Research
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Architecture and dynamics of protein
complexes
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It is a widely accepted concept in biology that proteins exert their
function in cells while being part of larger functional complexes. Our
resarch focuses on the characterization of protein complexes and their
dynamics during cell differentiation, cell cycle and upon stimulation by
external stimuli like UV irradiation, and how this influences protein
functioning.
Usually, the protein of interest is immunoprecipitated either by using in
vivoprotein tagging or by highly specific antibodies under relatively mild
conditions. Subsequently, the complex partners are separated by SDS-PAGE,
in-gel digested (or directly digested from the beads) and identified by
mass spectrometry. One of the major challenges is to differentiate between
bona fide interaction partners of your protein of interest that make up the
core protein complex and 'second-shell' interactors and non-specifically
binding proteins. By gradually changing the conditions under which the
protein complexes are isolated from cellular extracts, we are able to get a
rough idea about the relative strength of protein-protein
interactions.
Posttranslational modifications
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After translation, the posttranslational modification of particular amino
acid residues extends the range of functions of the protein by attaching to
it other biochemical functional groups such as acetate, phosphate, methyl,
ubiquitin, various lipids and carbohydrates, by changing the chemical
nature of an amino acid or by making structural changes, like the formation
of disulfide bridges. We try to answer questions such as how amino acid
modifications can affect protein complex composition and how they are
involved in cellular functioning. Mass spectrometry has proven to be an
excellent tool for the characterization of posttranslational modifications
such as acetylation, methylation, phosphorylation, ubiquitylation,
ISGylation, and so on. In order to analyze such modifications by mass
spectrometry, it is often necessary to enrich for proteolytic peptides
bearing the modification of interest. For example, phosphorylated peptides
can be enriched for using a protocol that involves trapping such peptides
on TiO2 chromatographic column material. Furthermore, we are developing
tools for the quantitative analysis of dynamic posttranslational
modifications and try to answer questions such as how the phosphorylation
state of a protein complex depends on its position in the cell cycle and
how modifications on histones and nucleosome associated proteins change
after DNA damage. Recently, we have characterized by mass spectrometry
multiple sites of ubiquitylation on Tramtrack, a protein which was found to
be the major substrate of the ubiquitin specific protease UBP64 in
Drosophila (link to MCB paper).
18O and SILAC labeling for quantitative proteomics
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We have optimized and implemented a method that uses 18O labeling of
tryptic peptides to account for quantitative differences in protein levels
between different samples. When all proteolytic peptides in e.g. a control
sample are labeled with 18O, we can easily differentiate between real
interactors of our protein of interest and non-specific background
proteins: in the case of background proteins, both heavily-labeled and
non-labeled peptides will appear in the same mass spectrum. In contrast, if
the protein is specific for the sample, only the light variant will be
observed. We have successfully applied this method for quickly subtracting
background proteins from real interactors in co-immunoprecipitations.
Another way to study changes in the proteome in a quantitative manner is
by using Stable Isotope Labeling of Amino acids in Cell culture (SILAC). In
this approach, one sample originates from cells that were grown under
normal conditions and the other sample is from cells that were grown in
media containing amino acids that were labeled with heavy carbon (13C),
nitrogen (15N) and/or hydrogen (D) isotopes. One of our reserach projects
involves investigation of changes in histone modifications after DNA damage
and we use SILAC to identify and quantitate such changes.
Characterization of complex protein mixtures
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For highly complex mixtures like complete cellular lysates, nuclear
extracts or body fluids, multi-dimensional separations of proteins and/or
proteolytic peptides should be applied to get the highest number of protein
identifications. Before reversed-phase separation on a C18 column in the
typical nanoflow LC-MS/MS setup that we use in the lab, peptides can be
separated and fractionated by either strong cation exchange (SCX), strong
anion exchange (SAX) or iso-electric focusing (IEF). Usually, 10 to 20
fractions for each preparation are collected and subjected to nanoflow
LC-MS/MS. Also, SDS-PAGE separation of proteins is used often as a first
fractionation step.
Generally, for complex biological mixtures, multiple separation steps are
required to obtain as many protein identifications as possible. We are
testing several combinations of fractionation steps in our approach to
study the proteome of human follicular fluid.
Bioinformatics
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We are currently developing tools for proteomics data storage, management
and retrieval, and for integrative analysis, including data mining and
visualization. Such databases can be questioned by using web-based
interfaces to give information about the experimental design as well as the
outcome of such experiments, like the composition of protein complexes,
gene ontology information of identified proteins and overlap with protein
interaction experiments in different organisms. See Bioinformatics section
for more detailed information.
