OptiMS Bruker MS Adapter
For Bruker Mass Spectrometers
Installation Guide

RUO-IDV-05-3314-C | 5302403

December 2023

This document is provided to customers who have purchased SCIEX equipment to use in the operation ofsuch SCIEX equipment. This document is copyright protected and any reproduction of this document or
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Software that may be described in this document is furnished under a license agreement. It is against thelaw to copy, modify, or distribute the software on any medium, except as specifically allowed in the license
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Contents

1 Overview.................................................................................................................................... 5
Bill of Materials..........................................................................................................................5
Regulatory Compliance.............................................................................................................5International Standards........................................................................................................ 5
Decommissioning and Disposal................................................................................................ 6
Hazard Symbols........................................................................................................................6
Occupational Health and Safety Symbols............................................................................6
Symbols, Indicators, and Labels.......................................................................................... 7
Documentation Symbols and Conventions.......................................................................... 7
2 Install the Adapter.....................................................................................................................9
Connect the Cables.................................................................................................................15
3 Align the System with the Mass Spectrometer.................................................................... 19
4 Prepare the Reagents............................................................................................................. 23
Prepare the 10% HAc Solution............................................................................................... 23
Prepare the 20% HAc Solution............................................................................................... 23
Prepare the 200 mM LE Buffer, pH 4.0................................................................................... 24
Prepare the Beta-Galactosidase Stock Solution..................................................................... 24
Prepare the Beta-Galactosidase Test Sample........................................................................ 25
Prepare the Peptide Test Sample........................................................................................... 25
5 Prepare the Buffer Trays........................................................................................................ 26
6 Prepare the System................................................................................................................ 27
Install the Cartridge................................................................................................................. 27
Remove and Install the Interface Plate.............................................................................. 32
Condition the Capillaries......................................................................................................... 34
Establish a Stable Spray......................................................................................................... 36
Optimize the ESI Voltage........................................................................................................ 39
Fine-Tune the Position of the Sprayer Tip...............................................................................43
7 Do a Test Separation...............................................................................................................46
Create the Mass Spectrometer Method.................................................................................. 46

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Contents
Manually Create the CESI-MS Separation Method.................................................................49
Load the Sample and Start the Run........................................................................................50
Analyze the Data.....................................................................................................................51
8 Shut Down and Disconnect................................................................................................... 56
Remove the Cartridge............................................................................................................. 56
Store the Cartridge..................................................................................................................60
Disconnect the CESI 8000 Plus System from the Mass Spectrometer...................................60
A Tighten the Screws that Hold the Stage...............................................................................62

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Overview

1

Overview

This document includes instructions to install an adapter for the mass spectrometer, connect
the CESI 8000 Plus system to the mass spectrometer, and perform a a test separation to verify
correct system operation.
Note: For instructions about how to use the system safely, refer to the document: User Guide.

Bill of Materials
Part Number Description
B82388Quantity
1

Adapter kit, containing:• Adapter• Chassis ground cable• Ground cable• OptiMS Bruker MS Adapter Installation Guide (thisdocument)• Relay cable

B45936

Ion source

1

Regulatory ComplianceThis system complies with the regulations and standards listed in this section. For dated
references, refer to the declaration of conformity included with the system and the individual
system components. Applicable labels have been affixed to the system.

International Standards
The OptiMS Bruker MS Adapter has been evaluated as part of the CESI 8000 Plus High
Performance Separation-ESI Module and found to be in compliance with the International
Electrotechnical Commission standard IEC 61010-1, 2nd edition: 2010 Safety requirements
for electrical equipment for measurement, control, and laboratory use - Part 1: General
requirements.

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Overview

Decommissioning and Disposal
WARNING! Environmental Hazard. Obey established procedures for disposalof biohazardous, toxic, radioactive, and electronic waste. The customer is
responsible for the disposal of hazardous substances, including chemicals,
waste oils, and electrical components, in accordance with local laws andregulations.
Before decommissioning, obey local regulations to decontaminate the entire system.
When the system is removed from service, obey national and local environmental regulations todivide and recycle different materials.
Note: SCIEX will not accept any system returns without a completed Decontamination Form.
Contact an FSE to get a copy of the form.
Do not discard system components or subassemblies, including computer parts, as unsorted
municipal waste.

Hazard Symbols
This section lists the hazard symbols and conventions used in the laboratory environment, on
the system, and in the documentation.

