Characterization
of hydrophobic peptides
by photoionization Mass
Spectrometry
Characterization of membrane proteins
is a challenging task owing to the presence
of detergent necessary to extract them.
These compounds are poorly compatible
with classical ionization methods used
in mass spectrometry. Here, the potential
of atmospheric pressure photoionization
(APPI) for mass spectrometric analysis
of membrane peptide and proteins has
been investigated and compared to classical
ionization methods. APPI allowed many
peptides distributed along the sequence
of the protein to be detected, in contrast
to electrospray ionization, which suffered
greatly from the presence of detergents.
Membrane proteins, which represent about
one-third of the genome [1], possess
a key role in cellular functions such as cell
adhesion, signal transduction, molecular
transport, endocytosis and trafficking.
Thus, this class of protein exhibits a high
potential for characterizing new biological
markers or targets for therapeutics [2].
In spite of its importance, the membrane
proteome has not been explored in
depth yet, owing to important challenges
pertaining to the characterization
of membrane proteins. The major issue
in studies on membrane proteins resides
in their high hydrophobicity, which requires
use of detergent and surfactant for their
extraction and solubilization. The intrinsic
hydrophobicity of membrane proteins
added with the presence of detergents
in membrane preparations remains
important limitations in further proteomic
and mass spectrometry analysis.
In the present work, potential of an
alternative ionization method, based
on photoionization has been investigated.
Atmospheric pressure photoionization
(APPI) has been coupled with synchrotron
radiation on the DISCO beamline [3, 4].
We focused on the tetraspanin CD9 and
the multidrug transporter BmrA. A set of
peptides from CD9, exhibiting a broad
range of hydropathicity, was investigated
using APPI and compared to electrospray
ionization (ESI)
It appeared, see figure
➊
, that most
hydrophobic peptides were hardly
ionized by ESI whereas all peptides,
including the highly hydrophobic one
that corresponds to the full sequence of
the first transmembrane domain of CD9,
were easily ionized by APPI. The native
protein BmrA purified in the presence of
the non-ionic detergent beta-D-dodecyl
maltoside (DDM) was digested in-solution
using trypsin. The resulting peptides
were investigated by flow injection
analysis of the mixture followed by mass
spectrometry. As seen in figure
➋
, upon
ESI, only detergent ions were detected
and the ionic signals from the peptides
were totally suppressed. In contrast, APPI
allowed many peptides distributed along
the sequence of the protein to be detected.
Furthermore, the parent ion corresponding
to the first transmembrane domain of the
protein BmrA was detected under APPI
conditions. Careful examination of the
APPI mass spectrum revealed a-, b-, c-
and y- fragment ions generated
by in-source fragmentation, see figure
➌
.
Those fragment ions allowed unambiguous
structural characterization of the
juxtamembrane peptide. In conclusion,
APPI–MS appears as a versatile method
allowing the ionization and fragmentation
of hydrophobic peptides in the presence
of detergent.
BIOLOGY AND HEALTH SCIENCES
56
SYNCHROTRON
HIGHLIGHTS
2013
