RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2003; 17: 672–677
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.958
The shielding effect of glycerol against protein ionization
in electrospray mass spectrometry
Maria Anita Mendes1, Jocelei Maria Chies2, Ana Christina de Oliveira Dias2,
Spartacos Astofi Filho3 and Mario Sergio Palma1*
1
Laboratory of Structural Biology and Zoochemistry, CEIS/Dept. Biology, Institute of Biosciences, São Paulo State University (UNESP),
Rio Claro, SP-Brazil
2
Laboratory of Development and Production, Cenbiotenzimas, Federal University of Rio Grande do Sul, Porto Alegre RS-Brazil
3
CAM Federal University of Manaus, AM-Brazil
Received 17 July 2002; Revised 22 January 2003; Accepted 27 January 2003
Most commercial recombinant proteins used as molecular biology tools, as well as many academically made preparations, are generally maintained in the presence of high glycerol concentrations
after purification to maintain their biological activity. The present study shows that larger proteins
containing high concentrations of glycerol are not amenable to analysis using conventional electrospray ionization mass spectrometry (ESI-MS) interfaces. In this investigation the presence of 25%
(v/v) glycerol suppressed the signals of Taq DNA polymerase molecules, while 1% (v/v) glycerol
suppressed the signal of horse heart myoglobin. The signal suppression was probably caused by
the interaction of glycerol molecules with the proteins to create a shielding effect that prevents the
ionization of the basic and/or acidic groups in the amino acid side chains. To overcome this difficulty the glycerol concentration was decreased to 5% (v/v) by dialyzing the Taq polymerase solution against water, and the cone voltage in the ESI triple-quadrupole mass spectrometer was set at
80–130 V. This permitted observation of a mass spectrum that contained ions corresponding to protonation of up to 50% of the ionizable basic groups. In the absence of glycerol up to 85% of the basic
groups of Taq polymerase became ionized, as observed in the mass spectrum at relatively low cone
voltages. An explanation of these and other observations is proposed, based on strong interactions
between the protein molecules and glycerol. For purposes of comparison similar experiments were
performed on myoglobin, a small protein with 21 basic groups, whose ionization was apparently
suppressed in the presence of 1% (v/v) glycerol, since no mass spectrum could be obtained even at
high cone voltages. Copyright # 2003 John Wiley & Sons, Ltd.
In recent years the advances in the technology of the manipulation of recombinant DNA have permitted the use of many
proteins as tools in molecular biology. Methods for cloning,
direct sequencing, clinical diagnosis and many other uses1
have proliferated, with the current ability to produce from
microgram to milligram quantities of particular proteins.
Thus, commercial insulin, growth hormones, cytokines,2
and DNA polymerases,3,4 among other recombinant proteins, have been used as therapeutic proteins and/or commercial biochemicals in contemporary biotechnological
processes.
The proteins resulting from these protocols are generally
submitted to sodium dodecyl sulfate/polyacrylamide gel
electrophoresis (SDS-PAGE) as the only criterion to check the
homogeneity of the preparations. However, some of these
proteins may present different molecular forms4,5 caused by
post-translational modifications, or may even suffer artifactual proteolytic cleavage.5,6 Taking into account the standard
*Correspondence to: M. S. Palma, Laboratory of Structural Biology and Zoochemistry, CEIS/IBRC-UNESP, Avenue 24A, 1515
Bela Vista, Rio Claro, SP-Brazil, CEP 13506-900.
E-mail: [email protected]
error of the electrophoretic methods, small changes in
molecular weight (MW) may not be revealed.