Occupational Health and Safety Symbols
This section describes some occupational health and safety symbols found in the documentationand laboratory environment.
Table 1-1 General Hazard Symbols
Safety Symbol

Description
Personal Injury Hazard

Table 1-2 Chemical Hazard Symbols
Safety Symbol

Definition
Biohazard

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Overview

Table 1-2 Chemical Hazard Symbols (continued)
Safety Symbol

Definition
Explosion Hazard

Toxic Chemical Hazard

Fire Hazard

Table 1-3 Electrical Hazard Warning Symbols
Safety Symbol

Definition
Electrical Shock Hazard

Table 1-4 Mechanical Hazard Symbols
Safety Symbol

Definition
Hot Surface Hazard

Symbols, Indicators, and Labels
Note: If any of the labels used to identify a component become detached, then contact a SCIEX
service representative.

Documentation Symbols and Conventions
The following symbols and conventions are used throughout the guide.
DANGER! Danger identifies an action that can cause severe injury or death.

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Overview

WARNING! Warning identifies an action that can cause personal injury if
precautions are not obeyed.
CAUTION: Caution identifies an operation that can cause damage to the system or
corruption or loss of data if precautions are not obeyed.
Note: Notes supply important information in a procedure or description.
Tip! Tips supply information that helps to apply the techniques in a procedure or gives ashortcut, but that is not essential to the completion of a procedure.

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Install the Adapter

2

Install the Adapter
Required Materials• NanoElectrospray capillary cap set (PN 8212915) (purchased from Bruker)• 1.5 mm hex key (supplied with the mass spectrometer)• SCIEX ion source (PN B45936)• Adapter (PN B10249)• Power-free gloves (nitrile or neoprene recommended)1.

In the otofControl software, click Standby to put the mass spectrometer in standby mode.
Figure 2-1 Standby Button in otofControl Software

2.

Turn off any solvent flow from the LC system and then shut off the nebulizer gas.
WARNING! Hot Surface Hazard. Beware of burns. The spray chamber becomes
hot during operation. Do not touch the part until it has had sufficient time to
cool.

3.

Disconnect the LC tubing and the tubing for the nebulizer gas from the ion source.

4.

Open the Source tab and then click Change to open the Change Source dialog.

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Install the Adapter
Figure 2-2 Change Source Dialog

5.

Click nano ESI on-line and then click Activate Source.

6.

Remove the Bruker ion source.

7.

a.

Open the door to the right of the source to provide access to the silver latch, and thenopen the latch.

b.

Pull the spray chamber cover to the left and then lift it out of the hinge cylinders toremove it. Put the cover in a safe place.

c.

Use the 1.5 mm hex key to loosen (but do not remove) the set screw at the top of thespray shield. Pull the spray shield away from the mass spectrometer to remove it.

d.

Pull off the capillary cap to remove it.

Install the black gas diverter from the NanoElectrospray capillary cap set and then secure itwith the set screw.
CAUTION: Potential System Damage. Tighten the screw until it is a small amountmore than finger-tight. To prevent damanage, do not overtighten the screw.

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Install the Adapter
Figure 2-3 Gas Diverter

8.

Put the metal cap on the capillary. The rear edge does not need to be flush with the gas
diverter.
CAUTION: Potential System Damage. For dry gas flow, use the lowest flow ratepossible for the experiment. Because the capillary cap is attached by friction, highflow rates can push the cap close enough to the sprayer tip to cause damage.

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Install the Adapter
Figure 2-4 Metal Cap

9.

Install the SCIEX ion source.
a.

Gently drop the ion source hinge pins into the hinge cylinders on the mass
spectrometer.

b.

Push the ion source to the right, toward the mass spectrometer, and then close thesilver latch to secure it.

c.

Close the door to the right of the ion source on the mass spectrometer.

10. Before installing the adapter, use the thumbscrews in the following figure to adjust theposition of the stage.

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Install the Adapter
Figure 2-5 Ion Source

Item

a.

Description

1

Thumbscrew for horizontal position

2

Thumbscrew for vertical position

3

Thumbscrew for forward and back position

4

Clamp

5

Clamp thumbscrew

Use thumbscrew 1 to move the stage horizontally until the two parts of the stage areflush with each other.

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Install the Adapter
Figure 2-6 Right Side View of Stage–Shaded Items Flushb.

Use thumbscrew 2 to move the stage vertically until the top portions of the stage areflush with each other.
Figure 2-7 Top View of Stage–Shaded Items Flushc.

Use thumbscrew 3 to move the stage as far from the ion source as possible.

