amaZon SL

trapControl Training Document

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1. APPLICATIONS DEFAULT METHODS FOR AMAZON SL WITHIN
TRAPCONTROL 4

1.1. Tuning Mix 4

1.2. Fast Chromatography/Small Molecules 4

1.3. Proteomics AutoMS/MS Methods 4

2. OPTIMIZATION OF THE ICC = ION CHARGE CONTROL 5

2.1. Principle 5

2.2. How to optimize the ICC Smart Target 7

3. TUNING – OPTIMIZATION OF SIGNAL INTENSITY 9

3.1 General Tuning Strategies 9

3.1.1. Smart Parameter Setting (SPS) 9

3.1.2. Adjusting all individual Voltages 10

3.1.3. Adjusting all voltages using “Optimize” 11

3.2. In-Source CID (ISCID) 12

4. AUTO MS(N) OPERATION 13

4.1. Principle 13

4.2. Main Parameters for Auto MS(n) 13

4.3. Parameters for Precursor Selection 14

4.4. Acquisition Parameters 15

4.5. Auto MS(n>2) Parameters 16

4.6. Neutral Loss 16

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 4.7. CID Parameters 16

5. MANUAL MS(N) OPERATION 19

5.1. Principle 19

5.2. Main Parameters for Manual MS(n) 19

5.3. CID Parameters 19

6. MRM OPERATION 20

6.1. Principle 20

6.2. Main Parameters for MRM(n) 20

6.3. CID Parameter 20

7. SPECIAL SCAN CALIBRATIONS 21

7.1. Low Mass Scan Calibration 21

7.2. High Mass Scan Calibration 21

7.1.1. High mass scan calibration using caesium perfluoroheptanoic acid cluster 22

8. ROLLING AVERAGING 23

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1. Applications Default Methods for amaZon SL within
trapControl

1.1. Tuning Mix

• tuning mix.m

Please use a 1:100 dilution in ACN/H2O (90/10) of the regular tuning mix (Agilent G2431A)
or the low concentration tuning mix (Agilent G1969-85000) without further dilution.

1.2. Fast Chromatography/Small Molecules

• Fast LC, 1 min Gradient, Alternating Polarity, MS only.m

• Fast LC, 1 min Gradient, Alternating Polarity, AutoMS (CID only).m

In case only one polarity is needed, switch off “Alternating Polarity”.

1.3. Proteomics AutoMS/MS Methods

• Proteomics, AutoMS/MS (CID only).m

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2. Optimization of the ICC = Ion Charge Control

2.1. Principle

In order to achieve good mass precision and resolution together with optimum sensitivity,
the ion trap has to be loaded properly. By activating the ICC = Ion Charge Control, the
number of ions entering the trap is regulated by either satisfying the Smart Target value
or the Maximum Accumulation Time, whichever is reached first.

ICC control using the Smart Target considers not only the number of ions but also their
distribution over the mass range. This is due to the fact that overloading occurs more likely
if ions of the same kind are collected in the trap. In this case, the ICC Actual is usually
lower than the Smart Target. If the ions are distributed more or less equally over the scan
range the ICC Actual reaches the Smart Target value.

Although the Target value is correlated to the number of ions in the trap it is a relative
value and not identical with the actual number.

Actual ICC Value

Actual Accumulation Time

ICC activated

Smart Target

Maximum Accumulation Time

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Too many ions in the trap lead to space charge effects. The ions “see” each other and are
therefore under the influence of more than just the ion trap field. As already mentioned, this
effect is most pronounced, when the trap is filled by ions of the same kind. The
consequences are:

• Poor mass resolution

• Poor mass accuracy
• Poor peak determination.

The ICC function should be especially used in

case of varying ion currents, e.g. in LC-MS.
ICC Target
Here, pure solvents lead to a low ion current and
therefore high accumulation times. When a Ion counts

compound elutes, the ion current increases, Accumulation

whereas the accumulation time goes down and Time

reaches a minimum value in the peak maximum.

