IMP Research Report 2002

Karl MECHTLER / Head of Facility
Jan-Michael PETERS / Scientific Coordinator
Mathias MADALINSKI / Technician
Ines STEINMACHER / Technician
PROTEIN CHEMISTRY FACILITY
The IMP Protein Chemistry Facility performs a large variety of mass spectrometry
experiments, including identification of proteins by peptide sequencing and
characterization of post-translational modifications such as phosphorylation. In addition
we develope new methods to increase sample throughput, sequence coverage and
sensitivity in mass spectrometry. Finally, our facility specializes in peptide synthesis and antibody purification.
Shotgun 2D Proteomics technology for the
analysis of post-translational
modifications in protein complexes
Tandem mass spectrometry (MS/MS) experiments are
capable of generating short stretches of sequence
of MS/MS spectral data we identify the sites and types
of modifications in large portions of the sequence of
our protein complexes. By using proteases with different
cleavage specificities we generate overlapping
peptides. The analyses of overlapping peptides
information but in most cases only a small fraction of
all generated peptides can be recovered and analyzed.
To overcome this problem we have developed the
following “shotgun” approach. First, protein complexes
are purified by immuno-precipitation and are subsequently
digested by different enzymes: one that
cleaves in a site-specific manner and one that cleaves
non-specifically. Next, the mixture of peptides is
separated by nano-HPLC or by the combination of
multiple chromatography steps (“multi-dimensional
liquid chromatography”) and is analyzed by tandem
mass spectrometry. By combining the high sensitivity
and the resolution of nanoscale multi-dimensional liquid
chromatography with the precise structural specificity
Figure: Shotgun Proteomics. Peptides were trapped on a SCX
column normally at pH~3. At this pH value, the peptides are
positively charged and the single charge is usually sufficient for the
binding. Peptides were eluted from the SCX column with increasing
ammonium acetate salt steps (from the top) onto the reversed
phase column. Peptides obtained by this separation procedure were
sequenced and analyzed using MS and the database search of
generated MS-MS spectra.
increase both the certainty with which modifications can
be identified and the likelihood of obtaining peptides,
from the “high-quality” MS/MS spectra can be obtained.
This approach is particularly important for analyzing
the complexity of multi-subunit protein complexes.
Peptide synthesis and antibody
purification
We are synthesizing about 150 peptides per year,
including an increasing number of branched peptides
containing acetylated, phosphorylated or methylated
amino acid residues. We employ a special protocol for
affinity purification of antibodies under mild conditions.
Contact: mechtler@imp.univie.ac.at
Protein chemistry facility
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