Electrospray ionization (ESI) mass spectrometry (MS) is a
rapid and precise method for determining molecular masses
of proteins and can be used to validate protein sequences;7 in
addition, it may be used as an important technique to
evaluate the protein purity/homogeneity. The mass accuracy
achievable using ESI-MS is generally within the range 0.01–
0.05% of the calculated masses,7,8 and it has been used to
characterize many recombinant proteins.9,10 No mutant or
post-translationally modified protein has been identified
only by comparison between theoretical and experimental
masses of the intact protein. However, ESI-MS/MS may be
used to characterize post-translational modifications,11 and
also to identify errors in cDNA sequences.12
Most commercial recombinant proteins used as molecular
biology tools, and also even many of those prepared in the
course of academic research, are generally maintained in the
presence of high glycerol concentrations after purification to
maintain the stability of their biological activity. The effects of
many salts, detergents and chaotropic agents are relatively
well documented to have a significant effect in decreasing the
Copyright # 2003 John Wiley & Sons, Ltd.
Glycerol shielding effect in ESI-MS of proteins
13,14
performance of ESI-MS protocols.
To our knowledge
there is only a single study in the literature characterizing the
effect of protein stabilization by glycerol leading in some
circumstances to the suppression of the protein signal in
EIS-MS.15 However, no detailed investigation appears to
have been performed to characterize this suppressive effect.
We describe here the suppression of protein signals in ESIMS analysis of both recombinant Taq DNA polymerase and
horse heart myoglobin in the presence of high concentrations
of glycerol, and also the use of an appropriate optimization of
mass spectrometric conditions to overcome this difficulty.
EXPERIMENTAL
Material and methods
All solvents used (HPLC quality) were purchased from Mallinckrodt. Horse heart myoglobin and glycerol were acquired
from Sigma Chemical Co. Bidistilled and ultra-purified water
used in all experiments was prepared using a Barnsted system.
Protein cloning, purification and treatment
Taq DNA polymerase was cloned from Thermus aquaticus,
expressed in Escherichia coli, as described by Lawyer et al.4
and purified according to the protocol of Engelke et al.3 After
purification a part of the protein preparation was maintained
in the presence of 25% (v/v) glycerol, and part was maintained soluble in the presence of bidistilled water and the
absence of glycerol. When necessary glycerol was added in
known concentrations to the Taq polymerase preparation.
To purify the Taq polymerase domain fragment (MW
46 270 Da) from the intact protein, part of the enzyme
preparation (250 mg) maintained in the absence of glycerol
was initially filtered through an AMICON-30 filter; the
protein material retained by the filter was dissolved in 5 mM
ammonium acetate (pH 6.8) and then submitted to gel
filtration chromatography on a Sephadex G-75 column
(30.0 1.5 cm), previously equilibrated with the same solvent. Elution was performed with 10 mM ammonium acetate
(pH 6.8) at a flow rate of 12 mL/h, and 3-mL fractions were
collected. Protein elution was monitored by measuring the
absorbance at 280 nm; the protein fractions were pooled and
concentrated by lyophilization.
Electrophoresis
SDS-PAGE was performed on a 10% gel at 30 mA for 2 h. Proteins were stained with Coomassie Brilliant Blue. The MW
markers used for SDS-PAGE were: phosphorylase B
(97.4 kDa), bovine serum albumin (66.2 kDa), glutamate
dehydrogenase (55.0 kDa), ovalbumin (42.7 kDa), aldolase
(40.0 kDa) and carbonic anhydrase (31.0 kDa), all purchased
from Promega.
N-Terminal amino acid sequencing
When necessary the primary sequence of the N-terminal
region of a specific protein was submitted to automated
Edman degradation sequencing using a gas-phase sequencer
PPSQ 21A (Shimadzu, Kyoto, Japan).
Determination of glycerol concentration
The protein solutions containing glycerol, after and before
dialysis, were collected and submitted to glycerol analysis
Copyright # 2003 John Wiley & Sons, Ltd.
673
by using an enzymatic assay based on the conversion of glycerol to dihydroxyacetone in presence of NAD. The reaction
was catalyzed by the enzyme glycerol dehydrogenase. The
NADH, which is formed in stoichiometric quantities by this
reaction, was estimated by UV absorption at 340 nm.
