Structural studies of proteins by hydrogen/deuterium exchange coupled to mass spectrometry (DXMS) require the use of proteases working at acidic pH and low temperatures. The spatial resolution of this technique can be improved by combining several acidic proteases, each generating a set of different peptides. Three commercial aspartic proteases are used, namely, pepsin, and proteases XIII and XVIII. However, given their low purity, high enzyme/protein ratios have to be used with proteases XIII and XVIII. In the present work, we investigate the activity of two alternative acidic proteases from Plasmodium falciparum under different pH and temperature conditions. Peptide mapping of four different proteins after digestion with pepsin, plasmepsin 2 (PSM2), and plasmepsin 4 (PSM4) were compared. PSM4 is inactive at pH 2.2 and 0°C, making it unusable for DXMS studies. However, PSM2 showed low but reproducible activity under DXMS conditions. It displayed no substrate specificity and, like pepsin, no strict sequence specificity. Altogether, these results show that PSM2 but not PSM4 is a potential new tool for DXMS studies

Fieschi, Franck

Forest, Eric

Marcoux, Julien

Signor, Luca

Thierry, Eric

Vivès, Corinne

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Investigating alternative acidic proteases for H/D exchange coupled to mass spectrometry: Plasmepsin 2 but not plasmepsin 4 is active under quenching conditions

