CESI-MS performance check and MS calibration using
PepCalMix standard for silica surface OptiMS cartridges
Featuring PepCalMix standard, CESI 8000 Plus ESI-MS System coupled to TripleTOF® 6600+ LC-MS/MSSystem
Zuzana Demianova1, Fang Wang1, Andreas Krupke2, Sahana Mollah2
1
SCIEX, Brea, CA; 2SCIEX, Redwood City, CA

Introduction
CE-MS methodology provides flexibility in addressing new andchallenging applications that sometimes cannot be accomplishedby LCMS. It also provides an orthogonal solution to LC-MSapplications. The OptiMS cartridge for the CESI 8000 Plussystem is directly connected to the mass spectrometer without
additional fluidic connections. Instead of having multiple methods
to check performance for each feature, it is desirable to have one
single analysis with a single set of standards. A group ofstandards can monitor the successful CESI-MS integration,
performance of the CESI-OptiMS cartridge and massspectrometer.
The system suitability test is an essential check of the CESI-MS
specifications; monitoring of the cartridges' separation
performance, optimizing CESI tip position, and calibrating themass spectrometer on MS and MS/MS level.
Here, we demonstrated using PepCalMix standards (a mixture ofpeptides) to monitor the silica surface OptiMS cartridge'sperformance. We showed excellent repeatability of thePepCalMix measure on the same cartridge, across different
batches of cartridges, and day-to-day measurements, totaling>100 sample injections across three random BFS cartridges.

CESI 8000 Plus coupled with Triple TOF® 6600+ system andPepCalMix standard

Furthermore, to ensure good mass spectrometer performance,
the PepCalMix was used for automatic calibration between
sample measurements.

Key features of CESI-MS performance check• A single system integration test with the PepCalMix standard
to evaluate the CESI and MS performances• The PepCalMix standard is robust and stable over time
• The cartridge performance can be monitored with two pairs ofclosely migrating peptides• The PepCalMix standard can be used for both manual andautomatic calibration of the MS

Figure 1. Electropherogram showing separation and m/z ions monitored in two key peptide pairs (blue/pink and orange/green). a) Separationof two pairs (1a/1b and 2a/2b; blue/pink and orange/green) of key peptides shown in XIC; m/z observed for each peptide are shown and marked as 1a,1b, 2a, and 2b.
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Materials and Methods
Sample preparation: The PepCalMix (SCIEX, P/N 5045759)
stock solution contains one pmol/µL of each of the 20 different
stable isotope-labeled peptides. The stock solution was aliquoted
into 5 µL and stored at -20°C.1 For the manual MS and MS/MS
calibration via CESI direct infusion, the PepCalMix (SCIEX, P/N5045759) was diluted 1:1 (v:v) with 10% acetic acid (backgroundelectrolyte solution, BGE). For the separation analysis and autocalibration, the PepCalMix was 10-fold diluted with leading
electrolyte (LE; 100 mM ammonium acetate, pH 4). Before
analysis, every sample was vortexed and centrifuged for 5 min at
12 000 g to remove any possible precipitant.
Table 1. CESI conditions were used to separate the PepCalMix.

EventTime Pressure Voltage
Direction Solution/Note(min) (psi)(kV)

1 Rinse2.5100Forward

0.1 M NaOH

2 Rinse2.5100Forward

0.1 M HCI

3 Rinse4100

Forwardwater4 Rinse

375Reverse

10% acetic acid

5 Rinse4100

Forward

10% acetic acid

6 Injection

60 s

5Forwardsample

7 Injection

25 s

0.5

Forward

10% acetic acid

8 Separation

30020

Forward

10% acetic acid

9 Separation

501

Forwardramp down

Instrument configuration was as follows, the CESI 8000 Plus
system with the silica surface OptiMS cartridge (SCIEX, P/NB07367) was coupled with the TripleTOF 6600+ System througha NanoSpray® III source and a CESI adapter (SCIEX, P/NB07363). 32 Karat™ and Analyst® TF Software 1.8.1 Software
were used to operate the CESI-MS.
Capillary electrophoresis and mass spectrometry: CESI
separation method parameters: The separation conditions arelisted in Table 1. Sample was injected hydrodynamically at 5 psifor 60 s, resulting in approximately 44 nL injected volume, which
is about 2.2 fmol of each peptide on the cartridge. The BGE(10% acetic acid) was prepared freshly on the analysis day.
Direct infusion of the PepCalMix: The standard was infusedthrough the cartridge using 5 psi to optimize the CESI sprayer
position, ion spray voltage, and to preform TOF MS and MS/MS
manual calibration.2 For the fine-tuning of the CESI tip sprayer
position, 20 kV voltage and 0.5 psi pressure on both cartridgeends were applied. The PepCalMix references ions for mass
calibration are listed in the SWATH acquisition performance
protocol1 (Table 2-3 and Table 2-4, page 16-17).
MS method parameters: The PepCalMix was measured using aTOF MS acquisition method with a looped MS/MS scan. The
acquisition TOF MS mass range was from 400 to 1250 m/z, witha cycle time of 0.8 s. Method details are listed in Table 2.
Data processing: SCIEX OS Software was used to visualize
and analyze measured data.

