A new approach to the analysis of intact MUPs (Major UrinaryProteins) from mice by CESI-MS
Dr. Stephen Lock1, Dr. Guadalupe Gómez-Baena2 and Prof. Robert Beynon2
1

SCIEX, United Kingdom and 2Centre for Proteome Research, Liverpool University, Liverpool, United Kingdom

Introduction

Materials and Methods

The Major Urinary Proteins (MUPs) of the house mouse, Mus musculus
domesticus, are 19 kDa beta-barrel lipocalins that are involved inchemical communication between individuals. Many of them areexcreted in urine where they play multiple roles, including coding ofowner identity and transport, and slow release of bound volatilepheromones. It has been shown that mass spectrometric analysis of
intact proteins, is capable of dissecting subtle structural differences
between the members of this class of proteins. Moreover, mass
spectrometric analysis of the intact proteins can contribute towards
molecular phenotyping of MUPs1, 2.

Chemicals: All chemicals were Reagent Grade and were purchasedfrom Sigma Aldrich.
Sample Preparation: The MUPs samples were provided by Centre forProteomeResearchinLiverpoolUniversity(https://www.liverpool.ac.uk/pfg). Samples were provided desalted into
50 mM ammonium acetate, pH 6.6 using Zeba columns and were at ahigh protein concentration (10 mg/mL). These extracts were further
diluted 1 in 10 into 100 mM ammonium acetate, pH 6.6 which helpedwith isotachophoresis focusing on the separation capillary.

A couple of common approaches have been used for the analysis ofintact MUPs by LC-MS. The first is direct infusion of desalted samples
which have been purified using molecular cut-off filters1. The second
used a C4 trap cartridge to first trap the proteins before eluting them withformic acid/acetonitrile at 10 µl/min2 into the MS. These two approaches
struggle to discriminate between proteins with small mass differences as
charge envelopes would overlap in the LC-MS analysis. Anotherapproach requires pre-fractionation by anionic LC to separate theproteins prior to LC-MS analysis3. This workflow is complex and canresult in protein loss, negatively impacting the quantitation of proteins inthe sample. Therefore, a technique which is able to separate proteinsdiffering by only a few mass units without the need for pre-fractionationwould be beneficial.
Capillary Electrospray Ionization (CESI) is the integration of capillaryelectrophoresis (CE) and electrospray ionization (ESI) into a single
process in a single device (Figure 1)4. CESI-MS operates at low nL/minflow rates and offers several advantages. These include increased
ionization efficiency and a reduction in ion suppression. CESI-MS
separates analytes by their charge and size and is, therefore, acomplementary separation mechanism to more traditional techniques,
such as reverse phase LC. The purpose of this study was to investigate
the use of CESI-MS in the separation of two desalted MUP extracts.
The study was designed to address two key questions. First, does theseparation of MUPs by their charge and size by CESI-MS allow thedetection of highly similar proteins by mass spectrometry. Second, canthe analysis be performed directly on urine samples without the need fordesalting to simplify the workflow and minimize the impact of the relative
quantitation of the MUPs.

CESI-MS method: Sample was injected by pressure (5 psi, 5 s) onto aNeutral OptiMS CESI cartridge (30 μm ID x 91 cm, polyacrylamidecoated) which was thermostatted using recirculating liquid coolant
regulated at 20oC. For this analysis, the SCIEX TripleTOF® 5600+ mass
spectrometer was fitted with the NanoSpray® III source. Gas 1 was not
used and the temperature of the interface was set at 60 ºC. Ionization atvery low flow rates occurs by simply applying the ionspray voltage (1800V). The curtain gas was set at 4.2 psi (set automatically). The CE
separation used the conditions shown in Table 1 with a backgroundelectrolyte (BGE) of 1% formic acid/10% isopropanol (pH 2.05) or 2%
acetic acid / 10% isopropanol (pH 2.55). Data was acquired by a split
period experiment. For the first period of 1 min the ionspray voltage wasswitched to 0 V (resulting in no ionspray), data was then collected at1800 V for the next 36 min. In both periods the DP voltage was set to
100 V and data was acquired in a TOF-MS scan mode over the mass
range 800– 2500 amu at a CE voltage of 10 V.
Action
Rinse
Rinse
Rinse
Rinse
Injection
Separation
VoltageTime(min)1.5355s
10s
35
2

Pressure(psi)100100100551.510Direction
ReverseForward
Forward
ReverseForward
Forward
ForwardVoltage(kV)
0000030
1Solution
10% Acetic acid
0.1 M HCl
BGE
Sample Vial
BGE
BGE
BGE

Table 1: CESI separation conditions used for the detection of MUPs.