11. Install the adapter.
a.

Loosen the thumbscrew on the clamp on the left side of the adapter mounting bracket.

b.

Install the adapter in the mounting bracket. Push gently until the adapter is flush against
the clamp (shown in blue in Figure 2-9).

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Install the Adapter
Figure 2-8 Inserting Adapter in Ion Source

Figure 2-9 Adapter Flush to Clampc.

Tighten the thumbscrew on the clamp (Figure 2-5, item 5).

Connect the Cables
1.

Put the CESI 8000 Plus system approximately 18 cm to 20 cm (7.1 inches to 7.9 inches)away from the ion source.

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Install the Adapter
2.

Connect the single connector on the end of the relay cable (Figure 2-11, item 3) to the relay
port on the CESI 8000 Plus system (labeled I/O in the following figure). Make sure that theconnector is as far to the right as possible.
Figure 2-10 Relay Cable Connection

3.

Connect the other end of the relay cable (Figure 2-11, item 1) to the Peripheral Interface
port on the mass spectrometer.
WARNING! Personal Injury Hazard. Route the relay cable where users cannot
trip on it.
Figure 2-11 Relay Cable

Item

Description

1

To Peripheral Interface port on mass spectrometer2

To port on mass spectrometer acquisition computer (refer to note below)

3

To CESI 8000 Plus system

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Install the Adapter

Note: As an alternative, the CESI 8000 Plus system can be configured so that the mass
spectrometer can use different acquisition methods in a single sequence. That configuration
uses 2 on the relay cable and requires additional software. Contact Bruker for instructions.
4.

Connect the ground cable.
a.

On the right side of the CESI 8000 Plus system, open the access door to show thehigh-voltage connections panel (Figure 2-12).

b.

Connect the white high-voltage cable to the Input port on the connections panel, andthen turn the cable clockwise to lock it in position.

c.

Connect the red banana plug to the red port on the connections panel.

Figure 2-12 High-Voltage Connections Panel

Item

Description

1

Input (red) port on connections panel

2

Red banana plug3

Output (black) port on connections panel

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Install the Adapter

Item

5.

Description

4Black banana plug

5

Ground cable

6High-voltage output cable to adapter to Output (black)Connect the high-voltage output cable (attached to the adapter) to the CESI 8000 Plus
system.
a.

Connect the white high-voltage cable to the Output port on the connections panel, andthen turn the cable clockwise to lock it in position.

b.

Connect the black banana plug to the black port on the connections panel.

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Align the System with the Mass Spectrometer

Align the System with the Mass
Spectrometer3

To prevent hydrodynamic (siphoning) flow in the capillaries and to let the coolant drain from the
cartridge before the cartridge assembly is removed, align the sprayer tip installed in the mass
spectrometer adapter with the height indicator on the CESI 8000 Plus system.
CAUTION: Potential System Damage. Move the adapter mounting stage as far away as
possible from the inlet on the mass spectrometer. If the sprayer is too close to the inlet,
then damage to the sprayer can occur.
1.

Make sure that the CESI 8000 Plus system is approximately 18 cm to 20 cm (7.1 inches to
7.9 inches) away from the ion source so that the coolant tubes can be extended fully.

2.

To adjust the height of the cart, press the buttons on the front of the cart until the indicatorline on the CESI 8000 Plus system is aligned with the inlet on the mass spectrometer.
WARNING! Personal Injury Hazard. Remove consumables from the mobile cart
before the height is adjusted.
Note: Do not press both the Up and Down buttons at the same time.

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Align the System with the Mass Spectrometer
Figure 3-1 Alignment of the CESI 8000 Plus System and the Ion Source Inlet

Note: If the cart cannot move to its lowest height or if it becomes locked in position, then the
cart must be programmed again. To set the home position for the mobile cart, refer to the
document: CESI 8000 Plus High Performance Separation-ESI Module User Guide.
3.

When the CESI 8000 Plus system is in visual alignment with the mass spectrometer, stepon the locking lever of each caster to lock each wheel of the mobile cart in position.

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Align the System with the Mass Spectrometer
Figure 3-2 Lock the Wheels of the Mobile Cart

4.

Install the sprayer tip in the adapter.
CAUTION: Potential System Damage. Do not move the sprayer tip too close to theinlet on the mass spectrometer. If the sprayer tip is < 2 mm from the inlet, then theaccidental suction of rinse solution into the mass spectrometer can occur and cause
damage.
a.

Remove the protective sleeve from the sprayer tip.
Figure 3-3 Protective Sleeve and Sprayer Tipb.

