Intact Casein - A Model System for the Separation of
Intact Phosphorylated Proteins by CESI-MS
Stephen Lock
SCIEX UKOverview
Who Should Read This: Senior Scientists and Lab Directors
with an interest in Proteomics Research.
Focus: Intact phosphorylated protein separation by CESI-MS.
Goals: To improve the detection and characterization of intact
protein phosphorylation by mass spectrometry.
Problem: Understanding the degree of phosphorylation of intact
proteins provides important insights into the regulation of many
biological processes. However, intact protein phosphorylationis challenging to analyse by LC/MS. This is because thephosphorylation isoforms do not separate well from other specieson the front-end LC, leading to loss of data through signal

CESI 8000 Plus High Performance Separation – ESI Module

suppression in the mass spectrometer. This reduced separation
also limits data dependent acquisition inhibiting the top down
sequencing of low abundant co-eluting protein isoforms.

Experimental DesignResults: The Sciex CESI 8000 Plus system was able toSample Preparation for CESI-MS

separate charge isoforms of phosphoproteins at which could

α Casein dephosphorylated (a protein which 20% phosphorylated

not be separated by LC/MS. Ultra-low flow CESI-MS with
1000 fold less material injected produces similar sensitivities
to LC/MS, making it an ideal technique for the analysis oflimited quantity samples.

Key Challenges:• Separation and detection of intact phosphorylated proteins.
• Analysis of limited amounts of sample.

Key Features:• CESI-MS was able to separate charge isoforms ofphosphoproteins at room temperature which co-eluted
in LC-MS.
• Sample consumption by CESI-MS was 0.1% of LC-MS
but provided the same sensitivity.

containing several phosphor-isoforms) was obtained from Sigma
Aldrich and dissolved in 1% formic acid before injecting onto theCESI 8000 Plus system (CESI-MS) or a standard LC-MS system.

CESI-MS Analysis
Samples were injected by pressure onto a 30 μm ID, 91 cm
long Polyethylenimine (positively) coated capillary.1 The
CE separation used reverse polarity (cathode at the sample
injection side) with a background electrolyte (BGE) consistingof acetic acid with water/organic solvent mixtures (Table 1).
The TripleTOF® 5600 mass spectrometer, fitted with aNanoSpray® III source, was used in full scan mode with anionspray voltage set in the range of 1600 – 2200 V which wasoptimized for the methanol content of the back ground electrolyte(BGE). Due to the low flow rates of CESI-MS, no source gas
1 was used and the curtain gas was set to 4.2 psi.

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LC-MS Analysis
The TripleTOF® 5600 system used was fitted with a DuoSpray™ Ion Source and used in full scan mode with the ionspray voltage andsource settings optimized for a 300 µl/min flow rate. Apart from the source conditions, the MS method was identical to the one usedfor CESI-MS. A C4 reverse phase column (Acquity UPLC BEH C4 1.7 µm 1.0 x 50mm column) was used for the LC-MS analysis.
A sample injection volume of 1 µl was used to produce a sharp peak on a Shimadzu Nexera HPLC system. The gradient conditions
employed are shown in Table 2.

ActionTime (min)Pressure (psi)Pressure Direction

Voltage (kV)Solution

Rinse

3100

Forward00.1 Molar HCl

Rinse

3100

Forward0BGE ϫRinse

175Reverse

010% Acetic acidInjection

10 sec0.5 ¥Forward0Sample VialBGE push

5 sec

0.5

Forward0BGE ϫSeparation30

2Forward and reverse

30

BGE ϫSeparation

2100

Forward and reverse

10BGE ϫVoltage Ramp down

210Forward

1BGE ϫ

¥ = Injection pressure was varied, Ϫ = 2.5% Acetic acid containing various % Methanol
Table 1. CESI method parameters used for separations using electrokinetic injections at 25° C.

Time

Flow rate (µl/min)% Water Containing
0.1% Formic Acid

% Acetonitrile Containing
0.1% Formic Acid

0.1

300

9010230090

1020.0300

4060

21.0300

4060

21.5300

1090

23.0300

109023.230090102530090

10Table 2. LC-MS gradient method used for the separation of samples at 40° C.

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The Effect of Organic Content of the BGE on the CESI-MS Separation
For this study the maximum separation voltage of (30 kV) was used for the separation. One factor that improves resolution is increasing
the separation time by keeping the sample plug longer in the capillary. A way to accomplish this is to adjust the BGE and in Figure 1 theamount of methanol was varied. By increasing the methanol content of the BGE the electroosmotic flow was reduced2 and the migration
times were extended and the protein isoform separation improved.

Figure 1. The effect of organic content in the BGE on the analysis of α casein dephosphorylated. Each color trace is the extracted ion chromatogram
for the major multiply charged species for an isoform.

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The Effect of Injection Volume on theCESI-MS Separation
One of the other factors which effects resolution is the injection conditions used in CESI-MS. In Figure 2 the separation conditions
remained constant but injection volume was decreased by decreasing the pressure of the injection. In this experiment the volume
loaded onto the capillary was reduced by reducing the injection pressure. The reduction in injection volume improved the separationof the isoforms but did not produce the corresponding drop in sensitivity with a 10 fold drop in capillary injection volume only producing
a 3 fold drop in peak intensity.

Figure 2. CESI–MS analysis of α casein dephosphorylated using a BGE of 2.5% acetic acid in 50% Methanol with a separation voltage of 30 kV.
Each color trace is the extracted ion chromatogram for the major multiply charged species for an isoform.

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Comparison of CESI-MS with LC-MS in
the Separation of a Phosphorylated ProteinStandard (Dephosphorylated α Casein)
Initially, the same on-column amount of protein (1 ng) wasinjected onto both the LC-MS and CESI-MS system. However,the proteins were not detected by LC-MS at this low level.
Therefore, the same sample concentration (1mg/mL) wasused on both systems and 0.7 ng was analyzed by CESI-MS
compared with 1 µg (over a 1000 times more sample) byLC-MS (Figure 3). In this comparison both approaches gavesimilar protein peak intensities. However, in the LC-MS analysis
the 5 major protein isoforms co-eluted while in CESI-MS the5 major isoforms [P1 (mw = 20457 Da), P2 (mw = 20377 Da),P3 (mw = 23136.5 Da), P4 (mw = 23057 Da) and P5 (mw =22977 Da)] were separated. The molecular weight of P3-P5
decreased (by a loss of a phosphate residue) while their CESIMS migration time increased. This was because the loss of thephosphate decreased the isoelectric point of the protein, slowing
it down in the reverse polarity field as the electrophoretic field isattracting the protein stronger to the injection side of the capillary.

Figure 3. 1 mg/mL of α casein dephosphorylated in 1% formic acid analysed by CESI–MS (0.7 ng) vs LC-MS (1 µg). In the CESI-MS analysis the BGE
was 2.5% acetic acid in 50% Methanol with a separation voltage of 20 kV.

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Reference
1. Santos, M. R., et al. “A covalent, cationic polymer coating
method for the CESI-MS analysis of intact proteins andpolypeptides,” SCIEX Separations application note, 2015.
2. Li, S. F. Y. “Capillary Electrophoresis: Principles, Practiceand Applications” 1992

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Publication number: RUO-MKT-02-7110-A 01/2018
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