure field, type 100.

d.

In the Duration field, type 5.

e.

Click Reverse.

f.

Click Pressure.

g.

Click OK.
To identify the direction of the pressure being applied, in the Direct Control window, lookat the Rinse Direction icon (a blowing face identified by a red arrow in the following
figure). The reverse direction refers to rinsing the conductive liquid capillary, and theblowing face shows on the right side of the window.

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Prepare the System
Figure 5-4 Rinse Direction (Blowing Face) Icon

When the conductive liquid capillary is filled with BGE, a droplet will show at the end of
the stainless steel needle.
Figure 5-5 Droplet at the End of the Stainless Steel Needle

4.

When the rinse is complete, to fill the separation capillary with BGE, do this:Instruction Guide
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Prepare the Systema.

Go to the Direct Control window.

b.

Click the Rinse field.
Figure 5-6 Pressure Settings Dialog to Fill the Separation Capillaryc.

In the Pressure field, type 100.

d.

In the Duration field, type 5.

e.

Click Forward.

f.

Click Pressure.

g.

Click OK.
When the separation capillary is filled with BGE, a droplet will show at the end of thesprayer tip.
Figure 5-7 Droplet at the End of the Sprayer Tip

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Prepare the System

Note: If the separation capillary is empty, then the first droplet of solution might notshow at the sprayer tip for as long as 7 minutes.
5.

To find the minimum ESI voltage, refer to the section: Optimize the ESI Voltage.

Optimize the ESI Voltage
1.

Fill the capillary with Protein Test Mix.

2.

Make sure that the separation capillary and conductive liquid capillary are filled with BGE.

3.

To set the values for voltage in the 32 Karat software, do this:a.

Go to the Direct Control window, and then double-click the Voltage field.
Figure 5-8 Voltage Settings Dialogb.

In the Voltage field, type 30.

c.

In the Duration field, type 60.

d.

In the Ramp time field, type 1.

e.

Select the With pressure check box.

f.

In the Pressure field, type 1.5.

g.

Click Normal.

h.

Click OK.

Note: If the BGE used is 10% HAc solution, then the electrical current should be 2 μA to
3 μA. If the BGE used is 50 mM AmAc buffer, pH 3.0, then the electrical current should be
0.5 µA to 1 µA.
4.

In the Analyst TF software, in the Navigation bar, under Tune and Calibrate, double-click
Manual Tuning.

5.

On the Source/Gas tab, do this:Instruction Guide
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In the Curtain Gas (CUR) field, type 10.
Note: In the Analyst TF software, the minimum value for curtain gas is 10. CESI-MS
runs use a value of 5 for curtain gas. To change the setting for gas for the Curtain Gas
interface, use the Curtain Gas patch application that is available on the computer for themass spectrometer.

b.

In the IonSpray Voltage Floating (ISVF) field, type 1.0.
The IonSpray Voltage Floating (ISVF) parameter sets the ESI voltage.

c.
6.

In the Interface Heater Temperature (IHT) field, type 50.0.

On the MS tab, do this:a.

In the Scan type field, select TOF MS.

b.

In the Accumulation time field, type 500.

c.

In the TOF Masses (Da) Min field, type 200.

d.

In the TOF Masses (Da) Max field, type 2000.

e.

In the Period Duration field, type 10.

7.

Click Start.

8.

If a non-SCIEX mass spectrometer is in use, then do this on the mass spectrometer
acquisition computer:9.

a.

Set the ESI voltage to zero.

b.

Set the scan range from m/z 200 to m/z 2000.

c.

Click Start.

Increase the value in the IonSpray Voltage Floating (ISVF) field in increments of 0.1 kV
until a continuous signal shows in the mass spectrum window.
Note: This is the minimum ESI voltage.

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Figure 5-9 Typical Spray Profile of a Cartridge that Uses 10% HAc Solution as the
BGE

10. In the Direct Control window, click Stop.
11. On the Source/Gas tab, in the IonSpray Voltage Floating (ISVF) field, type 0.0, and thenclick OK.
Make sure that there is no spray or background mass spectrum.
Note: If there is still spray when IonSpray Voltage Floating (ISVF) is 0.0, then there might
be an issue with the connection. Refer to the section: Fine-Tune the Position of the Sprayer
Tip.
12. In the IonSpray Voltage Floating (ISVF) field, type the minimum ESI voltage, and thenclick OK.
Make sure that electrospray starts again.
13. In the IonSpray Voltage Floating (ISVF) field, increase the value by 100 V.
14. To find the best position for the sprayer in relation to the inlet on the mass spectrometer, use
the XYZ-axes adjustment knobs to get a maximum total ion current signal.
Note: Keep the sprayer tip at least 2 mm away from the curtain gas plate.
15. To check the stability of the spray, continue to apply 20 kV (normal polarity) for 20 minutes.
16. In the Analyst TF software, do this:a.

Open the Advanced MS tab, and then click MCA.

b.

Open the MS tab.

c.

In the Scan type field, select TOF MS.

d.

In the Accumulation time field, type 1.

e.

In the TOF Masses (Da) Min field, type 70.

f.

In the TOF Masses (Da) Max field, type 2000.

g.

In the Period Duration field, type 5.

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17. Click Acquire, and then type a file name (for example, Baseline).
18. If a non-SCIEX mass spectrometer is in use, then do this on the mass spectrometer
acquisition computer:a.

Set the ESI voltage to 1.0 kV and continue to acquire data.

b.

Increase the ESI voltage in increments of 0.1 kV until there is a continuous signal in themass spectrum window.

c.

To find the best position for the sprayer in relation to the inlet on the mass spectrometer,maximize the XIC (eXtracted Ion Electropherogram) signal for the pI 7.0 or pl 10 marker
in use while minimizing its fluctuations.

d.

When the position of the sprayer is optimized, decrease the ESI voltage in incrementsof 0.1 kV until the spray stops.

e.

Increase the ESI voltage in increments of 0.1 kV until continuous spray is detected.

f.

Record this value as the minimum ESI voltage.

g.

