Krijgsveld Team
Quantitative proteomics
Generalized experimental workflow for the quantitative analysis of protein expression in treated vs. untreated cells. Cells that are labeled with heavy isotopes (SILAC) are mixed with non-labelled cells that have received a treatment (e.g. induction to differentiate). Quantitative analysis of peptide extracts by mass spectrometry results in the identification of thousands of peptides whose expression level can be quantified over time.
Previous and current research
Our research is centred on mass spectrometry-based proteomics.Mass spectrometry coupled to liquid chromatography has matured to the stage 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 targeting 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 approach combines biochemistry, analytical chemistry, mass spectrometry and bioinformatics. Specifically, we use 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, nanoflowHPLC, peptide isoelectric focusing (IEF)). Finally, in our newly equipped lab we have state-of-the-artmass spectrometers (aMaxis electrospray Qq-Tof and HCT ion trap) as well as bioinformatic data flows for protein identification and quantitation.
We have three main topics of biological interest. One is developmental biology, with a particular focus on stem cell biology. Over the past years, we have studied the dynamics of protein expression during differentiation of embryonic stemcells.We have also studied how protein phosphorylation changes as ES cells leave the pluripotent state, giving insight in (in)activation of phosphorylation networks. Currently, we are currently expanding our technology to iPS cells.
A second topic is in the area of transcriptional regulation.We are interested in the interaction of proteins with DNA, both with regards to the identification of transcription factors, along with their associated proteins, and to specific regulatory elements. More broadly, we are investigating the dynamics of chromatin, with a particular interest in the proteins that are not considered to belong to the core, but which might play an important regulatory role in the response to changing environmental conditions.
In a third research line, we investigate protein synthesis and turnover in yeast and mammalian cells, which is a missing link in our understanding of protein regulation.
Future projects and goals
Our future work can be divided into three major areas:
- We will apply quantitative proteomic techniques for global analysis of protein expression during embryonic development (particularly stem cells).
- We will focus on the identification of protein-protein and protein-DNA complexes to understand regulatory principles of transcription under various biological conditions.
- We will develop biochemical and bioinformatic tools to analyse and understand protein turnover.