TIC
In order to be independent of the accumulation
time the signal intensity is normalized relative to Time
the actual accumulation time.

Normalize to Accu Time

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2.2. How to optimize the ICC Smart Target

All amaZon SL systems are installed using as default ICC target values 100 000 in positive
mode and 35 000 in negative mode. However, the upper limit the individual instrument can
deal with can be determined by optimization of these target values.

In order to optimize the Smart Target infuse the pure compound (around 1 ng/µL for small
molecules or 100 fmol/µL for peptides). Start with a Target value of e.g. 50 000 in the
positive mode (25 000 in the negative mode) and increase the value until the isotopic
resolution gets worse and the mass begins to shift to higher m/z values (overloading of the
trap!). Then go back and decrease the Smart Target value until you see again proper
resolution and mass accuracy.

Example for ICC Target check in positive ion mode:

a) Smart Target = 500 000

ICC Actual = 393 000

Isotopes are resolved and mass precision

is o.k.

► Proper Target.

b) Smart Target = 800 000

ICC Actual = 772 000

Begin of space charge effects. The peaks

become broader, the position of the first
isotope starts to move and the isotopic
resolution starts to get worse.

► Upper limit for Target.

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 c) Smart Target = 2 000 000

ICC Actual = 1 532 000

The trap is overloaded. The mass shifts

to higher m/z values and the isotopic
resolution is lost.

► Target needs to be reduced.

However, the Smart Target should not be set too low because this leads to a bad
statistic, resulting in a deterioration of the isotopic pattern and the signal-to-noise ratio.

Apart from a proper Target value a correct multiplier voltage is mandatory for the ICC
regulation. Therefore a regular multiplier check is recommended.

In general, the ion trap is faster overloaded in the negative ion mode.
Therefore, lower Target values are required. When performing analysis
with alternating polarity, different Smart Targets for the positive and
negative mode can be set by checking the Unlink Edit function.

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3. TUNING – Optimization of Signal Intensity
The aim of TUNING is to obtain the maximum sensitivity for the compound of interest. This
is achieved by either using “Smart Parameter Setting” in the “Smart” mode or by adjusting
all individual voltages of the ion guide in “Expert” and additional “Optimize” mode.

“Smart Parameter Setting” is the method of choice to start with due to its simplicity. It is
sufficient in more than 90% of all samples and applications.

3.1 General Tuning Strategies

3.1.1. Smart Parameter Setting (SPS)

Use “Smart” view mode:

• Select compound specific “Target Mass” or in case of a mixture use mean m/z-
value.
• Set “Trap Drive Level” = 100%. Other values are necessary only in case the
compound mixture varies over an extreme m/z range.

As consequence, the voltages displayed on the Tune Expert page are adjusted for the
selected m/z.

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3.1.2. Adjusting all individual Voltages

Use “Expert” view mode:

Expert View

To obtain the maximum sensitivity for the compound of interest all voltages of the ion guide
can be optimized individually, namely Cap Exit, RF-Level, Funnel 1, Funnel 2, Octopole,
Focus 1, Multipole, Focus 4 and Trap Drive.

The most influential parameters are RF-Level, Funnel 2 Lens, Octopole 1 DC and Trap Drive.

Funnel 2 Lens, Octopole 1 DC and Trap Drive are interdependent; therefore it is

necessary to tune all 3 parameters. But, as the funnel transfer unit has a wide acceptance
of m/z-range in the majority of cases tuning of RF-Level and Trap Drive is satisfactory.

If ultimate sensitivity is required tuning of these parameters is recommended to be done in

the “Optimize function”.

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3.1.3. Adjusting all voltages using “Optimize”

For qualitative analysis Smart Parameter Setting is often sufficient. For further optimization
e.g. in quantitative analysis “Optimize” can be used to ramp the voltages displayed on the
Tune Expert page.