Increase the minimum ESI voltage by 0.2 kV.

19. Use the Y-axis alignment knob to maximize the baseline fluctuations.
Figure 5-10 Find the Baseline Fluctuationa.

If the baseline fluctuation is < 40% within 2 minutes to 5 minutes, then make a noteof the ESI voltage for the mass spectrometer methods (ion collection ESI voltage):
Baseline fluctuation (%) = [(highest value - average value)/average value] x 100.

b.

If the baseline fluctuation is > 40%, then do this procedure again until a good baseline isset. If a good baseline cannot be set, then refer to the section: Fine-Tune the Position of
the Sprayer Tip.

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20. If a non-SCIEX mass spectrometer is in use, then do this:a.

Monitor the spray stability for 20 minutes to 30 minutes to make sure that the baseline
fluctuation is ≤ 40%.
If the baseline fluctuation is > 40%, then condition the capillaries. Refer to the section:
Condition the Capillaries.

b.

Turn off the electrospray voltage on the mass spectrometer.

Fine-Tune the Position of the Sprayer Tip
CAUTION: Potential System Damage. Make sure that the sprayer tip is in position outsideof the curtain gas plate. If the sprayer tip is too close, then rinse solution can drip onto
the curtain gas plate and cause damage.
Before a separation is run, it is critical to optimize the position of the sprayer tip in front of thecurtain gas plate to get the correct ESI voltage. If the sprayer tip is too far away from the curtaingas plate, then a high ESI voltage can be required, which causes analyte fragmentation. For
intact proteins, electrochemical reactions such as oxidation can occur. The green area in thefollowing figure shows the recommended distance of the sprayer tip and the recommended ESI
voltage values. Avoid the red area.
Figure 5-11 Sprayer Tip Distance and ESI Voltage Correlation

Use this procedure to adjust the position of the sprayer tip and find the required ESI voltage formass spectrometer acquisition:• After installing a cartridge• If the signal separation voltage is changed• If a different BGE is used1.

Fill the separation capillary and the conductive liquid capillary with BGE.

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2.

Use the XYZ-axes adjustment knobs to move the sprayer tip 3 mm from the curtain gas
plate.

3.

In the 32 Karat software, use the Direct Control window to apply 30 kV voltage and 1.5 psi
forward pressure with a ramp of 1 minute.
• Make sure that the cartridge does not spray when the ESI voltage is 0 V during mass
spectrometer scanning.
• Monitor the Direct Control window to make sure that the electrical current is stable.

4.

Set the ESI voltage to 1,000 V and then increase the value in increments of 100 V until
electrospray is detected.

5.

Increase the ESI voltage by 200 V.

6.

Use the XYZ-axes adjustment knobs to move the sprayer tip and maximize the signal
intensity of the mass spectrometer.
Make sure that the sprayer tip is approximately 3 mm from the curtain gas plate.

7.

When the position of the sprayer is optimized, set the ESI voltage to zero.

8.

Set the ESI voltage to 1,000 V and then increase the value in increments of 100 V until
electrospray is detected.
Note: This is the minimum ESI voltage. This voltage is not high enough to maintain an
effective spray during separation.

9.

Increase the minimum ESI voltage by 200 V.
Note: This is the optimal ESI voltage. Use this voltage with the mass spectrometer method
to provide a stable electrospray during separation.

10. Set the ESI voltage to zero.
11. Turn off the separation voltage.

Manual Calibration
To get the most accurate mass determination, manually calibrate the mass spectrometer.
Note: The following procedures were made for the SCIEX 6600 system. For other SCIEX
mass spectrometers or mass spectrometers from other manufacturers, use the manufacturer's
recommendations.
After the mass spectrometer has been manually calibrated, include autocalibration in thesequence when data is acquired for samples. Refer to the section: About Autocalibration.

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For more information about calibration, refer to the document: AB SCIEX Mass Calibration
Tutorial. To open the document, click Start Menu > All Programs > AB SCIEX > Analyst TF
1.7 > Hardware and Software Guides.

Prepare for Manual Calibration
1.

In a sample vial, add 90 μL of PepCalMix and 10% HAc solution to make a 1:1 solution.

2.

Put the vial in position A1 (SI:A1) in the sample tray.

3.

In the 32 Karat software, go to the Direct Control window, and then click Inject.
Figure 5-12 Inject Parameters Dialog

4.

Select the injection parameters.
Note: To inject at 100 psi for 180 seconds, make sure that the For Capillary Fill check box
is selected.

5.

When the capillary is filled, go to the Direct Control window, and then click Voltage.
Figure 5-13 Voltage Settings Dialog

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6.

In the Voltage field, type 30.

7.

In the Duration field, type 20.

8.

Select the With pressure check box.

9.

In the Pressure field, type 1.5.

10. Make sure that the capillary inlet is in position BI:A1 and the outlet is in position BO:A1.
11. Click OK.

Create a Reference Table
1.

In the Analyst TF software, in the Navigation bar, click Acquire.

2.

Click Tools > Settings > Tuning Options, and then click Reference.
Figure 5-14 Reference Table Editor Window

3.

To open a new, empty reference table, click New.

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Figure 5-15 New Reference Table in the Reference Table Editor Window

4.

In the Name field, type a name that identifies the calibration solution (in this example,
PepCalMix).

5.

Make sure that Positive is clicked.

6.