While holding the body of the adapter to prevent it from moving towards the inlet, install
the sprayer in the adapter, aligning the arrow on the cartridge with the unlock positionon the adapter.

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Figure 3-4 Sprayer Tip Approaching the Adapter with the Adapter in the Unlocked
Positionc.

Push the sprayer into the adapter and turn it counterclockwise to lock it in position. The
triangle on the sprayer should line up with the lock icon on the adapter.
Figure 3-5 Sprayer in the Locked Position in the Adapter

5.

Use the thumbscrews on the ion source stage to move the sprayer tip 2 mm to 5 mm awayfrom the ion source inlet.
If a stage feels loose or the stage position is not maintained over time, then follow the
instructions to tighten it. Refer to the section: Tighten the Screws that Hold the Stage.

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Prepare the Reagents

4

Prepare the ReagentsThe following reagents are used with the silica surface OptiMS cartridge. The sample typecontrols which reagents are required to do a separation.
Note: The examples in this guide use beta-galactosidase stock solution. For information about
how to prepare and use PepCalMix, refer to the document: CESI 8000 Plus High Performance
Separation-ESI Module User Guide.
Table 4-1 Required Reagents for the Silica Surface OptiMS Cartridge
Reagent

Purpose

10% Acetic acid (HAc)

Background electrolyte (BGE) and conductive
liquid

20% HAc

pH adjustment of leading electrolyte (LE)LE buffer, 200 mM ionic strength in ammonium Sample and system suitability preparation
acetate (AmAc), pH 4.0
Beta-galactosidase stock solution, 1 μM

Test sample(Optional) PepCalMix

Test sample

Peptide test sample

Optimize electrospray ionization (ESI) voltage

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.

5.

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 LE buffer, pH 4.0 is prepared.
1.

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

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Prepare the Reagents
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 LE Buffer, pH 4.0
1.

To prepare 50 mL of 400 mM 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 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.

Prepare the Beta-Galactosidase Stock Solution
WARNING! Toxic Chemical Hazard. When handling, storing, and disposing ofchemicals, refer to the chemical product safety data sheets. Obey all of therecommended safety procedures . For health and safety precautions, refer to the
document: Overview Guide.
1.

Add 625 µL of CE Grade Water to the vial with beta-galactosidase stock solution.

2.

Mix the solution in a vortex mixer for at least 30 seconds.

3.

Use a centrifuge to spin the vial and move the liquid to the bottom.

4.

Divide the beta-galactosidase stock solution into aliquots of 50 µL.

5.

Keep the beta-galactosidase stock solution at –35 °C to –15 °C for as long as 1 year.

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Prepare the Reagents

Prepare the Beta-Galactosidase Test Sample
1.

If required, then remove 50 µL of beta-galactosidase stock solution from the freezer and let
it thaw at room temperature.

2.

In a 0.5 mL centrifuge tube, add 50 µL of beta-galactosidase stock solution and 50 µL of
1 μM 200 mM LE buffer, pH 4.0 to make a 1:1 solution.

3.

Mix the solution in a vortex mixer for at least 30 seconds.

4.

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

5.

Use a pipette to put 90 µL of the supernatant in a sample vial.
Keep the sample at 2 °C to 8 °C for as many as 3 days.

Prepare the Peptide Test Sample
1.

In a centrifuge tube, add 100 μL of BGE.

2.

Add 5 μL of a peptide marker, such as pI 9.5.

3.

Put the cap on the tube.

4.

Mix the solution in a vortex mixer for 3 seconds.

5.

Keep the sample at 2 °C to 8 °C.

6.

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

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Prepare the Buffer Trays

5

Prepare the Buffer Trays
1.

Use the following table to prepare the required reagents.
Table 5-1 Required Reagents2.

Content

Quantity

VolumeBGE (such as 10% HAc solution or 50 mM AmAc
buffer, pH 3.0)

3 vials

1.5 mL

Peptide test sample

1 vial

1.5 mLCE Grade Water

4 vials

1.5 mL

0.1 M NaOH

1 vial

1.5 mL

0.1 M HCl

1 vial

1.5 mLUse the following figure to put each reagent vial in the correct position in the buffer inlet andoutlet trays.
Note: This layout is the same for all five methods in the CESI 8000 Plus software.
Figure 5-1 Buffer Tray Layout

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Prepare the System

6

Prepare the System
This section describes additional steps to prepare the system for use.

WARNING! Electrical Shock Hazard. Do not touch the adapter when it is connectedto the CESI 8000 Plus system and the separation voltage is on.