Results
In CE, proteins are separated according to differences in their intrinsic
electrophoretic mobility. One of the ways to alter separations of proteinsis the pH of the BGE as this affects the charge state of the protein. Oneof the first development experiments tried was to test the effect of the pHby comparing BGE containing 2% acetic acid/10% isopropanol (pH 2.55)
with one containing 1% formic acid/10% Isopropanol (pH 2.05).

Figure 1: OptiMS® - Ultra low flow ESI Interface.

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2% (v/v) acetic acid

1% (v/v) formic acid

Figure 2: The effect of acid on the separation of MUPs.
Figure 2 shows the effect of changing the BGE pH. MUPs are acidic innature with isoelectric points ranging from 4.3 to 4.55 , In theory changing
the pH can improve separations and when the pH of the BGE wasdecreased, additional separation of the MUPs was observed. Theproteins also migrated faster to the mass spectrometer speeding up theanalysis. This combination of formic acid and isopropanol was used tocompare two different MUP extracts.

Figure 4: Spectra and de-convoluted protein profiles for sample BALB/c.

(A)

(B)

Figure 5: Spectra and de-convoluted protein profiles for sample(C)

C57BL/6.

Figure 3: Comparison of samples C57BL/ 6 (A), BALB/c (B) with asolvent blank (C).

Also oberved was a series of proteins of higher molecular weight,
examples of which is shown in Figure 6.

Figure 3 shows the total ion chromatogram of urine samples from twodifferent mouse strains compared with a solvent blank and highlighted
that CESI was capable of distinguishing the two different mouse strains.
When the spectra of the peaks BALB/c and C57BL/6 were compared(Figure 4 and Figure 5) different proteins were separated and identified
by CESI-MS (summarized in table 2). There are 5 MUPs which areidentical in mass (18964 Da) two were separated by CESI-MS in sample
BALB/c which would not have been possible by infusion analysis.
Protein
MW (Da)
18708
18645
18893
18694
18713

Sample C57BL/6
2Migration
MUP
time (min)
15.6
10
15.6
7
16.0
20 (Darcin)
16.0
9,11,16,18 or
19
16.0
14

Protein
MW (Da)
18708
18645
18693
18694

Sample BALB/c2
Migration
MUP
time (min)
15.8
10
16.0
7
16.0
1,12 or 2
16.3
9,11,16,1
8 or 19

Table 2: Major MUPs identified by CESI-MS experiments.

Figure 6: The identification proteins of higher molecular weights insample C57BL/6.
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The final variable investigated in this small study was the effect of salt onthe intensity and protein profiles. The samples had been provided
desalted so salt was added by mixing human urine with the previous
desalted extracts. Each sample contained the same level of protein. InFigure 5 sample A contained no urine, sample B contained 10% urine
and C 25% urine. The results show that the addition of human urine to asample had a very low effect on both the intensity of the spectra obtained
and the electropherograms observed.

(A)

Conclusions
A CESI-MS method for separation of intact MUPs has been developed.
These initial results have shown that CESI-MS is capable of separating
this class of proteins based on their charge and was capable of:•••

Separating MUPs with the same molecular weight.
Distinguishing different mouse strains by their MUP profiles.
Detecting new MUPs of higher mass.

Future work is planned to further evaluate CESI-MS analysis of more
complex crude extracts and to also evaluate the effect of salt
concentration and sample pH on the overall separation efficiency.

References
(B)

(C)

Figure 7: The effect of urine on MUP sample C57BL/6 protein profiles

1. Beynon, R. J et. al. “Characterization and Comparison of Major
Urinary Proteins from the House Mouse, Mus musculus
domesticus, and the Aboriginal Mouse, Mus macedonicus”. J
Chem. Ecol. (2007) 33, 613–630.
2. Beynon, R J., et al. “Mass Spectrometry for structural analysis andquantification of the major urinary proteins of the house mouse”.
International Journal of Mass Spectrometry 391, 146-156, 2015.
3. Hurst, J. L et. al. “Darcin: a male pheromone that stimulates female
memory and sexual attraction to an individual male's odour”. BMC
Biology, 8, 75-96, 2010.
4. Busnel, J-M., et. al., “High Capacity Capillary ElectrophoresisElectrospray Ionization Mass Spectrometry: Coupling a Porous
Sheath-less Interface with Transient-Isotachophoresis”, Anal.
Chem. 82, 9476-9483 2010.
5. Johnston, R., Müller-Schwarze, D. and Sorenson, P. W. “Advancesin chemical signals of vertebrates”. Springer Science & BusinessMedia, 361, 1999.

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