The Optimize window is available via the Options menu.

Keep the parameters in the same order as the ions pass the components of the ion guide. If
significant changes of the tune parameters occur, repeat the “Optimize” procedure.

Only for the target mass the intensity optimum is determined automatically. For the
monitor mass the software provides an optimization curve from which the best value can
be derived.

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If optimization is needed for two masses the optimum value has to be determined manually
from the two curves and to be set manually in the Tune Expert page.

In case several ion transfer parameters need to be optimized, “Ion optics” should be
selected as parameter. RF-Level, Trap Drive, Funnel 2 Lens and again Trap Drive are
then optimized.

3.2. In-Source CID (ISCID)

ISCID can be observed on the amaZon analogue to the ESI-qTOF systems. It takes place
between Funnel 1 and 2.

The following parameters can be changed manually in order to induce in-source CID:

• Funnel 1 Out & Lens 1:

Increase both by 30 to 80 V, e.g. from 35 to 95 V resp. 25 to 85 V.
(If the values are increased by 80 V, an increase of the CapExit voltage might be
required as well.)

If necessary, also the voltages of the second funnel might be changed:

• Funnel 2 In: From 12 decrease to 5.

• Funnel 2 Out: Should be adjusted it the same direction as Funnel 2 In.

Sometimes, undesired in-source CID can be observed for small labile compounds. A
decrease of the RF level should suppress ISCID.

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4. Auto MS(n) Operation

4.1. Principle

AutoMS(n) is used for fragmentation of precursor ions above a given intensity threshold.
The first step in an AutoMS/MS cycle is the acquisition of a full scan spectrum. Afterwards,
precursor ions that fulfil the specified criteria (intensity, mass range, etc.) are selected and
fragmented. A typical application is the qualitative analysis of unknowns.

4.2. Main Parameters for Auto MS(n)

• Include/Exclude/Scheduled Precursor List: Individual masses or mass ranges

can be included for or excluded from precursor ion selection. For instance, in order
to exclude impurities and solvent clusters in the lower mass range. Include and
exclude masses are complementary. The maximum number of entries is currently
restricted to 499. Targeted MS/MS experiments can be set up using a scheduled
precursor list.
• No. of Precursor Ions: Typical values for LC-AutoMS/MS are 2 to 20 precursor
ions, depending on the complexity of the sample. Maximum number of 30 is only
applicable in infusion experiments.
• Threshold Abs: The absolute intensity in the line spectrum that must be exceeded
by a potential precursor ion.
• Threshold Rel: The relative threshold is a dynamic value refering to the current
base peak and therefore needs to be calculated for each spectrum. Reasonable
values can avoid MS/MS of noise signals. If a broad dynamic range is required e.g.
for highly complex samples it should be decreased down to something like 0.1%.

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 • SPS: Transfer parameters for the selected precursor ion are adjusted using SPS in
order to enhance sensitivity. This option is recommended if SPS has been also
applied for the general tuning.
• High Resolution MS Scan: If selected, an additional Enhanced Resolution MS scan
is acquired for each precursor ion to determine higher charge states – a small mass
range of precursor m/z ± 10 m/z is scanned. This option is only useful in
combination with MS spectra acquired in UltraScan.
• Only: No MS/MS is performed, only a full scan MS plus Enhanced Resolution Scan
• Active Exclusion: After a given number of MS/MS spectra (in the above case: 2),
the precursor ion is excluded for a certain period of time (usually similar to the
chromatographic peak width, e.g. 1.0 min). If there is no other signal fulfilling the
criteria the “excluded” precursor ion will be selected again. This behaviour can be
changed with the option Strict active exclusion on the precursor selection
parameter page.