Install the Cartridge
Before a cartridge can be installed, the interface plate must be installed on the CESI 8000 Plus
system. Refer to the section: Remove and Install the Interface Plate.
1.

Open the cartridge cover.
Figure 6-1 Cartridge Cover

2.

Loosen the thumbscrews on the insertion bar, and then lift the insertion bar.

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Prepare the System
Figure 6-2 Insertion Bar

3.

Open the access panel.
Figure 6-3 Access Panel4.

Put the sprayer housing through the side panel, and then put the sprayer in the holster.

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Prepare the System
Figure 6-4 Sprayer in the Holster

Note: To prevent damage, make sure that the protective sleeve stays on the sprayer tip. Do
not let the sprayer tip touch any surfaces.
5.

To install the cartridge body, put the ends of the cartridge on the aligning blocks of theinterface plate at a 45-degree angle, and lightly push the cartridge in.
Figure 6-5 Aligning Blocks

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Prepare the System

Item

Description

1

Aligning blocks

2

T slider6.

Turn the cartridge up until it is parallel with the interface plate.

7.

Put the upper handles in the position shown in the figures: Figure 6-6 and Figure 6-7.

8.

Align the T slider in the groove of the interface plate and move the cartridge down. As the
cartridge moves down, the covers on the inlet and outlet sides retract and the capillaries canbe seen below the interface block within the ejectors.
Figure 6-6 T Slider

Item1

Description
Align the T slider in the groove

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Prepare the System
Figure 6-7 Capillaries Below the Interface Block

Item
1
9.

Description
Capillaries can be seen below the interface block within the ejectors

Put the coolant and capillary tubing through the notched arm.

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Prepare the System
Figure 6-8 Coolant and Capillary Tubing

10. Lower the insertion bar, and then tighten the thumbscrews.
Figure 6-9 Thumbscrews on the Insertion Bar

Remove and Install the Interface Plate
1.

To remove the interface plate, do this:a.

Remove the cartridge. Refer to the section: Remove the Cartridge.

b.

Turn off the power to the CESI 8000 Plus system.

c.

Loosen the thumbscrews on the sides of the interface plate.

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Prepare the System
Figure 6-10 Thumbscrews on the Interface Plate

2.

Item

Description

1

Thumbscrewsd.

Hold the handle on the top of the interface plate and pull it out.

e.

Keep the interface plate in a safe place.

To install the interface plate, do this:a.

Hold the handle on the top of the interface plate and install it. Make sure that theinterface plate is flush with the mounting surface of the CESI 8000 Plus system.

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Figure 6-11 Install the Interface Plateb.

Tighten both thumbscrews.
Figure 6-12 Tighten the Thumbscrews

Condition the Capillaries
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 the CESI-MS conditioning method to rehydrate the coating of the bare fused-silica
capillaries and to establish the electrical connection in the sprayer tip.

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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.

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.

4.

Install the protective sleeve on the sprayer.

5.

Put 10 mL of MeOH in a 50 mL Falcon tube, and then put the Falcon tube in the holster on
the side of the system.
CAUTION: Potential System Damage. Do not put more than 10 mL of MeOH in the
Falcon tube. If there is more than 10 mL in the tube, then the liquid can splash onto
the metal components of the sprayer and cause damage.

6.

Carefully immerse the sprayer tip in the Falcon tube.
Figure 6-13 Sprayer Immersed in Liquid

7.

Run the CESI-MS conditioning method.

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8.

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

9.

Use lint-free wipes to dry the sprayer carefully.
Figure 6-14 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.
11. Install the sprayer in the adapter on the mass spectrometer.
The cartridge is ready to set a stable spray.
12. 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 otofControl 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 6-15 Pressure Settings Dialog to Fill the Conductive Liquid Capillary

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In the Pressure field, type 100.

d.

In the Duration field, type 2.

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.
Figure 6-16 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.

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Figure 6-17 Droplet at the End of the Stainless Steel Needle

4.

When the rinse is complete, to fill the separation capillary with BGE, do this:a.

Go to the Direct Control window.

b.

Click the Rinse field.
Figure 6-18 Pressure Settings Dialog to Fill the Separation Capillaryc.

In the Pressure field, type 100.

d.

In the Duration field, type 2.

e.

Click Forward.

f.

Click Pressure.

g.

Click OK.

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When the separation capillary is filled with BGE, a droplet will show at the end of thesprayer tip.
Figure 6-19 Droplet at the End of the Sprayer Tip

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.