In the Reference Ions for TOF MS Calibration table, type the following values.
Table 5-1 Reference Ions for TOF MS Calibration
Compound Name

Precursor m/z (Da)CE forMS/MS

DP forMS/MS

AETSELHTSLK

408.55010

4080GAYVEVTAK

473.26020

4080

IGNEQGVSR

485.25302

4080

LVGTPAEER

491.26559

40

80

LDSTSIPVAK

519.79969

40

80

AGLIVAEGVTK

533.32333

40

80

LGLDFDSFR

540.27342

40

80

GFTAYYIPR

549.28633

4080

SGGLLWQLVR

569.83398

40

80

AVGANPEQLTR

583.31360

40

80

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Table 5-1 Reference Ions for TOF MS Calibration (continued)
Compound Name

Precursor m/z (Da)CE forMS/MS

DP forMS/MS

SAEGLDASASLR

593.80053

4080

VFTPLEVDVAK

613.34955

4080

VGNEIQYVALR

636.35273

40

80

YIELAPGVDNSK

657.34499

4080

DGTFAVDGPGVIAK

677.85827

40

80

YDSINNTEVSGIR

739.36148

40

80

SPYVITGPGVVEYK

758.91050

40

80

ALENDIGVPSDATVK

768.90340

4080

AVYFYAPQIPLYANK

883.47380

4080

TVESLFPEEAETPGSAVR

964. 97741

40

80

7.

For each compound, click the Use check box.

8.

In the Reference Ions for MS/MS Calibration table, type the following values.
Table 5-2 Reference Ions for MS/MS Calibration

9.

Fragment Name

Fragment m/z (Da)b2

185.09207

b3

348.15540

b4

560.30788

b5

661.35555

N/A

758.91050

y7

799.44398

y8

856.46544

y9957.51312

y101070.59719

y111169.66560

y12

1332.72893

For each fragment, click the Use check box.

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10. In the Use for MS/MS column, select the check box for the applicable compound in thecalibration solution (in this example, the peptide at m/z 758.91).
11. Make sure that the value in the Retention time tolerance +/- field is 30.
12. Click OK.
Figure 5-16 Tuning Options Dialog

13. Make sure that the name of the new reference table (in this example, PepCalMix) shows in
the Positive Reference field.
14. Click OK.

Manually Calibrate in TOF MS Mode1.

In the Analyst TF software, in the Navigation bar, under Tune and Calibrate, double-click
Manual Tuning.

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Figure 5-17 Tune Method Editor Window2.

On the Source/Gas tab, do this:a.

In the Ion Source Gas1 (GS1) field, type 0 (zero).

b.

In the Ion Source Gas1 (GS2) field, type 0 (zero).

c.

In the Curtain Gas (CUR) field, keep the default value, or type 10.
Note: To change the setting for gas for the Curtain Gas interface, use the Curtain Gas
patch application that is available on the computer for the mass spectrometer.

d.
3.

4.

In the Interface Heater Temperature (IHT) field, type 50.

On the MS tab, do this:a.

In the Scan type list, select TOF MS.

b.

In the Accumulation time field, type 1.000.

c.

In the TOF Masses (Da) Min field, type 350.

d.

In the TOF Masses (Da) Max field, type 1000.

e.

In the Period Duration field, type 1000.

Open the Advanced MS tab.

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Figure 5-18 Tune Method Editor Window: Advanced MS Tab

5.

Make sure that the MCA check box is cleared.

6.

In the IonSpray Voltage Floating (ISVF) field, type a value.
The IonSpray Voltage Floating (ISVF) parameter sets the ESI voltage.

7.

Adjust the position of the sprayer until the spray is stable.

8.

Click Start.
After approximately 1 minute, windows open to show the total ion chromatogram (TIC) andmass spectrum.
Figure 5-19 TIC and Mass Spectrum Windows

9.

To open windows that show the TIC and mass spectrum for the data file that was acquired,right-click the TIC window, and then click Open File.

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Figure 5-20 TIC and Mass Spectrum Windows from the Acquired Data File

10. To delete the mass spectrum pane, click the pane, and then on the toolbar, click the Delete
icon.
11. To get an average mass spectrum, click and drag the cursor to highlight the TIC window,
and then double-click the TIC window.

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Figure 5-21 Average Mass Spectrum Pane

12. Right-click the mass spectrum pane, and then click Re-Calibrate TOF.
Figure 5-22 TOF Calibration Window

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13. From the Reference Table list, select the reference table that was created for the calibrant(in this example, PepCalMix).
Note: If the correct reference file does not show in the list, then the reference table has notbeen created. Refer to the section: Create a Reference Table.
14. In the Tolerance field, type 0.2.
15. To calculate the average error for this new calibration, click Calculate new calibrations.
16. Make sure that the value for the Average Error is within the routine operating standards forthe mass spectrometer being calibrated.
17. Click Calibrate spectrum.
The new calibration values show.
18. Make sure that the Set As Instrument Default check box is selected.
Note: If an ion is not found during calibration, then right-click the missing ion in thereference table, and then click Delete. Click Calculate new calibrations.
19. To apply this calibration to all samples in the file, click Entire File.
20. Click Save.
21. Click OK twice.
22. To close the TOF Calibration window, click Close.
23. Close the TIC and mass spectrum panes.

Manually Calibrate in Product Ion Mode1.

In the Analyst TF software, in the Navigation bar, under Tune and Calibrate, double-click
Manual Tuning.

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Figure 5-23 Tune Method Editor Window: Compound Tab

2.

3.

4.

Open the Compound tab, and then do this:a.

In the Collision Energy field, type 42.

b.

In the Collision Energy Speed (CES) field, type 5.

On the MS tab, do this:a.

In the Scan type list, select Product Ion.

b.

In the Product Of field, type 758.91.

c.

In the TOF Masses (Da) Min field, type 100.

d.

In the TOF Masses (Da) Max field, type 1500.

e.

Make sure that High Sensitivity is selected.

Click Start.
Windows open to show the total ion chromatogram (TIC) and mass spectrum generated
during the product ion calibration.

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Figure 5-24 TIC and Mass Spectrum Windows

5.

Right-click the TIC window, and then click Open File.
Windows open to show the TIC and mass spectrum for the product ion calibration data filethat was acquired.
Figure 5-25 TIC and Mass Spectrum Windows from the Product Ion Calibration Data
File

Note: The spectrum that is shown cannot be used for calibration.

6.

Click the mass spectrum window, and then on the toolbar, click the Delete icon.

7.

To get an average mass spectrum, click and drag the cursor to highlight the TIC window,
and then double-click the TIC window.

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Figure 5-26 Re-Calibrate TOF

8.