4.3. Parameters for Precursor Selection

• Preferred Masses: Preferred masses also need to fulfil the criteria mentioned
above, but they are preferably selected even though they are not the most
abundant signals. Preferred masses are used by their order in the list. When
preferred masses are used, intensity threshold parameters must be properly
adjusted.
• Preferred Charge State: This option is used when analyzing multiply charged
ions, mainly peptides.

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 • Double or > Double: Favours certain charge states, e.g. for CID or ETD in
combination with appropriate digestion enzymes. Other charge states are not
excluded.
• Exclude singly charged ions: Signals with a charge state of 1 are excluded from
precursor ion selection. This option can be efficiently used in proteomics
experiments to exclude background ions as they are typically singly charged.
• Usually, precursors are selected for MS/MS by decreasing intensity unless Sort
Precursors by mass is selected.
• Group Length of 5 ensures that all isotopes of a compound are considered as one
precursor.

4.4. Acquisition Parameters

• MS(n) Parameters: In AutoMS/MS mode different parameters can be used for MS

and MS/MS spectra acquisition regarding
o Use UltraScan – UltraScan will be activated for MS/MS to enhance the duty
cycle when Enhanced Resolution mode is used for MS spectra acquisition
o Scan Range
o ICC Target
o Max. Accu Time
o Averages
• Isol Width: Represents the isolation window that is used for the precursor prior to
fragmentation.
• MS/MS Fragmentation Amplitude: The fragmentation amplitude should be
adjusted to give good fragmentation for the compound of interest. The SmartFrag
option on the CID parameter page should be activated to achieve optimal
fragmentation of all compounds with varying fragmentation stability.

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4.5. Auto MS(n>2) Parameters

Parameters comparable to the Main Auto MS(n) dialog are available for subsequent MS/MS
stages.

4.6. Neutral Loss

In case of significant neutral loss fragments occurring in MS/MS, a MS(3)CID of the neutral
loss fragment can be triggered, e.g. in phosphopeptide analysis.

4.7. CID Parameters

This parameter dialog is used in all three MS/MS modes Auto MS(n), Manual MS(n), and
MRM.

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The Default setting for Cut-Off Selection is 27%. With this setting most of the precursor
ions can be easily fragmented. The Cut-Off used can be changed for all precursor ions with
Set to XX% or for individual precursor ions in Manual MS(n) and MRM experiments by
selecting Manual.

The default fragmentation Width is 4 m/z.

SmartFragTM: In order to obtain satisfactory MS/MS data from different compounds of

varying stability SmartFrag should be activated.

• SmartFrag causes a ramping of the fragmentation amplitude, here from 30 to

200% of the specified value. For quantitative analyses a narrower range like 50 to
150% is recommended.
• Switching SmartFrag off uses the specified fragmentation amplitude only without
any ramping.

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Page 18
5. Manual MS(n) Operation

5.1. Principle

In Manual MS(n) the precursor ions can be defined by user for MS/MS up to MS(11).

5.2. Main Parameters for Manual MS(n)

• All Off: Sets all Isolation and Reaction checkboxes off.

• Precursor: Desired precursor for isolation and fragmentation.
• Isolation/Width: Checkbox for ion isolation, width in Da.
• Reaction: Switches on or off the CID fragmentation. The “CID CutOff” can be set,
when “manual Cut-Off selection” was checked in the CID subpage. The “CID Ampl”
can be additionally controlled by the SmartFrag option within the CID subpage.

5.3. CID Parameters

Please refer to Auto MS(n) CID page.

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6. MRM Operation

6.1. Principle

The MRM (Multiple Reaction Monitoring) mode alternates between the acquisition of
different MS(n) spectra. MS/MS or MS(3) experiments can be set up for up to ten ions. MRM
uses always CID.

6.2. Main Parameters for MRM(n)

• All Off: Sets all Isolation and Reaction checkboxes off.