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

2.

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.

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Figure 6-20 Voltage Settings Dialogb.

In the Voltage field, do this:• For the silica surface OptiMS cartridge, type 20.
• For the neutral OptiMS cartridge, type 30.

c.

In the Duration field, type 60.

d.

In the Ramp time field, type 1.

e.

Click Normal.

f.

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.
3.

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

4.

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

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. If the mass spectrometer is connected to the CESI
8000 Plus system, then the factory setting of 5 for curtain gas is applied.

b.

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

c.
5.

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.

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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.

6.

Click Start.

7.

If a non-SCIEX mass spectrometer is in use, then do this on the mass spectrometer
acquisition computer:8.

a.

Set the ESI voltage to zero.

b.

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

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.
Figure 6-21 Typical Spray Profile of a Cartridge that Uses 10% HAc Solution as the
BGE

9.

In the Direct Control window, click Stop.

10. 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.
11. In the IonSpray Voltage Floating (ISVF) field, type the minimum ESI voltage, and thenclick OK.
Make sure that electrospray starts again.

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12. In the IonSpray Voltage Floating (ISVF) field, increase the value by 100 V.
13. 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.
14. To check the stability of the spray, continue to apply 20 kV (normal polarity) for 20 minutes.
15. 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.

16. Click Acquire, and then type a file name (for example, Baseline).
17. 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.

18. Use the Y-axis alignment knob to maximize the baseline fluctuations.

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Figure 6-22 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.

19. 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. Do not move the sprayer tip too close to the inlet
on the mass spectrometer. If the sprayer tip is < 2 mm from the inlet, then the accidental
suction of rinse solution into the mass spectrometer can occur and cause damage.
Before a separation is run, it is critical to optimize the position of the sprayer tip in front of
the inlet on the mass spectrometer to get the correct ESI voltage. If the sprayer tip is too far
away from the inlet on the mass spectrometer, then a high ESI voltage can be required, whichcauses analyte fragmentation. For intact proteins, electrochemical reactions such as oxidation
can occur.

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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 used
For troubleshooting information, refer to the document: CESI 8000 Plus High Performance
Separation-ESI Module User Guide.
1.

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

2.

Put the sprayer tip 2 mm to 5 mm from the inlet on the mass spectrometer.

3.

In the 32 Karat software, use the Direct Control window to apply 20 kV voltage.
• 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.

Adjust the position of the sprayer tip to maximize the mass spectrometer signal intensity.
a.

In the otofControl software, open the Source tab and type 500 in the Capillary field to
set the ESI voltage.
Figure 6-23 Source Tabb.

Increase in the value in the Capillary field in increments of 100 V until electrosprayionization is detected.

c.

Increase the value in the Capillary field by 200 V.

d.

Adjust the position of the sprayer tip to maximize the signal intensity while minimizing
fluctuations in signal intensity.
Make sure that the sprayer tip is 2 mm to 5 mm from the inlet on the mass
spectrometer.

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5.

In the Capillary field, type 0 to set the ESI voltage to 0 V.

Find the ESI voltage for the acquisition method:a.

In the Capillary field, set the voltage to 500 V, and then increase it in increments of
100 V until ionization is detected.
This value is the minimum voltage and it is not high enough to maintain an effective
spray during the CESI-MS separation method.

b.

Increase the value in the Capillary field by 200 V.
Note: Use this ESI voltage in the MS acquisition method to provide a stable spray
during the CESI-MS separation method.

6.

In the otofControl software, in the Capillary field, set the ESI voltage to 0 V.

7.

In the 32 Karat software, go to the Direct Control window, and then set the voltage to 1.0 kV
with a ramp of 5.0 minutes and a duration of 5.0 minutes.

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7

Do a Test Separation

This section gives the instructions to do a test separation with beta-galactosidase test sample to
make sure that the CESI 8000 Plus system and the mass spectrometer operate correctly.
As an alternative, PepCalMix can be used as the test sample. PepCalMix has 20 different
stable isotope-labeled peptides and is available from SCIEX. For instructions about how to use
PepCalMix, refer to the document: CESI 8000 Plus High Performance Separation-ESI Module
User Guide.
Note: Before a test separation is run, make sure that a stable spray has been establishedand the optimum ESI voltage has been set. Refer to the section: Fine-Tune the Position of theSprayer Tip.

Create the Mass Spectrometer Method
Use the otofControl software to create an MS method with three segments.
1.

Click Method > Application Methods > Proteomics > Protein quantification - Label-free
MS.n.