Right-click the mass spectrum window, and then click Re-Calibrate TOF.
Figure 5-27 TOF Calibration Window

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9.

From the Reference Table list, select the reference table that was created for the calibrant(in this example, PepCalMix).
Note: If the correct reference file does not show in the list, then the reference table has notbeen created. Refer to the section: Create a Reference Table.

10. In the Tolerance field, type 0.2.
11. To calculate the average error for this new calibration, click Calculate new calibrations.
12. Make sure that the Average Error is within the routine operating standards for the mass
spectrometer being calibrated.
13. Click Calibrate spectrum.
The new calibration values show.
14. Make sure that the Set As Instrument Default check box is selected.
15. To apply this calibration to all samples in the file, click Entire File.
16. Click Save.
17. Click OK twice.
18. To close the TOF Calibration window, click Close.
19. Close the TIC and mass spectrum windows.

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Methods for the Neutral OptiMS Cartridge

Methods for the Neutral OptiMS
Cartridge

6

The methods and sequences for the neutral OptiMS cartridge have been updated forrobustness. Save the methods to the C:\32karat\projects\CEMS\Methods folder.
Table 6-1 Methods for the Neutral OptiMS Cartridge
File Name

Method Description

Neutral CESI electrical conditioning method

Used to create the electrical path in thesprayer of a new cartridge or a cartridge thathas been in long-term storage.

Neutral CESI peptide separation method

Used to inject and separate a peptide mixture.

Neutral CESI protein separation method

Used to inject and separate a protein sample.

Neutral CESI rest method

Used to clean the separation capillary andthe conductive liquid capillary at the end of asequence before the cartridge is removed forlong-term storage.

Neutral CESI washing method

Used to rehydrate the neutral coating of a new
cartridge or a cartridge that has been in longterm storage.

CLC conditioning method

Used to condition the conductive liquid
capillary

If the methods are missing, then use the following parameters on the Initial Conditions and Time
Program tabs to create them manually.

Initial Conditions Tab
All of the methods for the neutral OptiMS cartridge use the initial conditions in the following
figure.

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Methods for the Neutral OptiMS Cartridge
Figure 6-1 Initial Conditions Tab

Time Program Tab
Each of the methods for the neutral OptiMS cartridge uses a different time program.
Figure 6-2 Neutral CESI Washing Method

For the neutral CESI electrical conditioning method, use the following parameters:• In steps 1 and 2, use 50 mM AmAc buffer, pH 3.0 for the BGE.
• In steps 3 and 4, use a pressure of 5 psi at both ends.
Figure 6-3 Neutral CESI Electrical Conditioning Method

For the neutral CESI protein separation method, use the following parameters:• In step 7, use a forward pressure of 0.5 psi and a reverse pressure of 0.5 psi to prevent the
conductive line needle from drying.
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Methods for the Neutral OptiMS Cartridge• In step 9, use a forward pressure of 1.5 psi and a reverse pressure of 1.5 psi to prevent the
conductive line needle from drying.
• In step 10, use a forward pressure of 50 psi and a ramp time of 5 minutes to decrease thevoltage.
Note: The ramp down in step 10 can be omitted. Line 11 would be at time 21.
Figure 6-4 Neutral CESI Protein Separation Method

For the neutral CESI peptide separation method, use the following parameters:• In step 7, use a forward pressure of 0.5 psi and a reverse pressure of 0.5 psi to prevent
drying.
• In step 9, use a forward pressure of 2.0 psi and a reverse pressure of 2.0 psi to prevent
drying.
• In step 10, use the values in the Separate dialog to decrease the voltage. This step ismandatory for the method.
Note: The ramp down in step 10 can be omitted.

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Figure 6-5 Neutral CESI Peptide Separation Method: Separate Dialog

Figure 6-6 Neutral CESI Peptide Separation Method

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Figure 6-7 Neutral CESI Peptide Separation Method: Separate Dialog with Parameters toRamp Down the Separation Voltage

If the cartridge will be used again within 24 hours, then to prevent blockage in the sprayer tip, instep 3 of the neutral CESI rest method, change the rinse pressure to 5 psi and the duration to999 min.
Figure 6-8 Neutral CESI Rest Method

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Do a Cartridge Performance Test

7

Do a Cartridge Performance Test

After the mass spectrometer and CESI 8000 Plus system are set up, do the following procedure
to make sure that the neutral OptiMS cartridge operates correctly.
1.

In a microvial, add 100 µL of Protein Test Mix sample.

2.

Put the sample vial in the inlet sample tray in position S1:A1.

3.

If required, then update the sample location in the sequence, and then save the sequence.

4.

Do a check of the coolant level in the CESI 8000 Plus system.
If required, then add coolant. Refer to the section: Add Capillary Cartridge Coolant.

5.

Start the mass spectrometer batch.

6.

Make sure that the mass spectrometer is ready to acquire.

7.

Start the neutral CESI protein separation method.

Analyze the Data (Protein Test Mix Sample)
When the separation of the Protein Test Mix is complete, look at the detection times to makesure that all three proteins are in the 8 minute to 15 minute range.
If the proteins are not separated, then refer to the section: Troubleshooting.
The following figure shows a total ion electropherogram (TIE) for a typical separation of Protein
Test Mix and 50 mM AmAc buffer, pH 3.0.

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Figure 7-1 TIE of Protein Test Mix in 50 mM AmAc, pH 3.0 Buffer

The following figure shows the mass spectrum for each protein in the Protein Test Mix.

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Do a Cartridge Performance Test
Figure 7-2 Mass Spectra of Cytochrome C, Lysozyme, and Ribonuclease A Protein Test
Mix

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Separation of Peptide Mixtures

8

Separation of Peptide Mixtures
For a separation run with the neutral OptiMS cartridge, do this:• Use the neutral CESI peptide separation method with 10% HAc solution.
• If required, then optimize the neutral CESI peptide separation method.
• Make sure that the sample has 100 mM LE buffer, pH 4.0.