• Precursor : The desired precursor for isolation and fragmentation can be selected.
• Isolation/width: Checkbox for precursor ion isolation, width in m/z.
• Reaction: CID is applied.
• CID: The “CID CutOff” can be set, when “manual Cut-Off selection” was checked on
the CID subpage. The “CID Ampl” can be controlled by the SmartFrag option on
CID subpage.

6.3. CID Parameter

Please refer to Auto MS(n) CID page.

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7. Special Scan Calibrations

7.1. Low Mass Scan Calibration

For some applications, e.g. the detection of fragments with low m/z it is necessary to
calibrate the mass range below m/z 100. This is possible for the Enhanced Resolution mode.
Either the procedure described for the “PAN calibration” can be applied or, if the required
chemicals are not available solvent clusters generated from a very simple mixture of
solvents and volatile acids can be used instead. However, since the solvent clusters are
rather unstable they can be only used for the scan calibration and are not suited for
isolation and fragmentation calibration.

Procedure:

• First, calibrate the standard mass range between 118 m/z and 2122 m/z using tune
mix as usual.
• Prepare a mixture of H2O, ACN, formic acid, and acetic acid (80/10/5/5).
• From this mixture the following signal can be used for the low mass calibration:
o m/z 61.03 = [CH3COOH + H]+
• Infuse the solvent/acid mixture.
• Start a manual scan calibration and accept all of the tune mix calibration points
without changing the cursor position.
• Then, add the value of 61.03 m/z, adjust the calibration cursor position and accept
the point.
• Save the scan calibration.

7.2. High Mass Scan Calibration

When operating in the extended mass range it is necessary to extend scan calibration up to
4000 m/z. Caesium perfluoroheptanoic acid (CsPFHA) clusters are a suited calibrant for this
purpose. They can be used for a mass range from 600 m/z up to 6000 m/z (S. König, H.M.
Fales, J Am Soc Mass Spectrom 1999, 10, 273-276). If the required chemicals are not on-
site, tuning mix dimers can be used as described in the following.

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4.1.1. High mass scan calibration using caesium
perfluoroheptanoic acid cluster

Compared to tuning mix this procedure provides more calibration points and is less
dependent on the spray parameters.

Required stock solutions and solvents:

• 1 M CsI stock solution: 259.80 mg/ml H2O

• 5 M or 3.58 mg perfluoroheptanoic acid (tridecafluoroheptanoic acid, mp 30°C)
• H2O/i-PrOH (50/50)

Preparation of 10 mM CsPFHA solution

• 988 µl solvent
+ 10 µL acqueous 1 M CsI
+ 2 µL tridecafluoroheptanoic acid

The calibration solution is introduced by direct infusion at a flow rate of 3 µl/min.

Calibrant [M+H]+ average

Cs(C7F13O2Cs)1 628.9

Cs(C7F13O2Cs)2 1124.8

Cs(C7F13O2Cs)3 1620.8

Cs(C7F13O2Cs)4 2116.7

Cs(C7F13O2Cs)5 2612.7

Cs(C7F13O2Cs)6 3108.7

Cs(C7F13O2Cs)7 3604.6

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8. Rolling Averaging
Rolling Averaging improves S/N ratios. On the other hand, during LC operations high values
( ≥ 3) can result in peak broadening.

Example: Averages = 5 and Rolling Averaging = 1

50% 50%

5 5 5 5 5 5

Ø Ø Ø Ø Ø

Final Spectra

Example: Averages = 5 and Rolling Averaging = 2

25% 50% 25%

5
5 5

Ø Ø Ø Ø

Final Spectra

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For Rolling Averaging > 2 the previous spectra gain bigger influence.

Example: Averages = 5 and Rolling Averaging = 3 resp. 4

50% 50%
67% 33%

5 5

50%
33%

Ø 5

50%
33%

Ø 5

Ø
……..

Rolling Averaging = 3:

Average of previous and current spectrum with a relative weighting of 50%.

Rolling Averaging = 4:

Previous spectrum contributes 2/3 and the new one 1/3.

And so on ….

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