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Probing transferrin-metal

metal interactions by means of travelling-wave


travelling
ion mobility mass spectrometry
Charlotte A. Scarff1, Frances D. L. Kondrat1,2, Claire Booyjzsen2, Arindam Mukherjee2, Peter J. Sadler2, James H. Scrivens1

OVERVIEW

of Biological Sciences, University of Warwick, Coventry, U.K. and 2 Department of Chemistry, University of Warwick, Coventry, U.K.
METHODS

Aim

Protein dialysis and purification

To investigate the gas-phase conformations of transferrin-metal complexes under varying


pH conditions by means of travelling wave ion mobility-mass spectrometry (TWIM-MS).

Methods
Recombinant human transferrin (hTF) (Novozymes, UK) was purified by means of chromatography
approaches. hTF was subjected to tryptic digestion prior to analysis by reversed-phase (RP) liquid
chromatography (LC) MSE to confirm protein identity. A Synapt HDMS G2 system (Waters,
Manchester, UK) was used to perform TWIM-MS experiments on apo-hTF, holo(Fe)-hTf and
holo(Mn)-hTF under controlled non-denaturing solvent conditions from pH 7.4-5.5 and under
denaturing conditions. Estimated collisional cross-sections for various protein charge states were
obtained by calibration of TWIM-MS data. These were compared to those calculated from published
X-ray crystallographic structures.

Results
Sequence analysis confirmed protein identification with 86% sequence coverage. The loss of ferric
and manganese ions from holo-transferrin were observed by incubation under mildly acid
conditions. Arrival time distributions, along with estimated collisional cross-sections, of the holo and
apo-transferrins, were similar for all species studied. Estimated collisional cross-sections agreed
with values calculated from published structures.

Conclusions
TWIM-MS experiments suggest that no significant conformational change in hTF global structure
occurs upon ferric ion loss. holo(Mn)-hTF and holo(Fe)-hTf exhibit similar conformations.

INTRODUCTION
Human serum transferrin (hTF) is an ~80 kDa monomeric, bilobal, glycoprotein that plays an
integral role in iron homeostasis. Transferrin has the ability to bind Fe3+ at neutral pH and functions
to solubilise and transport iron(III) in the bloodstream, which would otherwise precipitate as
insoluble iron(II) hydroxides. Transferrin releases Fe3+ at pH 5.5 and delivers iron to cells via
receptor-mediated endocytosis [1], Figure 1.
pH 7.4

Transferrin is able to bind one ferric cation,


in each of the Venus fly-trap clefts
present in each lobe, Figure 2. The metal
is held in a distorted octahedral binding
site by two tyrosinate, an aspartate and a
histidine residue. The final two coordination sites are filled by a synergistic
anion, usually carbonate, essential for
metal binding [1].

Recombinant human transferrin, gifted from Novozymes UK, was purified by fast performance liquid
chromatography and anion exchange chromatography. holo(Fe)-hTf samples were exchanged into
10 mM ammonium bicarbonate, using 30K MWCO spin columns (Millipore, MA, USA). apo-hTf
samples were generated by dialysing iron-containing hTf against ca. 250 mM ammonium citrate
pH5.0-5.5, with multiple exchanges of buffer to remove all ferric ions. holo(Mn)-hTF was produced
by the addition of potassium permanganate to an aliquot of apo-hTf. Excess salts were removed on
a PD10 desalting column equilibrated with 10 mM ammonium bicarbonate.

ESI-spectra obtained for apo-hTF analysed under denatured conditions indicated the presence of
two protein species, Figure 4. Deconvolution of this data by MaxEnt (maximum entropy modelling
algorithm contained in MassLynx) onto a true molecular mass scale indicated that the two species
had molecular masses of 75098 and 75260 Da ( 2Da) respectively. This suggested that these
protein species were recombinant hTF(S415A, N611
611D,V612P,T613A) with and without the addition
of a single O-linked glycosylation at S32.

2Fe3+ + 2CO32- = 231.7 Da


231.7/18 = 12.9 Da

Denatured
Spectrum
Transferrin

12 Da
2 species are
present

ApoTransferrin

To confirm the sequence of the recombinant transferrin, hTf was subjected to tryptic digestion prior
to analysis by reversed-phase (RP) liquid chromatography (LC) - MSE. Data were acquired in a
data-dependent manner by alternating between low and elevated energy scan functions, from
which intact precursor and peptide fragmentation information were obtained. The data were
processed and searched against an in-house database including the putative recombinant hTf
sequence by use of ProteinLynx Global Server v.2.4.

