Improving the Detection Limits for Highly Basic
Neuropeptides Using CESI-MS
Stephen Lock
SCIEX UKOverview
Who Should Read This: Senior Scientists and Lab Directors
with an Interest in Protein Analysis and Proteomics Research.
Focus: High sensitivity neuropeptide detection in biologicalsamples by mass spectrometry.
Goals: To improve the detection limits for highly basic
neuropeptides by mass spectrometry.
Problem: Important neuropeptides include Vasoactive IntestinalPeptide (VIP), Pituitary Adenylate Ayclase-Activating Polypeptide(PACAP) and Parathyroid Hormone (PTH). These peptides
are present at low physiological levels, so analysis requires
low limits of detection. In addition, sample availability is often
limited, so analysis from small sample volumes is needed.

CESI 8000 Plus High Performance Separation – ESI Module

Experimental DesignHowever, these very basic peptides (isoelectric points > 10)

Sample Preparation for CESI-MS

are difficult to analyze by LC-MS methods because they bind

All chemicals were Reagent Grade and were purchased fromto LC auto-sampler components and columns resulting in very

Sigma Aldrich, including standards of VIP and PACAP-38.

poor chromatographic results.

Standard stocks were prepared by dissolving the solid peptides
into water to make 0.1 mg/mL concentration standards. TheseResults: The SCIEX CESI 8000 Plus system enabled the MS
detection of the target neuropeptides with over 20 fold moresensitivity than a traditional LC-MS approach. The method

stocks were serially diluted into different solvents.

CESI-MS Analysis

was successfully applied to spiked plasma extracts which

Samples were separated using a Neutral OptiMS cartridge

had been purified by offline solid phase extraction.

(30 μm ID, 91 cm long) thermostatically controlled at 25º C
with a background electrolyte (BGE) of 1% formic acid: 20%

Key Challenges:Methanol: 79% water. Two injection modes were used; either
isotachophoresis (tITP, 5 psi, 99s of a sample containing 100 mM• Separation and detection of neuropeptides inbiological samples

ammonium acetate) or electrokinetic supercharging (EK, 99 s,• Carry-over from one sample to the next using LC-MS

water shown in Table 1). Both methods used the same rinse and• Low limits of detection required10 kV injection after solvent plugs of ammonium acetate andseparation conditions (Table 1). A SCIEX QTRAP® 6500+ system,
fitted with the NanoSpray® III source was used for peptide

Key Features:detection. The source conditions were optimized for low nano-• Neuropeptides were detected in biological samples using aSCIEX CESI 8000 Plus connected to a SCIEX QTRAP® 6500+

and curtain gas = 5 psi) with just the ion-spray voltage (1800 V)• 100 times less carry-over compared with a traditional LC-MS
approach using the same Mass Spectrometer.
• 20 x more sensitivity than with a traditional LC-MS approach.

flow rates (source temperature = 50° C, Gas 1 and 2 = 0 psi
used to generate spray. The other MRM conditions are shownin Table 2. The MS method was split into 3 periods to cover theCE separation. In the first and last periods (1 minute each) theionspray voltage was set to zero so that no spray occurred.
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ActionTime (min)Pressure (psi)Pressure Direction

Voltage (kV)Solution

Rinse

2.5

100

Forward00.1 Molar HCl

Rinse

3100

Forward0BGE

Rinse

0.7575

Reverse010% Acetic acid

Solvent plug

60 sec0.5

Forward0250 mM
Ammonium Acetate

Solvent plug

10 sec0.5

Forward0WaterInjection

99 sec0Forward

10Sample Vial

Separation

255

Forward and Reverse

30

BGE

Separation

2100

Forward and Reverse

10BGE

Voltage Ramp down

210Forward

1BGE

Table 1. CESI method parameters used for separations using electrokinetic injections at 25° C.
Curtain gas

5psi

PeptideQ1 Mass

Q3 Mass

CE (V)

Gas 10 psi

PACAP

567.6

671.8

23

Gas 20 psi

PACAP

567.6

647.7

23

ISV1600 V

VIP

666663

24DP80 V

VIP666771.527EP10 VResolution

Q1 Unit

Q3 UnitCXP

13 VHeater50°C

Table 2. MRM conditions used for both LC-MS and CESI-MS methods.
Time [min]Flow rate (µl/min)% Water Containing 0.1%
Formic Acid

% Acetonitrile Containing
0.1% Formic Acid

130095510300505012300

595

13300

595

13.1300

95

51630095

5Table 2. LC-MS gradient method used for the separation of samples at 40° C.