Mass spectrometric analysis
The determination of the homogeneity of the protein preparation and of molecular mass was performed by mass spectrometry using some adaptations to the system described
elsewhere.16 Samples were dissolved in 50% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid (TFA), and
analyzed using a QUATTRO II triple-quadrupole mass
spectrometer (Micromass, Altrincham, UK) equipped with
a standard electrospray probe; solutions were infused at
ca. 5 mL/min. During all experiments the source temperature
was maintained at 808C and the needle voltage at 3.6 kV, the
drying gas flow (nitrogen) was 200 L/h, and the nebulizer gas
flow was 20 L/h. The mass spectrometer was calibrated with
intact horse heart myoglobin and its typical cone-voltage
induced fragments.
The cone-to-skimmer voltage controlling the ion transfer to
the mass analyzer was manually varied from 30 to 130 V.
About 50 pmol of each sample were continuously infused
into the electrospray transport solvent using a microinfusion
pump (KD Scientific), connected to a 500-mL microsyringe
(Hamilton) which in turn was connected to the ESI probe
using a silica microcapillary. The ESI spectra were obtained in
the continuous acquisition mode, scanning from m/z 500–
2500 with a scan time of 7 s.
The mass spectrometer data acquisition and processing
system was equipped with MassLynx and Transform software for handling and deconvoluting spectra.
RESULTS AND DISCUSSION
The purified recombinant Taq DNA polymerase was apparently homogeneous in the presence of 25% (v/v) glycerol,
since it was recorded as a single protein band of MW
94 kDa in SDS-PAGE (Fig. 1(a)).
When 50 pmol of the fresh enzyme preparation were
analyzed in the presence of 25% (v/v) glycerol no ESI-MS
spectrum could be obtained for the enzyme, probably
suppressed by the high glycerol concentration (results not
shown). However, in the presence of 5% (v/v) glycerol and
using 50% (v/v) acetonitrile containing 0.1% (v/v) TFA as
solvent at a cone voltage of 32 V, ESI-MS analysis produced a
bimodal envelope of peaks from m/z 590 to 1921 (Fig. 2(a));
deconvolution of this spectrum resulted in two apparent MW
values of 22 580 17and 24 330 12 Da. The molecular form
with MW 22 580 Da resulted from a series of peaks in this
envelope corresponding to ionized protein molecule populations containing from 20 to 31 positive charges (designated
‘A’ in Fig. 2(a)), and the form with MW 24 420 Da resulted
from an envelope of peaks corresponding to molecules
containing from 13 to 19 positive charges (designated ‘B’ in
Fig. 2(a)).
When the sample cone voltage was increased to 80 V, a
second bimodal envelope of peaks appeared in the mass
spectrum from m/z 634 to 1972 (Fig. 2(b)). The deconvolution
Rapid Commun. Mass Spectrom. 2003; 17: 672–677
674
M. A. Mendes et al.
Figure 1. SD-PAGE of purified recombinant Taq DNA
polymerase (a) in the presence of 25% (v/v) glycerol and
(b) in the absence of glycerol; standard protein MW markers
appear in the left lane.
yielded two molecular forms with MW 46 880 34 and 93
324 57 Da. The form with MW 46 880 Da results from an
envelope of peaks corresponding to protein molecules
containing from 40 to 68 positive charges (designated ‘C’ in
Fig. 2(b)); the form with MW 93 324 Da, which seems to
represent the intact Taq polymerase, results from a population of molecules containing from 47 to 67 positive charges
(designated ‘D’ in Fig. 2(b)).
However, this bimodal envelope was still very complex,
and did not provide a reliable deconvolution result for MW
determination. Therefore, the cone voltage was increased to
130 V, resulting in another bimodal envelope of peaks from
m/z 764 to 2059 (Fig. 2(c)); under these experimental
conditions, the first envelope of peaks was deconvoluted as
the molecular form ‘A’, already observed in Fig. 2(a). The
deconvolution of the second envelope of peaks resulted in a
molecular mass of 93 320 Da, already designated as molecular
form ‘D’ in Fig. 2(b).