18+
2Mn3+

Figure 2- Crystal structure of apo-hTF, 2HAU. Clobe (blue) and N-lobe (purple) are shown in the
open conformation [2].

5550

Apo-Transferrin

5500

Fe(2)-Transferrin

5450

Mn(2)-Transferrin

5400
5350
5300
5250
5200
5150
5100
16

17

18

19

20

21

Charge state

Figure 9 - Estimated collisional cross-sections for the three transferrin


species at varying charge states.

Apo-transferrin
N-lobe

Fe-transferrin
N-lobe

MOBCAL
PA 2401 2
EHSS 3060 2

MOBCAL
PA 2301 2
EHSS 2941 2

10 Da

Figure 4- Denatured ESI spectrum of recombinant hTF 5 M in 30% acetonitrile, 0.1% formic acid. Insert
shows the deconvoluted spectrum. Molecular masses obtained for the two species suggest they are
recombinant hTF with and without an O-linked glycosylation at S32.

Mn2Transferrin

ApoTransferrin

(partially manganese-loaded)

To confirm the sequence of the hTf an MSE experiment was performed on the purified sample,
Figure 3. Sequence analysis confirmed the sequence was human transferrin (S415A, N611D,
V612P, T613A). The mutations present within the sequence prevent N-linked glycosylation
occurring at N413 and N611, but do not interfere or change metal-binding properties. O-linked
glycosylation at S32 can however still occur.

ESI-spectra obtained for holo(Fe)-hTf and holo(Mn


Mn)-hTF, at pH 7.4, and apo-hTF, at pH 5.5, show
similar charge-state distributions, Figure 5. As no differences in the protein ion charge state
distributions between holo(Fe)-hTf and apo-hTF are observed this suggests that iron release does
not cause a global conformational change within the protein.

Figure 10 - Overlay of crystal structures for apo-transferrin N-lobe ( PDB ID 1BTJ) and Fe-transferrin N-lobe ( PDB ID
1A8E) with MOBCAL rotationally-averaged collision cross-section estimations.

CONCLUSIONS

18+

Fe2-Transferrin
pH 7.4

17+

Figure 7- Overlaid ESI spectra of Mn2-transferrin and apo-transferrin. Insert focuses on the 18+ charge state and
reveals a mass-to-charge difference of 10 Da.

19+

Extracted arrival time distributions for the 17+, 18+ and 19+ charge states of holo(Fe)-hTf, holo(Mn)hTF and apo-hTF respectively are shown in Figure 8.
18+

Fe3+
binding site

2- =

+ 2CO3
229.9 Da
229.9/18 = 12.8 Da

RESULTS

Recombinant human transferrin: Average MW 75098.5 Da

A conformational change within the clefts from a


closed to an open conformation allows the
release of the metal ions. The conformational
change is thought to be initiated by the drop in
the environmental pH resulting in the protonation
and dissociation of the synergistic anion.
Although Fe3+ has the highest affinity for the
binding cleft, other metal ions, of biological or
even therapeutic importance, such as zinc,
nickel and manganese also have the ability to
bind.

5600

MOBCAL estimations for the rotationally-averaged collision cross-sections of the transferrin N-lobe
in apo- and holo-form are similar, Figure 10. This indicates that although a local structural change
is known to occur in the transferrin structure upon iron release this does not have a significant
effect on global protein conformation. This observation is supported by work conducted by
Gumerov and Kaltashov [4].

Figure 6- Overlaid ESI spectra of Fe2-transferrin (pH 7.4) and apo-transferrin (pH 5.5). Insert focuses on the 18+
charge state and reveals a mass-to-charge difference of 12 Da.

Sequence analysis

pH 5.5

Figure 1- The internalisation and release of Fe3+ via


transferrin receptor-mediated endocytosis.

No significant differences are


observed between the arrival time
distributions, for each of the charge
states, for different metal-bound
species. The estimated crosssections for these species, for
various charge states, are shown in
Figure 9. Estimations of crosssection for apo-hTF charge states
17+, 18+ and 19+ obtained are
between the MOBCAL PA and
EHSS calculated values of 4143 2
and 5384 2 respectively.