Figure 2. Example spectrum from silica surface OptiMS cartridge infusion. a) Example Data for TOF-MS Spectrum; b) Example Data for TOFMS/MS Spectrum for Peptide m/z 758.91 in high-resolution (HR) and high sensitivity (HS) mode. Arrows highlight the m/z values corresponding to thepeptides in the mixture and values used for TOF-MS and MS/MS calibration.
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Table 2. Summary of mass spectrometry parameters.
ParameterValueMS mass range

400-1 250 m/zMS accumulation time

250 msMS/MS mass range

150-2 250 m/z

MS/MS accumulation time

500 msCurtain gas:

5 psiPolarity:

positive

Source temperature:

50 °C

Collision energy (CE)42 V

CE spread±5VIon spray voltage

1 850 V*

*Value is tuned during direct infusion of the PepCalMix.

Results and Discussions
When the peptide solution in BGE is infused through theseparation capillary, a spray variation should be less than 40%
within 2-5 minutes time frame. The example data for the infusion
TOF MS spectrum from the silica surface OptiMS cartridge, andfor the MS/MS high-resolution and MS/MS high-sensitivity
spectrum for peptide m/z 758.91 are shown in Figure 2. The
peptides used for the calibration of MS and MS/MS are

highlighted with arrows (Figure 2). Peptide standard infusion isan easy and quick way to perform manual MS and MS/MS tuningand calibration.
In addition to direct infusion, the peptide standards can be
diluted into 100 mM leading electrolyte (LE) solution and use
10% acetic acid in water as BGE to perform the high-resolution
separation. The separation method utilizes the tITP (transientIsotachophoresis) technique to concentrate and separate
peptides in the mixture. These peptides are scanned with TOF
MS, and peptide 758.91 m/z is selected for further MS/MS
fragmentation. Overlay of peptides extracted ion chromatogram(XIC) from three consecutive injections is shown in Figure 3.
The migration time of peptides can be different when multiple
cartridges are used. They may also shift when the sample andBGE used for other injections are different from those used forthe peptide standard separation. Nevertheless, the relative
separation of the key peptides should not be affected (Figure 4and Figure 5).
An extensive evaluation of the PepCalMix and silica surface
cartridges was executed. Overall, >100 measurements wereconducted to cover the inter-day, intra-day, and cartridge lot-lot
performance. The RTs of 20 standard peptides remained
consistent among the entire study.

Figure 3. Example of inter-day cartridge separation repeatability. Overlay of three injections of CESI-MS separation results of the PepCalMix on asingle silica surface OptiMS cartridges.

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Figure 4. Example of batch-to-batch cartridge separation repeatability. Overlay of three injections of CESI-MS separation results of the PepCalMix
on three different batches of silica surface OptiMS cartridge.

Figure 5. Separation of the two pair selected key peptides of three CESI-MS separation injections on three different batches of silica surface
OptiMS cartridges. XIC of four key peptides with difference in the migration time for the corresponding pair (blue/pink and orange/green) is more orequal to 0.08 and 0.25 minutes, respectively.

p4peptides remained consistent among the entire study.
The RT variation of performance was on average the inter-day
1.8% ±0.12 per cartridge, the intra-day 3.1% ±0.21 and ancartridge lot-lot variation was 3.9% ±0.15 for three cartridges.
The intra-day RT variation is higher compare to inter-day due toother complex samples were measured between the PepCalMix
which influence the RT shift.
The separation of two pair peptides is selected as an indicator of
the silica surface OptiMS cartridge performance (Figure 1). The
first pair migrates at the beginning of the entire peptide train,
while the second pair migrates towards the end. The efficiency ofcartridge performance can be gauged by identifying and clearing
separation between the two sets of peptides, 485.25 m/z (bluetrace) with 491.25 m/z (pink trace) and 739.35 m/z (orangetrace)with 768.90 m/z (green trace), Figure 1 and 5. The
difference in the migration time between each peak in thecorresponding pair should be more significant than 0.08 minutes
and 0.25 minutes, respectively (Figures 1 and 5).

Conclusions
This technote introduces an approach for performing bothcalibrations of TOF MS and MS/MS and gauges the silica
surface OptiMS cartridge's performance. This type of calibration
approach can be performed easily over a wide mass range via
direct infusion or separation with the silica surface OptiMS
cartridge. The performance of the silica surface OptiMS cartridge
is gauged by the relative separation between two pairs ofpeptides of similar migration times.

References
1.

SWATH acquisition performance protocol, Protocol,
RUO-IDV-05-3363-A May 2016
https://sciex.com/Documents/protocols/SWATH_Acquisi
tion_Performance_Kit_Protocol.pdf

2.

CESI 8000 High Performance Separation System, Usermanual, RUO-IDV-05-3897-C March 2020
https://sciex.com/Documents/manuals/cesi8000_uManu
al_B11949AD.pdf

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