Therefore, high glycerol concentrations (25% v/v) seem to
prevent ionization of Taq polymerase, since no spectrum was
obtained under these conditions. However, on decreasing the
glycerol concentration to 5% (v/v), different MW forms were
detected at different cone voltages. Thus, at 32 V, two forms of
MW 22 580 and 24 420 Da were detected, while, at 80 V, two
other MW forms were identified at 46 880 and 93 324 Da.
When 130 V was applied to the sampling cone, both the
smallest and the largest forms were detected. These results
suggest that an intact form of Taq polymerase plus three
smaller fragments of this enzyme co-exist in the preparation.
The fragments may result from proteolytic action after
gene expression and/or during the purification protocol.
Probably, glycerol interacts strongly with Taq polymerase
and all its fragments, promoting strong hydrogen bonding
between glycerol molecules and the surface of each protein
form in a way that keeps these fragments tightly united in a
larger cluster similar to the intact Taq polymerase (form ‘D’);
at least in SDS-PAGE these fragments were not detected in
the presence of glycerol (Fig. 1(a)).
Glycerol at a concentration of 5% (v/v) seems to prevent
the detection of the forms with MW 46 880 and 93 320 Da
Copyright # 2003 John Wiley & Sons, Ltd.
Figure 2. ESI-MS spectra of Taq DNA polymerase in the
presence of 5% (v/v) glycerol at different cone voltages: (a)
32 V; (b) 80 V; and (c) 130 V.
under low cone-voltage conditions. Probably, the intact
protein and its largest fragment interact more strongly with
glycerol, partially preventing the ionization of the basic
amino acid residues in these molecular forms.
Taq polymerase was prepared in the absence of glycerol,
and 50 pmol of this enzyme were analyzed in presence of 50%
(v/v) acetonitrile containing 0.1% (v/v) TFA. A rather
complex ESI-MS spectrum was obtained when the cone
voltage was 45 V (Fig. 3(a)). Since different envelopes of peaks
overlapped one another, the deconvolution and the assignment of each series of peaks, followed by their respective
charge determinations, were performed using centroided
data (Figs. 3(b)–3(e)). The insert in Fig. 3(a) reveals that
four different MW forms were detected in the absence of
glycerol at a relatively low cone voltage (45 V), corresponding to the same forms already detected in the presence of
5% (v/v) glycerol at high cone voltages (Figs. 2(a)–2(c)), i.e.,
22 066 18, 24 080 14, 46 290 36 Da, and the intact Taq
polymerase with 93 416 61 Da. SDS-PAGE of this glycerolfree Taq polymerase preparation revealed the presence of a
band of MW 46 kDa, while the smaller forms (22–24 kDa)
were not detected by this method (Fig. 1(b)). The form
presenting MW 46 kDa was isolated from the Taq polymerase
preparation in the absence of glycerol (results not shown), as
described in the Experimental section. The N-terminal region
of this Taq fragment was sequenced by Edman degradation,
yielding the sequence EGERLL. Thus, taking into account this
N-terminal sequence and the molecular mass value obtained
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Glycerol shielding effect in ESI-MS of proteins
675
Figure 3. (a) ESI-MS spectra of Taq DNA polymerase in the absence of glycerol, acquired
in the continuum mode, with the cone voltage adjusted to 45 V; (b)–(e) decomposition of the
ESI-MS spectrum in (a) into different envelopes of peaks, represented in centroid mode.
after purification (46 290 Da), this protein was identified as
the DNA polymerase domain of the Taq enzyme, which
corresponds to the Taq polymerase fragment 420–835 (with
expected MW 46 270.76 Da); this fragment is still active
(results not shown). These results also corroborate the
proposal that somehow glycerol interacts strongly with
protein fragments, keeping them so tightly bound (probably
through hydrogen bonds between glycerol molecules and
protein fragment surfaces) that they cannot be separated/
detected from each other by using mild experimental
conditions.
