ayout 1 - EMBL Grenoble

EMBL Research at a Glance 2009
Jeroen Krijgsveld
PhD 1999, University of
Amsterdam, The Netherlands.
Postdoc at Utrecht University,
The Netherlands and Harvard
Medical School, Boston, USA.
Assistant Professor, Utrecht
University, The Netherlands
Team leader at EMBL since
2008.
Quantitative proteomics
Previous and current research
Our research is centred around mass spectrometry-based proteomics. Mass spectrometry coupled
to liquid chromatography has matured to the extent that thousands of proteins can be identified,
so for simple organisms we can now start thinking of studying entire proteomes. For more
complex organisms, including humans, complementary strategies are still required targeted at
specific classes of proteins/peptides by pre-fractionation or selective enrichment. Our interest is
in the expansion of this ‘proteomic toolbox’ and its integration into the larger domains of molecular
biology and biochemistry. We focus particularly on quantitative techniques in mass spectrometry
using stable isotope-labelling to study protein dynamics in a biological context.
Our tools include stable isotope-labelling for protein quantitation (e.g. SILAC and chemical approaches),
enrichment strategies for specific classes of proteins (membrane proteins, phosphopeptides)
and separation techniques for detailed coverage of even very complex samples (SCX,
nanoflow HPLC, peptide isoelectric focussing (IEF)). Finally, in our newly equipped lab we have
state-of-the-art mass spectrometers (a Maxis electrospray Qq-Tof and HCT ion trap) as well as
bioinformatic data flows for protein identification and quantitation.
Our biological interest is in developmental biology and in the underlying mechanisms in transcriptional
regulation. Currently we are studying the dynamics of protein expression during fruit
fly development by comparing distinct embryonic stages. To do this in a quantitative fashion, we have developed tools to metabolically label
flies with stable isotopes. In conjunction with gene expression data this provides mechanistic insight in regulation of individual or functionally
related proteins and helps explain regulatory mechanisms in early embryonic development. Another major effort is in application of
quantitative proteomic techniques to stem cell pluripotency and differentiation. Currently we are studying the dynamics of phosphorylation
upon inducing differentiation of human stem cells (see figure).
This should benchmark an initial blueprint of the
phosphorylation network activated during differentiation,
while providing a starting point for further exploration in
other cell lines and organisms.
Future projects and goals
Our future work can be divided in three major areas:
• We will apply quantitative proteomic techniques
for global analysis of protein expression during
embryonic development (stem cells, fruit flies).
• We will focus on the identification of protein-protein
and protein-DNA complexes to understand
regulatory principles of transcription under various
biological conditions.
• In addition we will use targeted mass-spectrometric
approaches tracking defined sets of proteins
over time employing MRM (multi-reaction monitoring)
experiments. We will be specifically focussed
on applying this to the dynamics of
posttranslational modifications, particularly phosphorylation.
Experimental workflow for the quantitative study of protein phosphorylation
during stem cell differentiation. hESC that are labelled with heavy isotopes
(SILAC) are induced to differentiate and mixed with non-labelled hESC.
Selective isolation of phosphopeptides and mass spectrometric
analysis results in the identification of thousands of peptides whose
phosphorylation status can be quantified over time.
Selected references
Gouw, J.W., Tops, B.B., Mortensen, P., Heck, A.J. & Krijgsveld, J.
(2008). Optimizing identification and quantitation of 15N-labeled
proteins in comparative proteomics. Anal Chem., 80, 7796-7803
Van Hoof, D., Pinkse, M.W., Oostwaard, D.W., Mummery, C.L.,
Heck, A.J. & Krijgsveld, J. (2007). An experimental correction for
arginine-to-proline conversion artifacts in SILAC-based quantitative
proteomics. Nat. Methods, , 677-678
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Van Hoof, D., Passier, R., Ward-Van Oostwaard, D., Pinkse, M.W.,
Heck, A.J., Mummery, C.L. & Krijgsveld, J. (2006). A quest for human
and mouse embryonic stem cell-specific proteins. Mol. Cell
Proteomics, 5, 1261-1273
Krijgsveld, J., Ketting, R.F., Mahmoudi, T., Johansen, J., Artal-Sanz,
M., Verrijzer, C.P., Plasterk, R.H. & Heck, A.J. (2003). Metabolic
labeling of C. elegans and D. melanogaster for quantitative
proteomics. Nat. Biotechnol., 21, 927-931