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APPLICATION NOTE
Investigating Alternative Acidic Proteases for
H/D Exchange Coupled to Mass Spectrometry:
Plasmepsin 2 but not Plasmepsin 4 Is Active
Under Quenching Conditions
Julien Marcoux,a,b Eric Thierry,a Corinne Vivès,b Luca Signor,a
Franck Fieschi,b and Eric Foresta
a Laboratoire de Spectrométrie de Masse des Protéines, Institut de Biologie Structurale, Grenoble, France
b Laboratoire des Protéines Membranaires, Université Joseph Fourier, Institut de Biologie Structurale (IBS),
Grenoble, France
Structural studies of proteins by hydrogen/deuterium exchange coupled to mass spectrometry
(DXMS) require the use of proteases working at acidic pH and low temperatures. The spatial
resolution of this technique can be improved by combining several acidic proteases, each
generating a set of different peptides. Three commercial aspartic proteases are used, namely,
pepsin, and proteases XIII and XVIII. However, given their low purity, high enzyme/protein ratios
have to be used with proteases XIII and XVIII. In the present work, we investigate the activity of
two alternative acidic proteases from Plasmodium falciparum under different pH and temperature
conditions. Peptide mapping of four different proteins after digestion with pepsin, plasmepsin 2
(PSM2), and plasmepsin 4 (PSM4) were compared. PSM4 is inactive at pH 2.2 and 0 °C, making it
unusable for DXMS studies. However, PSM2 showed low but reproducible activity under DXMS
conditions. It displayed no substrate specificity and, like pepsin, no strict sequence specificity.
Altogether, these results show that PSM2 but not PSM4 is a potential new tool for DXMS
studies. (J Am Soc Mass Spectrom 2010, 21, 76–79) © 2010 American Society for Mass
SpectrometryDXMS has recently been widely used to improvethe structural knowledge of protein complexesthat are too large for NMR [1] or too flexible for
crystallographic [2] studies, and to obtain complemen-
tary information on ligand binding [3] or protein dy-
namics [4]. One of the main time-consuming steps of
this method is the peptide mapping of the protein of
interest, after digestion with a nonspecific protease.
Acidic proteases are used since their proteolytic activity
is maintained under hydrogen/deuterium exchange
mass spectrometry (DXMS) experimental conditions,
i.e., between pH 2 and 3 and at 0 °C where H/D
back-exchange is minimal [5]. To reduce back-exchange,
one must also work with proteases able to digest
proteins within a fewminutes. Recently, new insights in
fragmentation techniques, limiting the intramolecular
migration of hydrogens, have allowed reaching single-
residue resolution. However, so far these techniques
have been tested only with synthetic peptides or small
proteins such as myoglobin [6]. The combined use of
alternative acidic proteases with different specificities is
Address reprint requests to Dr. E. Forest, Institut de Biologie Structurale,
Laboratoire de Spectrométrie de Masse des Protéines, 41 Avenue des
Martyrs, 38027 Grenoble, France. E-mail: eric.forest@ibs.fr
© 2010 American Society for Mass Spectrometry. Published by Elsevie
1044-0305/10/$32.00
doi:10.1016/j.jasms.2009.09.005another way to improve the method’s resolution. Our
laboratory has developed the use of commercial-type
XIII and XVIII fungal proteases [7, 8] that are now
routinely used to improve the resolution of DXMS
studies [9, 10]. However, the presence of impurities
requires high enzyme/protein ratios (10.5:1 (wt/wt) for
protease type XIII and 17:1 for protease type XVIII) [7].
Recently, we developed recombinant type XVIII pro-
tease with higher purity and activity [11].
In the present study, the use of two alternative
recombinant aspartic proteases from the parasite Plas-
modium falciparum, plasmepsins 2 (EC 3.4.23.39) and 4
(EC 3.4.23.B14), is evaluated with the goal of improving
protein mapping and spatial resolution in DXMS exper-
iments. Plasmepsins play an important role in the
intra-erythrocytic digestion of hemoglobin responsible
for the tropical disease malaria. Structural and enzy-
matic studies were previously reported [12, 13]. How-
ever, very little is known about their sequence and
substrate specificities. This study shows for the first
time that PSM2 can be used to digest nonerythrocytic
proteins. We obtained peptide maps and were thus able
to compare plasmepsin activity under DXMS conditions
with other proteolytic results (pepsin), not only on their
natural substrate (hemoglobin), but also on myoglobin
Published online September 17, 2009
r Inc. Received June 3, 2009
Revised September 8, 2009
Accepted September 8, 2009
77J Am Soc Mass Spectrom 2010, 21, 76–79 STUDY OF NEW PROTEASES UNDER DXMS QUENCHING CONDITIONSand the PX domain of p47phox (p47phox-PX), a cytosolic
factor of the neutrophil NADPH oxydase [4].
Experimental
Materials
All proteins were from Sigma-Aldrich (St. Louis, MO,