The centroided representation permitted individual
deconvolution of the ESI-MS spectral envelopes for each
form of different MW. Figure 3(b) shows that the envelope of
peaks corresponding to the form of MW 22 066 Da was
produced by a population of molecules containing from 23 to
Copyright # 2003 John Wiley & Sons, Ltd.
33 positive charges (designated the ‘A’ series of centroid
peaks in Fig. 3(b)), i.e., in the same range of charges as that
observed in the presence of glycerol. Figure 3(c) shows that
the envelope of peaks corresponding to the Taq fragment of
MW 24 083 Da was produced by molecules containing from
18 to 33 positive charges centered around 25 charges
(designated the ‘B’ series of centroided peaks in Fig. 3(c)).
In the presence of glycerol this fragment produced an
envelope of peaks corresponding to molecular species
containing from 12 to 19 positive charges (Fig. 2(a)). Thus,
the presence of glycerol seems to be partially shielding some
basic residues of this protein fragment.
In the absence of glycerol the form corresponding to Taq
fragment 420–835 yielded an envelope of peaks produced by
protein molecules with from 49 to 58 positive charges
(designated the ‘C’ series of centroided peaks in Fig. 3(d))
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M. A. Mendes et al.
when a cone voltage of 45 V was used. In contrast, in the
presence of 5% (v/v) glycerol, no signal was detected for this
protein form at the same cone voltage, although an envelope
of peaks interpreted as arising from this form was detected
using 80 V cone voltage. This envelope was produced by
protein molecules containing from 43 to 68 positive charges.
By setting the sample cone voltage to 45 V in the absence
of glycerol, the intact Taq polymerase produced an envelope
of peaks which corresponded to molecules containing from
47 to 117 positive charges (designated the ‘D’ series of
centroided peaks in Fig. 3(e)), while, in the presence of 5%
(v/v) glycerol, the envelope of peaks for this protein was
detected only at sample cone settings 80 V. Under these
conditions the envelope was produced by protein molecules
containing from 47 to 69 positive charges, centered around 59
charges. Taking into account the total number of the basic
amino acid residues present in the intact Taq polymerase
molecule (76 arginines, 42 lysines, 18 histidines) in addition to
the amino terminal residue, one might expect that a
maximum of 137 positive charges is possible for this protein.
This means that, in the absence of glycerol, up to 85% of the
basic groups were ionized at relatively low cone voltage,
while, in the presence of 5% (v/v) glycerol, only up to 50% of
these groups were ionized in the mass spectrum, even at high
cone voltage. Thus, it is very clear that the shielding effect
caused by glycerol also required higher cone potentials to
allow detection of the protein signal.
Of course the cone potential has no effect on the
electrospray process itself, i.e., the nature and distribution
of the ions emerging from the electrospray source and
presented to the API interface are entirely independent of the
potentials within the interface. A possible explanation for this
and related observations described above is that glycerolbound dimers (or even higher oligomers) can be formed by
electrospray in the presence of glycerol for Taq DNA
polymerase and its larger fragment, but not for the smaller
(20 kDa) fragments. At lower cone voltages these glycerolbound oligomers survive the collision conditions in the API
interface, but possess m/z values too high to be detected by the
instrument used in this work. At higher cone voltages the
collision conditions are more energetic and protein monomers are formed and detected, although with a lower chargestate distribution than that formed in the absence of glycerol
due to the glycerol shielding effect that operates in the
electrospray process itself.
In order to corroborate these observations, a similar
experiment was performed with a smaller protein, horse
heart myoglobin. In the absence of glycerol the ESI-MS
spectrum of this protein, obtained using a cone voltage of
35 V, revealed a single (unimodal) envelope of peaks from m/z
645 to 1540, corresponding to protein molecules containing
from 11 to 21 positive charges. The deconvolution of this
spectrum resulted in a MW value of 16 924 12 Da (Fig. 4(a)).