The following figure is an example of a total ion electropherogram (TIE) for a separation of an E.
coli cell lysate digest. The protein concentration was 3 μg/μL. The sample was injected at 5 psifor 60 seconds, and then separated with the neutral CESI peptide separation method.
Figure 8-1 TIE of E. coli Cell Lysate Digest

The following figure is an example of a TIE for a separation of beta-galactosidase digest. The
protein concentration was 1 μM.

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Separation of Peptide Mixtures
Figure 8-2 TIE of 1 μM Beta-Galactosidase Digest Diluted 50:50 in 200 mM AmAC LE
buffer, pH 4.0 with 10% HAc Solution as the BGE

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About Autocalibration

About Autocalibration

9

Small variations in temperature that occur during usual operation can cause incorrect masses tobe reported. To increase the mass accuracy, we recommend that autocalibration be done every
5 hours of data acquisition.
Note: Do manual calibration before autocalibration is done. If the reference ions are outside of
the 100 ppm tolerance for peak identification, then autocalibration will not be successful.
Note: The following procedures are for the SCIEX 5600 and 6600 systems. For other SCIEX
mass spectrometers or mass spectrometers from other manufacturers, use the manufacturer's
recommendations.
The following figure shows the sample tray layout for autocalibration.
Figure 9-1 Sample Tray Layout for Autocalibration

Autocalibration uses the CESI-MS autocalibration method and the following time program.

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About Autocalibration
Figure 9-2 Time Program for Autocalibration

Analyze the autocalibration data to make sure that the peaks were identified and their calculatedmasses are satisfactory. Refer to the section: Analyze the Autocalibration Data.

Create a Reference Table
1.

In the Analyst TF software, in the Navigation bar, click Acquire.

2.

Click Tools > Settings > Tuning Options, and then click Reference.
Figure 9-3 Reference Table Editor Window

3.

To open a new, empty reference table, click New.

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About Autocalibration
Figure 9-4 New Reference Table in the Reference Table Editor Window

4.

In the Name field, type a name that identifies the calibration solution (in this example,
PepCalMix).

5.

Make sure that Positive is clicked.

6.

In the Reference Ions for TOF MS Calibration table, type the following values.
Table 9-1 Reference Ions for TOF MS Calibration
Compound Name

Precursor m/z (Da)CE forMS/MS

DP forMS/MS

AETSELHTSLK

408.55010

4080GAYVEVTAK

473.26020

4080

IGNEQGVSR

485.25302

4080

LVGTPAEER

491.26559

40

80

LDSTSIPVAK

519.79969

40

80

AGLIVAEGVTK

533.32333

40

80

LGLDFDSFR

540.27342

40

80

GFTAYYIPR

549.28633

4080

SGGLLWQLVR

569.83398

40

80

AVGANPEQLTR

583.31360

40

80

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About Autocalibration
Table 9-1 Reference Ions for TOF MS Calibration (continued)
Compound Name

Precursor m/z (Da)CE forMS/MS

DP forMS/MS

SAEGLDASASLR

593.80053

4080

VFTPLEVDVAK

613.34955

4080

VGNEIQYVALR

636.35273

40

80

YIELAPGVDNSK

657.34499

4080

DGTFAVDGPGVIAK

677.85827

40

80

YDSINNTEVSGIR

739.36148

40

80

SPYVITGPGVVEYK

758.91050

40

80

ALENDIGVPSDATVK

768.90340

4080

AVYFYAPQIPLYANK

883.47380

4080

TVESLFPEEAETPGSAVR

964. 97741

40

80

7.

For each compound, click the Use check box.

8.

In the Reference Ions for MS/MS Calibration table, type the following values.
Table 9-2 Reference Ions for MS/MS Calibration

9.

Fragment Name

Fragment m/z (Da)b2

185.09207

b3

348.15540

b4

560.30788

b5

661.35555

N/A

758.91050

y7

799.44398

y8

856.46544

y9957.51312

y101070.59719

y111169.66560

y12

1332.72893

For each fragment, click the Use check box.

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About Autocalibration
10. In the Use for MS/MS column, select the check box for the applicable compound in thecalibration solution (in this example, the peptide at m/z 758.91).
11. Make sure that the value in the Retention time tolerance +/- field is 30.
12. Click OK.
Figure 9-5 Tuning Options Dialog

13. Make sure that the name of the new reference table (in this example, PepCalMix) shows in
the Positive Reference field.
14. Click OK.

Create the MS Acquisition Method for
Autocalibration
1.

In the Analyst TF software, in the Navigation bar, under Acquire, double-click Build
Acquisition Method.

2.

In the Acquisition method pane, click TOF MS (+).

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About Autocalibration
Figure 9-6 Acquisition method Pane: TOF MS

3.

On the MS tab, click Edit Parameters.
The Parameter Settings dialog opens.

4.

On the Source/Gas tab, do this:a.

In the Curtain Gas (CUR) field, type 10.

b.

In the IonSpray Voltage Floating (ISVF) field, type 1500 or the optimized voltage.
The ISVF parameter sets the ESI voltage.

c.

In the Interface Heater Temperature (IHT) field, type 50.0.

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About Autocalibration
Figure 9-7 TOF MS (+) Parameter Settings Dialog: Source/Gas Tab

5.

Open the Compound tab, and then do this:Instruction Guide
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About Autocalibration
Figure 9-8 TOF MS (+) Parameter Settings Dialog: Compound Tab

6.

a.

In the Declustering Potential (DP) field, type 100.

b.

In the Collision Energy (CE) field, type 10.

In the Acquisition method pane, click Product Ion (+) 758.9 (item 1).
Figure 9-9 Acquisition method Pane: Product Ion

7.

On the MS tab, click Edit Parameters.
The Parameter Settings dialog opens.

8.

On the Source/Gas tab, do this:a.

In the Curtain Gas (CUR) field, type 10.

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About Autocalibrationb.

In the IonSpray Voltage Floating (ISVF) field, type 1600.
The ISVF parameter sets the ESI voltage.

c.
9.

In the Interface Heater Temperature (IHT) field, type 50.0.

Open the Compound tab, and then do this:a.