USA). The reversed-phase C18 column Jupiter (50 
1.00 mm; 5 m, 300 Å) was from Phenomenex (Tor-
rance, CA, USA) and the protein MacroTrap C8 was
from Michrom Bioresources (Auburn, CA, USA).
Protein Expression and Purification
PSM2 (residues 78–453) and PSM4 (residues 74–448)
clones were the kind gifts of Professor Ben M. Dunn
(University of Florida). Plasmepsin expression and pu-
rification were carried out according to Westling et al.
[14]. p47phox-PX was expressed in E. coli BL21(DE3) and
purified according to the protocol used for the entire
p47phox described in Durand et al. [15].
Protein Digestion and Protease Cleavage
A total of 338 pmol of protein were digested for 2 min
at a 1:1 enzyme/protein ratio (wt/wt). Digestions were
carried out in 10 mM glycine pH 2.2 or in 50 mM citrate
pH 4.5. PSM4 was activated for 40 min in 50 mM citrate
pH 4.5.
LC-MS and LC-MS/MS
Before MS analysis, peptides were desalted for 1 min on
a MacroTrap column at a 400-L/min flow rate with a
0.03% TFA water solution. Peptides were then eluted
with an 8-min step gradient (0-10-15-20-25-30-35 and
45% of 95% acetonitrile, 0.03% TFA) for peptide map-
ping, which was performed on a quadrupole ion trap
mass spectrometer (ESQUIRE 3000; Bruker Daltonics,
Billerica, MA, USA) equipped with an electrospray
ionization source (ESI). For MS/MS experiments, the
three most intense ions were fragmented and then
excluded after two spectra had been obtained. Data
processing was done using Bruker Data Analysis 3.0
software and Mascot server. MS/MS spectra were
checked manually and confirmed by accurate mass
measurement on an ESI-time-of-flight (TOF) mass spec-
trometer (6210; Agilent Technologies, Santa Clara, CA,
USA). The Agilent software MassHunter and MagTran
were used for data processing. Peptide maps were
made with scripts available at http://ms.biomed.cas.
cz/MSTools/.
Results and Discussion
PSM2 and PSM4 are generally studied under physio-
logical conditions (37 °C, pH 4.5). Maturation of the
43-kDa PSM2 precursor into the 38-kDa activated formwas achieved most often at 37 °C or room temperature
for 40 min to 2 h and pH 4.5 to pH 5 [16–18]. Maturation
of PSM4 was shown to be efficient after 5 min at pH 4.5
and 37 °C [19]. We determined the optimal time for
plasmepsin maturation by following their auto-cleav-
age on ESI-TOF MS (Figure S1 available as supplemen-
tary material, which can be found in the electronic
version of this article). PSM2 auto-cleavage after F112
was complete after 20 min in 10 mM citrate pH 4.5
(Figure S1 available as supplementary material, which
can be found in the electronic version of this article).
After 10 min at room temperature in 10 mM citrate pH
4.5, PSM4 was partly cleaved after Phe109 (38,218 Da),
Phe110 (38,071 Da), or Phe81 (41,508 Da); however,
cleavage was not complete, even after 7 h (data not
shown). We finally used a 40-min maturation time for
further experiments with PSM4. PSM2 and PSM4 activ-
ity on bovine hemoglobin was then tested before and
after maturation at pH 4.5, at two different acidic pHs
and two different temperatures (Figure 1a). Table S1
(available as supplementary material, which can be
found in the electronic version of this article) lists the
identified cleavage sites. Reproducibility of the diges-
tion was confirmed by triplicate ESI-TOF MS accurate
mass measurement. PSM2 was active in all tested
conditions, but at pH 4.5, the number of cleavage sites
was lower at 0 °C than at room temperature (13 versus
19 cleavage sites for the nonmature form). PSM2 pro-
duced more cleavage sites at pH 4.5 than at pH 2.2 (13
versus 10 cleavage sites, at 0 °C for the nonmature
form). Interestingly, PSM2 was less active after matura-
tion (19 versus 12 cleavage sites at room temperature).
At pH 2.2, PSM2 activity was surprisingly observed
only without maturation (intact plasmepsin was iden-
tified at the end of the LC-MS run). Contrary to PSM2,
PSM4 was more active after maturation. It was able to
digest bovine hemoglobin in different pH and temper-
ature conditions, but its activity was considerably low-
ered at pH 2.2 (five cleavage sites at room temperature
and only one at 0 °C), making it unusable for DXMS.
Proteolytic activity was also tested on human hemoglo-
bin with nonmature PSM2 at pH 2.2 and 0 °C. More
than 70% of sequence coverage was obtained with 16
identified cleavage sites (Figure 1b). Similar results
were obtained with mature PSM4 but at pH 4.5 and
room temperature (11 identified cleavage sites). In
conclusion, these first experiments using hemoglobin as
substrate showed PSM2 as a candidate for DXMS
experiments since it was active at pH 2.2 and 0 °C for 2
min of digestion time, at an 1:1 enzyme/protein (wt/
wt) ratio, in contrast with PSM4.
To investigate the substrate specificity of PSM2 and
PSM4, we tested their activity on nonerythrocytic pro-