2.

Open the Sample Info tab and update the sample information, file name, and data folder.
Figure 7-1 Sample Info Tab

3.

To create segments for the method, do this:a.

Open the Chromatogram tab.

b.

Right-click the Chromatogram tab, and then select Insert New Segment Limit.

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Figure 7-2 Chromatogram Tab–Right-click Menuc.

In the Edit Segment Limit dialog, in the New Value field, type 1, and then click OK.
Figure 7-3 Edit Segment Limit Dialog

The new segment shows on the Chromatogram tab.
d.
4.

Do steps 3.b and 3.c again to create another segment at 39 minutes.

Open the Source tab, and then in the Dry Gas field, type 1.2.
CAUTION: Potential System Damage. For dry gas flow, use the lowest flow ratepossible for the experiment. Because the capillary cap is attached by friction, highflow rates can push the cap close enough to the sprayer tip to cause damage.

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Figure 7-4 Source Tab

5.

In the Ion Polarity pane, clear the All Segments check box.

6.

To edit the capillary voltage for each segment, do this:a.

Open the Chromatogram tab, and then click the number of the first segment to select it.
Figure 7-5 Chromatogram Tab: Selected Segment in Red

7.

b.

Open the Source tab and then in the Capillary field, type 0.

c.

Click the second segment and then in the Capillary field, type the value that was found
for the maximum signal intensity in the section: Fine-Tune the Position of the Sprayer
Tip.

d.

Click the third segment, and then in the Capillary field, type 0.

In the Ion Polarity pane, select the All Segments check box.

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Figure 7-6 Ion Polarity Pane

8.

Click Method > Save As, and then type a name for the method.

Manually Create the CESI-MS Separation Method
The cesi-ms_separation_Bruker_MS.met method file is installed with the 32 Karatsoftware 10.3 or later in the folder C:\32Karat\projects\CE MS\Method. If the method
is missing, then use this procedure to create it manually.
1.

In the 32 Karat software, click File > Method > New.

2.

Use the initial conditions in the following figure.
Figure 7-7 Initial Conditions for the CESI-MS Separation Method

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3.

Use the time program in the following figure.
Figure 7-8 Time Program for the CESI-MS Separation Method

4.

Click File > Method > Save As, and then save the method as cesims_separation_Bruker_MS.met.

Load the Sample and Start the Run
1.

To configure the mass spectrometer so that it is controlled by the CESI 8000 Plus software,
do this:a.

In the otofControl software, open the Mode tab.

b.

Click Configure.
Figure 7-9 Configure Dialogc.

Click Remote Control, and then click Close.

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The Remote Control status turns green. The mass spectrometer is now controlled by the
CESI 8000 Plus software.
Figure 7-10 Status Section in the otofControl Software2.

Put the vial with beta-galactosidase test sample in position SI:A1 of the sample inlet tray.

3.

In the 32 Karat software tool bar, click the Single Run icon.
The Single Run Acquisition dialog opens.

4.

Make sure that the CESI-MS Separation for Bruker MS method file shows in the
Sequence name field.
If the correct method file does not show, then click the folder icon and browse to the
CESI-MS Separation for Bruker MS method file.

5.

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

6.

When the carriers have moved forward, open the front cover of the CESI 8000 Plus systemand put the inlet and outlet buffer trays in the correct carrier.

7.

Put the sample inlet tray in the back of the inlet carrier (left).

8.

Click Start.

Analyze the Data
Use the Bruker Data Analysis software to analyze the data from the test run.
1.

Click File > Open, and then select the file from the test run.
The base peak chromatogram opens.

2.

Create two extracted ion chromatograms:a.

Click Edit Chromatogram.

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Figure 7-11 Edit Chromatogram Traces Dialogb.

From the Type list, select Extracted Ion Chromatogram.

c.

In the Masses field, type 714.8649.

d.

From the Width list, select 0.3.

e.

Click Add.
The extracted ion chromatogram (EIC) shows in the Traces field.

f.

Create the second EIC with a mass of 729.3652 and a width of 0.3.

g.

Click OK.
The EICs show in the Analysis List pane.

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Figure 7-12 Analysis List Pane

3.

Integrate the peaks to find the peak area.
a.

In the Analysis List pane, select the EIC check boxes.

b.

Click and drag to select the two large peaks in the EIC.
Figure 7-13 Chromatogram Pane: Peaks Selectedc.

Click Find > Integrate > Chromatogram.

The results show in the Compound List pane.
4.