In the Declustering Potential (DP) field, type 100.

b.

In the Collision Energy (CE) field, type 42.

c.

In the Collision Energy Spread (CES) field, type 5.

d.

In the Ion Release Delay (IRD) field, type 67.

e.

In the Ion Release Width (IRW) field, type 25.

Add Autocalibration to the Sequence
Use this procedure to edit the sequence table to include autocalibration.
Note: The sequences in the 32 Karat software and on the mass spectrometer must be the
same.
1.

In the 32 Karat software, open the sequence table.

2.

Click to highlight the first run, right-click the highlighted run, and then click Insert Line.

3.

At the bottom of the Method column, click the green arrow icon and then click CESI-MS
Auto-calibration_ABSciex.met.

4.

In the Sample ID and File name fields, type the applicable values.

5.

To add another line for autocalibration, click to highlight the run on line 7, right-click thehighlighted run, and then click Insert Line.

6.

Do steps 4 and 5 again.
The following figure shows an example of the sequence.

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About Autocalibration
Figure 9-10 Sample Sequence Example

7.

On the toolbar, click Sequence > Properties.
Figure 9-11 Sequence Properties Dialog

8.

Make sure that the File paths for Method and Data show the correct folder to save the data,
and then click OK.
Note: If required, then create a folder.

9.

Click File > Sequence > Save As.
In this example, the sequence name is PepCalMix_Installation.

Analyze the Autocalibration Data
For the autocalibration to be successful, the following criteria must be met:

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About Autocalibration• The intensity of the reference ions is at least 10 counts per second (cps) in the mass
spectrometer data and 3.3 cps in the MS/MS data.
• The reference ions are within a mass tolerance of 100 ppm.
• There are ≥ 80% of the selected ions in the reference table.
If autocalibration is successful, then a green check mark is shown. If the sample was acquired
but the calibration failed because one or more of the reference ions did not meet the calibration
criteria, then a red circle with a diagonal line through it is shown. To see information about the
failure, double-click the circle icon.
If autocalibration fails, then an error message is shown in the Sample Details dialog. Click OK,
and then refer to the section: Troubleshoot an Autocalibration Failure.
1.

In the Analyst TF software, in the Navigation bar, under Explore, double-click Open Data
File.
If the Keep calibration data file check box was selected in the Queue Options dialog,
then data for each autocalibration is saved as a separate data file in the Cal Data
subfolder. The autocalibration data file names start with Cal and include the time stamp
and calibration sample index. For example, Cal20130910162907040.wiff.

2.

To extract the mass spectra from the TIC, highlight the region and then double-click
the region. Extract one mass spectrum between 2 minutes to 2.5 minutes (MS before
calibration) and another between 4 minutes to 4.5 minutes (MS after calibration).
Figure 9-12 Data Analysis

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About Autocalibration

Item

3.

Description

1

MS before calibration

2

MS/MS before calibration

3

MS after calibration

4

MS/MS after calibration

To get a list of ions in the mass spectrum, right-click the mass spectrum and then click List
data. Do this for both MS before calibration and MS after calibration.
Figure 9-13 Obtaining List Data

4.

To show all the reference ions used for calibration, in each data list table, open the
Calibration Peak List tab.

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About Autocalibration
Figure 9-14 Reference Ions

5.

To make sure that the correct reference table has been selected, right-click the referencetable, click the PepCalMix_CE-MS_Calibration Ref reference table, and then click Use as
reference.
Figure 9-15 Reference Table Verification for MS Before Calibration

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About Autocalibration
Figure 9-16 MS After Calibration Analysis

The tables show the Target Mass (or theoretical mass) and the Found At mass (orexperimental mass) for all eight reference ions selected for autocalibration. The mass shift
between the Target Mass and the Found At mass is given as Mass Shift (ppm).
The mass shift is higher before calibration and lower after calibration for all ions.
6.

To evaluate the MS/MS mass accuracy, do steps 3 and 4 for MS/MS before and after
calibration.
For the MS/MS experiment, the mass shift after calibration is lower than it was before
calibration.

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About Autocalibration
Figure 9-17 Evaluation of Peak Data in MS/MS Experiment

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Shut Down and Disconnect

10

Shut Down and Disconnect
Remove the Cartridge

If the CESI 8000 Plus system will be shut down and the power turned off, then remove thecartridge.
1.

Make sure that the ESI voltage on the mass spectrometer is off.

2.

Do the applicable procedure in the section: Stow the Cartridge.

3.

In the 32 Karat software, go to the Direct Control window, and then click Load.

4.

Open the cartridge cover.
Figure 10-1 Cartridge CoverThe coolant pump for the CESI 8000 Plus system turns on and releases the coolant fromthe cartridge coolant lines. Approximately 30 seconds are required. Wait for the pump toturn off.
5.

Loosen the thumbscrews on the insertion bar.

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Shut Down and Disconnect
Figure 10-2 Thumbscrews on the Insertion Bar

6.

Lift the insertion bar fully.

7.

To prevent damage to the sprayer tip, retract the stage as far away from the inlet on themass spectrometer as possible.

8.

To loosen the end of the sprayer from the adapter, turn the arrow on the sprayer to the
Unlock position.
WARNING! Hot Surface Hazard. The surfaces of the ion source become hot
during operation. Let the ion source cool for at least 30 minutes before thesprayer is removed from the adapter on the mass spectrometer.
Figure 10-3 Unlock the Sprayer from the Cartridge

Item1

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Description
Sprayer in the Unlock position

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Shut Down and Disconnect

Item
2
9.

Description
Turn to lock the sprayer from the adapter

Remove the sprayer from the adapter.
Figure 10-4 Sprayer Removal

10. Put the protective sleeve on the sprayer tip.
11. Put the tubing with the sprayer end through the access panel.
Figure 10-5 Sprayer and Tubing Through the Access Panel

12. Remove the coolant tubing from the notched arm.

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Shut Down and Disconnect
Figure 10-6 Coolant and Capillary Tubing

13. Hold the cartridge sprayer with one hand and the midsection of the cartridge with the other
hand, then lift the cartridge up and pull it out.
Figure 10-7 Remove the Cartridge

Note: As the cartridge moves up, the protective sleeves on the inlet and outlet sides godown over the capillary tips.
Note: Drops of liquid coolant usually fall from the cartridge tips, and do not cause damage
to the hardware.
14. Refer to the section: Stow the Cartridge.