The presence of 0.1% (v/v) glycerol did not change
significantly the spectrum observed in its absence; however,
it was necessary to set the cone voltage to 45 V in order to
obtain a useful protein signal (Fig. 4(b)). In the presence of
0.5 % (v/v) glycerol the ESI-MS spectrum became partially
suppressed, presenting a small envelope of peaks from m/z
942 to 1210, corresponding to protein molecules containing
Copyright # 2003 John Wiley & Sons, Ltd.
Figure 4. ESI-MS spectrum of horse heart myoglobin under
different experimental conditions with inserts representing
the deconvolution of each spectrum: (a) in the absence of
glycerol and with the cone voltage set to 35 V; (b) in the
presence of 0.1% (v/v) glycerol with the cone voltage set to 45
V; (c) in the presence of 0.5% (v/v) glycerol with the cone
voltage set to 60 V; and (d) in the presence of 1% (v/v)
glycerol with the cone voltage set to 95 V.
from 14 to 19 positive charges (Fig. 4(c)). However, it must be
emphasized that with 0.5% glycerol present, no signal was
observed at cone voltages lower than 60 V. Nonetheless, in
spite of this effect, the deconvolution of these data resulted in
a consistent MW value (16 918 14 Da). When the mass
spectrometric analysis of myoglobin was performed in the
presence of 1% (v/v) glycerol, no ESI-MS signal was
observed, even by setting high cone voltages (up to 95 V;
Fig. 4(d)). Once more it seems that glycerol is interacting with
the protein, preventing the ionization of the basic side chains
of arginine and lysine residues. The present observations for
the effect of glycerol on the charge-state distribution of
myoglobin are qualitatively similar to those reported
previously15 for other small proteins (lysozyme and cytochrome c).
CONCLUSIONS
The addition of glycerol after the final purification step, for
both natural and recombinant proteins, has the purpose of
preventing these molecules from denaturation.15 However,
Rapid Commun. Mass Spectrom. 2003; 17: 672–677
Glycerol shielding effect in ESI-MS of proteins
in addition to the stabilizing effect on the protein molecules,
high glycerol concentrations strongly influence the electrospray mass spectrometric performance, since they suppress
the protein signal resulting in a flat and noisy base line. Other
authors have also reported that protein samples containing
high glycerol concentrations are not amenable to ESI interfaces, requiring the use of high nozzle potential in an ESITOFMS instrument.15
An explanation for this effect is proposed, based on strong
interactions between glycerol molecules and the side chains
of the basic amino acid residues in the proteins, thus
interfering with protonation of some of these residues. For
some larger proteins these interactions with glycerol are
sufficiently large that glycerol-bound dimers (or higher
oligomers) are formed by the electrospray process, and these
species have m/z values too high to be detected by the mass
spectrometer. However, by increasing the voltage in the API
interface, it is possible to create collision conditions sufficiently violent that some protein ions without any clustered
glycerol molecules are formed and detected, although with a
lower charge-state distribution than that observed in the
absence of glycerol. This same proposal can also account for
the fact that our Taq polymerase preparation, when analyzed
by SDS-PAGE in the presence of glycerol, appeared to contain
only the intact protein although both mass spectrometric and
SDS-PAGE analysis in the absence of glycerol clearly
indicated the presence of a fragment of the native enzyme
with about half the correct MW. It is thought that glycerol
could bind the fragments together to form dimers that cannot
be resolved from the intact protein by SDS-PAGE.
Finally, similar experiments on a much smaller protein
(myoglobin) yielded evidence for the effects of added
glycerol on both suppression of ionization and a shift in the
charge-state distribution that were qualitatively similar to
those reported previously15 for other small proteins. Thus,
the present observations on our Taq DNA polymerase
Copyright # 2003 John Wiley & Sons, Ltd.
677
preparation cannot be attributed to instrumental effects, but
reflect different phenomena that become apparent only for
larger proteins.
Acknowledgements
This work was supported by grants from FAPESP (CAT/
CEPID and SMOLBNET) and Instituto do Milênio (CNPq/
MCT). MAM is a post-doctoral fellow of FAPESP; MSP is a
researcher of the National Research Council (CNPq,
500079/90–0).
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