LC-MS Analysis

Factors Effecting EK Injections with CESI-MS

For the LC-MS analysis an IonDrive™ Turbo V source was used

When using electrokinetic (EK) injections the pH, salt levels

as the separations were run at 300 µL/min. The source conditions

and organic content of the sample have an effect on the peakwere optimised for this flowrate (source temperature = 550° C,

height. It was found that when the acid concentration was too

curtain gas = 30 psi, gas 1 = 50 psi and gas 2 = 60 psi) with thehigh, the sensitivity dropped dramatically. So for this application

ionspray voltage at 4500 V. The other MRM conditions were theit is recommended that low levels of acetic acid (e.g. 0.05%) besame as used for the CESI-MS analyses. The LC system wasused in the sample solvent (Figure 1). Also the organic contenta Nexera system from Shimadzu and samples (10 µL) wereand type of solvent present in the sample impacted sensitivity. In

injected onto a Phenomenex AERIS PEPTDIE 100 x 2.1mm

Figure 2 three organic solvents were compared and acetonitrile

1.7 µM XB-C18 HPLC column held at 40° C.

produced the best response. The level of organic solvent in thesample had an effect on the response and a level of 50 -75%was found to be the best. If 100% organic solvent was used theconductivity of the sample was low and the peak height of aninjection dropped.

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Figure 1. The effect of acid content on CESI-MS sensitivity [PACAP-38 (first peak) and VIP (second peak)].

Figure 2. The effect of organic solvent in the sample on the sensitivity [PACAP-38 (first peak) and VIP (second peak)].

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Comparison of CESI-MS Separation withLC-MS for Neuropeptide Quantitation
When EK injection was compared with the standard LC-MSapproach (Figure 3), the first noticeable difference was that theEK injection was significantly more sensitive than the LC-MSinjection. When a blank was injected, after a 10 ng/mL standard,

0.87% with LC-MS (both systems used standard conditioningmethods). The small amount of carryover seen in CESI-MS
was later attributed to PACAP-38 on the outside of the capillary.
When the EK injection was compared with the tITP injection
the tITP injection gave a similar response to the LC-MS analyses(Figure 4) but was slower than the EK approach.

the carryover with CESI-MS was only 0.0069% compared toFigure 3. A carryover comparison of CESI-MS vs LC-MS all peaks are labelled with signal to noise for PACAP-38.

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Figure 4. Comparison of a LC-MS with EK and a tITP injection by CESI-MS using a 10 ng/mL (all peaks are labelled with signal to noise for PACAP-38).

Linearity and Robustness of CESI-MSEK Injections
The EK method was further shown to give a linear responseover the range 1 – 100 ng/mL (Figure 5) even though nointernal standard was used. When it was tested on an SPE
extract of a 1 ng/mL spiked plasma sample (produced using apreviously developed protocol,1 the response for PACAP andVIP was high (>1 million cps). In these initial experiments, the CVfor the analysis of the same SPE extract was <10% (Figure 6)even without the use of a labelled internal standard. Even though
the current sample preparation method is still under development
and recoveries are only in the range of 10 - 20% this approach
was shown to be reproducible and when compared with thepreviously published study,1 which used CESI-MS tITP injections,was 10 times more sensitive.

Figure 5. Calibration Line for CESI-MS EK injection approach for
PACAP-38 range was 1 – 100 ng/mL (no internal standard was used).

Figure 6. . Three SPE extracts of human serum samples spiked with
1 ng/mL of VIP analyzed by CESI-MS by separate EK injections(no internal standard was used).

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Reference
1. Lock, S. ‘The application of Capillary Electrospray Ionization
to the detection of Neuropeptides’. Conference Proceedings
ASMS 2016, San Antonio, June 5-9, 2016.

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Publication number: RUO-MKT-02-7150-A 01/2018
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