Configure the Compound List pane.
a.

Click the Compound List pane to select it, and then right-click and select Layout.

b.

Open the Layouts tab, and then click Compound List Layout.

c.

Click the New icon (circled in red in the following figure).

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Figure 7-14 Method Parameters Dialogd.

Select Peak Area and Signal-to-Noise Ratio, and then click OK.

e.

Click OK to close the Method Parameters dialog.

The Compound List pane changes to show the selected items.
Figure 7-15 Compound List Pane

5.

Examine the results.

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• The difference between the two retention times (RT) should be > 0.05 minutes.
If the results are not similar to these values, then set the home position on the mobile cart.
Refer to the document: CESI 8000 Plus High Performance Separation-ESI Module User
Guide.

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8

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: Store the Cartridge.

3.

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

4.

Open the cartridge cover.
Figure 8-1 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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Figure 8-2 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 8-3 Unlock the Sprayer from the Cartridge

Item1

Description
Sprayer in the Unlock position

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Item
2
9.

Description
Turn to lock the sprayer from the adapter

Remove the sprayer from the adapter.
Figure 8-4 Sprayer Removal

10. Install the protective sleeve on the sprayer tip.
11. Put the tubing with the sprayer end through the access panel.
Figure 8-5 Sprayer and Tubing Through the Access Panel

12. Remove the coolant tubing from the notched arm.

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Figure 8-6 Coolant and Capillary Tubing

13. Hold the cartridge sprayer with one hand and the midsection of the cartridge in the other,
then lift the cartridge up and pull it out.
Figure 8-7 Remove the Cartridge

Note: As the cartridge is moved up, the sheaths on the inlet and outlet sides go down overthe capillary ends.
Note: Drops of liquid coolant that might fall from the cartridge ends are normal and do notcause damage to the hardware.
14. Refer to the section: Store the Cartridge.

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Store the Cartridge
WARNING! Puncture Hazard. Be careful when handling the cartridge. The tips ofthe capillaries 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 stored for less than 3 days, do this:a.

Run the CESI-MS cleaning method.

b.

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

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

Run the CESI-MS storage method.
Note: The CESI-MS storage method cannot be run as part of a sequence because
methanol is volatile and can evaporate before the sequence is complete. Vials filledwith methanol must be used immediately. The ESI needle can stay in air but not inliquid. The ESI needle can be dried by vacuum.

b.

Disconnect the sprayer from the ion source.

c.

Remove the cartridge from the system.

d.

Install 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 Parafilm.

g.

Keep the cartridge in a safe place at room temperature.

Disconnect the CESI 8000 Plus System from theMass Spectrometer
Disconnect the CESI 8000 Plus system from the mass spectrometer when:• The mass spectrometer will be used without the CESI 8000 Plus system.
• The mass spectrometer or the CESI 8000 Plus system will be moved.
1.

Make sure that the cartridge has been removed.
Refer to the section: Remove the Cartridge.

2.

Turn off the CESI 8000 Plus system.

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3.

On the CESI 8000 Plus system high-voltage connections panel, disconnect the high-voltage
cable, ground cable, and black and red banana plugs.
Refer to the figure: Figure 2-12.

4.

Disconnect the relay cable from the CESI 8000 Plus system and the mass spectrometer.

5.

Adjust the height of the mobile cart to the lowest position.

6.

Disconnect the power cables from the CESI 8000 Plus system and the mobile cart.

7.

To unlock the wheels on the mobile cart, lift the lever at each caster.
Figure 8-8 Unlock the Wheels of the Mobile Cart

8.

Carefully move the CESI 8000 Plus system away from the mass spectrometer.

9.

Remove the adapter from the ion source and then remove the ion source from the mass
spectrometer.

10. Keep the adapter, ion source, and cables in a safe place.

Installation Guide
RUO-IDV-05-3314-C | 5302403

OptiMS Bruker MS Adapter For Bruker Mass
Spectrometers
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Tighten the Screws that Hold the Stage

Tighten the Screws that Hold the
Stage

A

Required Materials• 3/32 inch hex key• Use a 3/32 inch hex key to turn the set screw clockwise, but do not tighten it so much that thestage cannot move.
Tip! The following figure shows the three set screws. The blue arrow identifies the direction toturn the screw.
Figure A-1 Set Screw Locations

Item

Description

1

Top view

2

Location of the arrow

3Side view

OptiMS Bruker MS Adapter For Bruker Mass
Spectrometers
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Installation Guide
RUO-IDV-05-3314-C | 5302403