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Shut Down and Disconnect

Stow the Cartridge
WARNING! Puncture Hazard. Touch the cartridge carefully. The capillary tips are
extremely sharp.
CAUTION: Potential System Damage. To prevent contamination of the internal surface ofthe separation capillary, do not use the pressure rinse function to blow air through thecapillaries. Use the vacuum function to dry the capillaries.
1.

2.

To prepare the cartridge to be stowed for less than 3 days, do this:a.

Run the neutral CESI rest method.

b.

Keep the cartridge in the system for as long as 3 days.

To prepare the cartridge to be stowed for more than 3 days, do this:a.

Run the neutral CESI rest method.

b.

Disconnect the sprayer from the ion source.

c.

Remove the cartridge from the system.

d.

Put the protective sleeve on the sprayer tip.

e.

Put the sprayer tip in a 2 mL microcentrifuge tube of water.

f.

Loosely seal the tube with a film cover.

g.

Keep the cartridge in a safe area at 2 °C to 8 °C.

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Add Capillary Cartridge Coolant

Add Capillary Cartridge Coolant

11

Required Materials• Capillary cartridge coolant (PN 359976)• Coolant fill tool (PN 144647)

CAUTION: Potential System Damage. To prevent damage, do not use the plunger inthe coolant fill tool when coolant is added. Gravity supplies sufficient force to pull thecoolant into the system.
1.

Open the sample cover.
Figure 11-1 Sample Cover (Outer Door)

2.

Connect the coolant fill tool to the coolant fill port.

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Add Capillary Cartridge Coolant
Figure 11-2 Coolant Fill Tool Connected to the Coolant Fill Port

3.

Fill the syringe with 120 mL of coolant.

4.

Make sure that the CESI 8000 Plus system is on and a cartridge is installed.

5.

Slowly add coolant until the fill indicator is between the yellow lines in the coolant sightglass.
Figure 11-3 Coolant Sight Glass

6.

Remove the coolant fill tool, and then close the sample cover.

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Troubleshooting

12

Troubleshooting

For more information about troubleshooting, refer to the document: CESI 8000 Plus High
Performance Separation-ESI Module User Guide.
Symptom

Possible Cause

Electrospray is detected when 1.
the ESI voltage is zero andseparation voltage is applied2.
with forward pressure

A cable is missing or is
not fully connected.

3.

The cartridge capillaries
are not filled withseparation buffer.

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Corrective Action
1.

Make sure that all of
the required cables areattached fully. Refer tothe document: CESI 8000
Plus High Performance
Separation-ESI Module
User Guide.

2.

Remove the sprayer fromthe adapter and pull up
on the metal contact forthe sprayer. Install thesprayer in the adapter,and make sure that thesprayer is in the locked
position.

3.

To make sure that the
separation capillary andconductive liquid capillary
are filled with separationbuffer, rinse each one at
100 psi for 3 minutes.

The adapter on the mass
spectrometer does nottouch the cartridge.

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Troubleshooting

Symptom

Possible Cause

Corrective Action

No flow through theseparation capillary1.

The capillary is blocked.

1.

2.

The source temperature
is too high, which causes
the precipitation of protein
at the emitter.

Refer to the section:
Clean Blockage fromthe Sprayer Tip. If
the blockage is at the
inlet side, then apply avacuum.

3.

The capillary is broken.

2.

To increase solubility, add
an organic such as 10%
isopropanol. To remove
the blockage, run theneutral CESI washingmethod with 10% IPA
while the sprayer tip is
immersed in a 50 mL
Falcon tube filled with
10 mL of 10% IPA.

3.

Replace the cartridge.

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Troubleshooting

Symptom

Possible Cause

Corrective Action

Electrospray is not stable

1.

The ESI voltage is not
optimized.

1.

2.

The sprayer position is
not optimized in front of
the inlet on the mass
spectrometer.

Find the minimum
ESI voltage, and thenincrease its working value
by 0.2 kV.

2.

At the same time,
optimize the sprayer
position, and apply
separation voltage andforward pressure at
1.5 psi.

3.

Increase the pressure to
1.5 psi or more.

3.

The forward pressure in
the separation capillary is< 1.5 psi.

4.

The sprayer needle is not
fully filled.

5.

The buffer concentration4.
and pH are not optimized.

6.

The value for curtain gas
is too high.

7.

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The cartridge was
exposed to temperatures< 2 °C.

Make sure that the
conductive liquid capillary
is filled fully with solution.

5.

Optimize the bufferconcentration and pH.

6.

Decrease the curtaingas to the recommended
value.

7.

Condition the capillaries.
If the electrospray is still
not stable, then replace
the cartridge.

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Troubleshooting

Symptom

Possible Cause

Corrective Action

Carryover occurs between
runs

1.

The buffer vials
are contaminated withsample.

1.

2.

The separation capillary
was not rinsed sufficiently
between separations.

Replace the buffer vials.
To prevent contaminationof the buffer vials, makesure that a water dip stepis included after sample
injection in the separation
method.

2.

Do this:Sample peaks are tailing in
the TIE

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a.

Do a rinse with
0.1 M hydrochloric
acid (HCl) at the startof each separation.

b.

Increase the rinse
times between
separations.

1.

The separation capillary1.
was not rinsed sufficiently
between separations.

Do a rinse with 0.1 M
HCl at the start of eachseparation.

2.

The buffer concentration 2.
and pH are not optimized,
and high electro-migration
dispersion occurred.

Do this:3.

The forward pressure
value is too high, whichcaused a distorted peak
shape.

4.

The neutral coating isdamaged.

a.

Increase the rinse
times between
separations.

b.