Fe2Transferrin
(partially iron-loaded)

Mn2-Transferrin
pH 7.4

Fe3+
binding site

18+

Transferrin + O-linked
Glycosylation

Mass spectrometry and ion mobility mass spectrometry


To investigate the conformational change in hTf upon the removal of Fe3+ and Mn3+ , the protein
was exchanged into 200 mM ammonium acetate adjusted to pH 7.4-5.5 in 0.5 pH unit increments.
Samples were further desalted and buffer-exchanged using centrifugal-filter devices (Biomax-10,
Millipore, MA, USA). A Synapt HDMS G2 system (Waters, Manchester, U.K) was used to perform
all TWIM-MS experiments. The following instrumental conditions were used: backing pressure 8.5
mBar, capillary voltage 1.2-1.8 kV, cone voltage 90 V, helium cell gas flow 180 mL/min, IMS cell
gas flow 90 mL/min, travelling-wave height 40V and travelling wave velocity 700 m/s. Sperm whale
myoglobin was also analysed under these conditions to allow calibration of ion mobility data.
Rotationally-averaged collision cross-sections were calculated from X-ray structures using
MOBCAL, a program to calculate mobilities [3,4].

ESI-spectra obtained for holo(Fe)-hTf and holo(Mn)-hTF under controlled non-denaturing solvent
conditions from pH 7.4-6.0 showed no significant differences. At pH 5.5 a shift in m/z values
observed was seen indicating that the previously bound metal ions had been released, Figures 6
and 7. The shift in mass observed suggests that neither the holo(Fe)-hTf sample nor the holo(Mn)hTF sample were fully metal-loaded. This is consistent with observations that Tf is only ~30%
loaded in nature [1].

Estimated cross-section ( 2)

1Department

19+

18+

Monomeric and dimeric species of hTF were observed by ESI-MS for holo(Fe)-hTf and holo(Mn)hTF and apo-transferrin. No shifts in the charge state distributions of holo(Fe)-hTf and holo(Mn)hTF were observed as the pH was lowered to pH 5.5 and the metal released. Ion mobility extracted
arrival time distributions were very similar for both the metal-bound species and apo-transferrin for
a range of charge states.

17+

The local conformational change which occurs in transferrin as Fe3+ is released from a
closed to an open conformation does not appear to have a significant impact on global
protein conformation.

19+
17+

MSE run
sequence
coverage 86%

Recombinant human transferrin was confirmed as hTF( S415A, N611D, V612P, T613A) by LCMSE analysis. ESI-MS analysis under denaturing conditions determined that the sample contained
two different transferrin species, un-gycosylated and O-linked glycosylated recombinant hTF.

REFERENCES
1.
2.

18+

Apo-Transferrin
pH 5.5

3.
4.

19+
17+

5.

Sun, H., Li, H. and Sadler, P.J. Chem. Rev, 1999, 99: 2817-2842.
Wally, J., Halbrooks, P.J., Vonrhein, C., Rould, M.A., Everse, S.J., Mason, A.B., Buchanan, S.K. J Biol. Chem.,
2006, 281: 24934-24944.
Shvartsburg, A. A. and Jarrold, M. F. Chem. Phys. Let., 1996, 261: 86-91.
Mesleh, M. F., Hunter, J. M., Shvartsburg, A. A., Schatz, G. C. and Jarrold, M. F. J Phys. Chem., 1996, 100:
16082-16086.
Gumerov, D.R. and Kaltashov, I.A. Anal. Chem., 2001, 73: 2565-2570.

ACKNOWLEDGEMENTS
Figure 3- Recombinant human transferrin sequence with displayed disulphide bonds, accompanied by
sequence coverage data, determined from an MS E analysis.

Fe3+

Mn3+

Figure 5- ESI spectra of the


and
bound transferrin at pH 7.4 and an apo-transferrin spectra at pH 5.5.
Each spectrum shows a range of charge states corresponding to the monomer, centred around the 18+ peak, along
with a charge state distribution indicating the presence of dimer.
dimer

Figure 8- Arrival time distributions corresponding to the 17+, 18+ and 19+ charge states for the apo (red), Fe2 (green)
and Mn2 (purple) transferrin species.

Charlotte would like to thank, Claire and Fran for all their help with this work, her supervisor, Prof.
Jim Scrivens for support and Waters for funding. We thank Phil Morton from Novozymes for helpful
sample information.

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