Synchrotron
radiation - tandem
mass spectrometry
for proteomic and
structural biology
We introduce a method based upon
the coupling of mass spectrometry and
VUV-synchrotron radiation for biological
investigations. This new approach provides
information both on the primary structure
of full proteins and on the localization
of their noncovalent binding sites.
This new methodology has been developed
through the study of a human intrinsically
disordered protein, namely IB5, and its
noncovalent interactions with the tannin
procyanidin B2 3’OG.
An important part of the proteome is
composed of functional proteins that lack
ordered structure, under physiological
conditions [1]. The peculiar structures
and conformations of these proteins,
referred to as intrinsically disordered
protein (IDP), enable them to fulfill an
important repertoire of vital biological
functions. Some IDPs have the ability to
bind to different and/or several partners,
thus producing protein-ligand complexes
with distributions of stoichiometries and
conformations [2]. Investigations on such
systems by classical high-resolution
structural methods remain extremely
challenging if not intractable, owing the
flexibility and the heterogeneity of the
coexisting objects. In contrast, the unique
ability offered by mass spectrometry to
manipulate
m/z
resolved species reveals
a clear advantage when dealing with
such biological objects[2]. Tandem mass
spectrometry (MS2) is a widely used
method in structural biology in order to
determine the sequence of biopolymers,
such as proteins, DNAs, oligosaccharides...
In MS2, an ion of interest is isolated,
activated, and brought to dissociation.
The analysis of the generated fragments
provides structural information on the
precursor ion. Recently, synchrotron
radiation (SR) has been introduced as a
new activation technique for MS2[3].
In the present work, the potential of VUV-
SR to provide structural information is
probed through the study of a human IDP,
namely IB5[4]. The only described function
of this salivary proline-rich protein (PRP)
is to bind and scavenge tannins. However,
the binding site of tannins on IB5 has
never been precisely determined.
The coupling of an ion trap with the
DESIRS beamline shows that SR provides
an easy access to a wide variety
of photon activations regimes, ranging
from photodissociation (PD) to dissociative
photoionization (DPI) (figure
➊
).
The sequence coverage of IB5, obtained in
DPI regime, was higher than with classical
activation techniques (figure
➋
). It makes
SR-MS2 an efficient sequencing method.
Moreover, DPI has allowed for the first
time to determine the binding site of the
tannin, B2 3’OG, on IB5 (figure
➌
). The
comparison between the IB5
7+
and IB5•B2
3’OG
7+
MS2 spectra obtained at 16 eV has
allowed to identify and interpret more than
fifty peaks as fragments
of IB5 noncovalently bound to B2 3’OG.
It appears that all these fragments from
both N- and C-terminal series contain the
KPQGPPPPPQGG segment of the sequence,
indicating a strong interaction between
B2 3’OG and this part of the protein.
This sequence with a cluster of five
prolines very likely adopts a PPI
or a PPII helix conformation in solution.
Such structural elements are thought
to be crucial for IDPs in the binding with
their partner, as this stable segment might
provide an initial contact point. The role
of the proline clusters in PRPs sequence,
which were thought to be the tannin-
binding site on PRP, is thus unequivocally
confirmed.
Therefore, this new method should
open new perspectives in the growing
and challenging fields of proteomics,
structural biology and IDPs studies.
BIOLOGY AND HEALTH SCIENCES
60
SYNCHROTRON
HIGHLIGHTS
2013