teins. Horse myoglobin was chosen because of its
structural similarity with hemoglobin and p47phox-PX
because of its nonrelated structure. Horse myoglobin
and p47phox-PX were digested by pepsin and PSM2
(Figure 2) but not by PSM4, even at pH 4.5 and room
ets.
78 MARCOUX ET AL. J Am Soc Mass Spectrom 2010, 21, 76–79temperature. These results confirm the broad substrate
specificity of PSM2, which is able to digest proteins not
only from erythrocyte cytoskeleton [18], but also from
muscle cells and neutrophils. On the contrary, PSM4
seems to be only able to digest its physiologic substrate
(human hemoglobin) or species homologues.
A broad-spectrum substrate does not exclude any
sequence motif recognition or at least any preference for
Figure 1. Peptide map of bovine (a) and hum
pepsin, non-mature PSM2, and mature PSM
percentages of coverage are indicated in the ins
Figure 2. Peptide map of (a) horse myoglobin
PSM2 at pH 2.2 and 0 °C. Percentages of coverage arcertain amino acids. Acidic proteases used for H/D
exchange such as pepsin and proteases type XIII and
XVIII are not really specific even though pepsin was
described to cleave preferentially on the C-terminal end
of Met, Phe, and Leu [20]. Protease type XIII was
recently shown to favor cleavage after basic amino acids
[10]. Specific acidic proteases would considerably re-
duce the experimental and data analysis time by avoid-
b) hemoglobin  subunit after digestion with
ifferent pH and temperature conditions and
(b) p47phox-PX after digestion with pepsin andan (
4. Dand
e indicated in the insets.
79J Am Soc Mass Spectrom 2010, 21, 76–79 STUDY OF NEW PROTEASES UNDER DXMS QUENCHING CONDITIONSing the MS/MS peptide identification step. However,
low specificity is generally considered to be an advan-
tage for DXMS experiments because it leads to a large
number of peptides [20], which increases the spatial
resolution of the analysis. We identified here different
cleavage sites on various proteins to evaluate plasmep-
sin specificity. Digestion of bovine hemoglobin  sub-
unit, at pH 2.2 and 0 °C, with either pepsin or PSM2,
generated peptides by cleavage after eight different
types of residues. This large number of recognized
residues seems to indicate low sequence specificity. The
proportions of cleavage sites that are obtained with
PSM2 but not with pepsin were 30%, 50%, 47% and 45%
for bovine and human hemoglobin, myoglobin, and
p47phox-PX, respectively (Figures 1 and 2), showing the
complementarities of both enzymes. Finally, the aver-
age size of peptides generated by digestion of the four
proteins under DXMS conditions was 12 versus 15.5 for
pepsin and PSM2, respectively.
Conclusions
Recombinant PSM2 and PSM4 from Plasmodium falcipa-
rum were studied for the first time as new tools for
structural biology studies. PSM2 activity does not seem
to be enhanced upon maturation. However, it was still
active at pH 2.2 and 0 °C, contrary to PSM4, which
therefore cannot be used for DXMS experiments.
Moreover, in these conditions, PSM2 was able to
digest not only bovine and human hemoglobin (its
physiologic substrate), but also horse myoglobin and
p47phox-PX, chosen as representatives of nonerythro-
cytic and structurally different proteins. Despite close
sequence specificity of PSM2 with pepsin, both pro-
teases were able to generate unique cleavage sites,
thus showing their complementarity. The ability of
PM2 to work under quenching conditions and its low
substrate and sequence specificities positions it as a
potential novel protease to increase DXMS resolution,
even though it was less active than pepsin under
quenching conditions. Further studies need to be
conducted in this field to develop an increasing
number of proteolytic tools for DXMS. Comparison of
plasmepsin activity and specificity from different
Plasmodium species could be interesting, since some
of them have been described as more efficient in
acidic conditions [19].
Acknowledgments
The authors are grateful to Professor Ben M. Dunn and Melissa
Marzahn (University of Florida) for their kind gift of Plasmo-
dium falciparum clones expressing PSM2 and PSM4. The authors
also thank Daniel Kavan (Institute of Microbiology, Prague) for
the development of scripts facilitating data processing and
interpretation.Appendix A.
Supplementary Material
Supplementary material associated with this article
may be found in the online version at doi:10.1016/
j.jasms.2009.09.005.
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Investigating Alternative Acidic Proteases for H/D Exchange Coupled to Mass Spectrometry: Plasmepsin 2 but not Plasmepsin 4 Is Active Under Quenching Conditions 

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Investigating Alternative Acidic Proteases for H/D Exchange Coupled to Mass Spectrometry: Plasmepsin 2 but not Plasmepsin 4 Is Active Under Quenching Conditions

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