Optimize the bufferconcentration and pH
for the sample that isanalyzed.

3.

Decrease the forward
pressure to 1.5 psi.

4.

Run a Protein Test Mix
sample to make sure thatthe separation is correct.
If the separation fails,
then replace the cartridge.

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Troubleshooting

Symptom

Possible Cause

Corrective Action

No sample is detected

1.

The sample vial is in anincorrect position.

1.

2.

The sample volume is not
sufficient.

Make sure that the
sample vial is in thecorrect position in thesample tray, sequence,and method.

3.

The injection plug is too
short.

2.

4.

The sample is too dilute.

5.

An incorrect polarity isused in the method.

Increase the sample
volume to > 50 μL for
microvials or > 5 μL for
nanoVials.

3.

The separation method is
too short.

Increase the injection time
and/or injection pressure.

4.

Use a more concentrated
sample.

6.
7.

Low sensitivity due to highly
oxidized proteins

1.

The ESI voltage is too
high.

2.

The sprayer tip is too
close to the inlet on themass spectrometer.

3.

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The mass spectrometersettings are not optimized. 5.

Use the normal polarity.

6.

Increase the separation
time in the CE and mass
spectrometer methods.

7.

Make sure that the mass
spectrometer settings arecorrect and the ionization
parameters have been
optimized for the sample
that is analyzed.

1.

Decrease the ESI voltage
and optimize the positionof the sprayer tip.

2.

Move the sprayer tip awayfrom the inlet on the mass
spectrometer. Refer to thesection: Fine-Tune thePosition of the Sprayer
Tip.

3.

Decrease the entrance
temperature for the mass
spectrometer.

The entrance temperature
for the mass spectrometeris too high.

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Troubleshooting

Symptom

Possible Cause

Corrective Action

Capillary coating is frozen

The cartridge was exposed totemperatures < 2 °C.

Run the neutral CESI washingmethod to rehydrate theneutral coating, and then keep
the cartridge in the instrumentfor 24 hours. Continued
flushing is not required. After
24 hours, run the neutral CESI
electrical conditioning method.

Troubleshoot an Autocalibration Failure
1.

Do the procedure in the section: Analyze the Autocalibration Data.

2.

Make sure that the intensity of the reference ions is correct:• Mass spectrometer data: > 10 cps• MS/MS data: > 3.3 cps

3.

In the Calibration peak list table, if the value in the Mass shift (ppm) field is > 100 ppm,
then do manual calibration.
Refer to the section: Manual Calibration.

4.

Make sure that the threshold for peak detection is 1% in the spectrum.
a.

In the Navigation bar, under Tune and Calibrate, click Tools > Settings > Appearance
Options > Other Graph.

b.

In the Default Threshold for the Spectrum field, type 1.

Conductive Liquid Capillary Contamination
If the baseline mass spectrum shows a series of peaks with a difference in m/z of 98, thenthe conductive liquid capillary might be contaminated with phosphate. To remove phosphate
contamination, condition the conductive liquid capillary.

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Troubleshooting
Figure 12-1 Profile of Phosphate Contamination

Condition the Conductive Liquid Capillary1.

Use the time program in the following figure.
Figure 12-2 Time Program for the Conductive Liquid Capillary Conditioning Method2.

Use the following figure to put each vial in the correct position in the buffer inlet and outlet
trays.
Note: This method uses reverse rinses. Only one vial is required in the inlet buffer tray, at
position A1.

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Troubleshooting
Figure 12-3 Tray Layout for the Conductive Liquid Capillary Conditioning Method

Clean Blockage from the Sprayer Tip
CAUTION: Potential System Damage. Do not put more than 10 mL of CE Grade water inthe tube. If there is more than 10 mL in the tube, then the liquid can splash onto the metalcomponents of the sprayer and cause damage.
1.

Put 10 mL of CE Grade water in a 50 mL Falcon tube, and then put the tube in the holster
on the side of the system.

2.

Carefully immerse the sprayer tip in the CE Grade water.

3.

In the 32 Karat software, go to the Direct Control window.

4.

Do a forward rinse with BGE at 100 psi for 5 minutes.

5.

If required, then run the neutral CESI washing method and then the neutral CESI electricalconditioning method to condition the capillaries.

6.

After 5 minutes, remove the sprayer from the CE Grade water.

7.

Use lint-free wipes to dry the sprayer carefully.

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Troubleshooting
Figure 12-4 Dry the Sprayer

8.

Install the sprayer in the adapter on the mass spectrometer.

9.

Remove the Falcon tube of CE Grade water from the holster, and discard the contents.

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Contact Us

Contact Us
Customer Training• In North America: NA.CustomerTraining@sciex.com• In Europe: Europe.CustomerTraining@sciex.com• Outside the EU and North America, visit sciex.com/education for contact information.

Online Learning Center• SCIEX Now Learning Hub

Purchase Supplies and Reagents
Reorder SCIEX supplies and reagents online at store.sciex.com. To set up an order, use theaccount number, found on the quote, order confirmation, or shipping documents. Currently,
customers in the United States, Canada, United Kingdom, Belgium, Netherlands, France,
Germany, and Switzerland have access to the online store, but access will be extended to othercountries in the future. For customers in other countries, contact a local SCIEX representative.

SCIEX Support
SCIEX and its representatives have a global staff of fully-trained service and technicalspecialists. They can supply answers to questions about the system or any technical issues
that might occur. For more information, go the SCIEX website at sciex.com or use one of thefollowing links to contact us.
• sciex.com/contact-us• sciex.com/request-support

Cybersecurity
For the latest guidance on cybersecurity for SCIEX products, visit sciex.com/productsecurity.

Documentation
This version of the document supersedes all previous versions of this document.
To find software product documentation, refer to the release notes or software installation guidethat comes with the software.

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Contact Us
To find hardware product documentation, refer to the documentation that comes with the systemor component.
The latest versions of the documentation are available on the SCIEX website, at sciex.com/
customer-documents.
Note: To request a free, printed version of this document, contact sciex.com/contact-us.

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