Krijgsveld Team
Quantitative proteomics
Previous and current research
Proteins fulfil most of the functions that are crucial for cellular survival. In addition, it is becoming increasingly clear that proteins rarely act alone, but that they constitute intricate networks, both among themselves and with other biomolecules. This system is both robust and dynamic, allowing a cell to respond to external cues and an organism to develop from an embryonic to a mature state. Our interest is to understand cellular behaviour from this perspective, realising that one needs to study proteins collectively rather than in isolation, and dynamically rather than under a static condition.
Our research is centred on quantitative proteomics, combining biochemistry, analytical chemistry, mass spectrometry and bioinformatics. Mass spectrometry coupled to liquid chromatography has matured to the stage that thousands of proteins can be analysed simultaneously, encompassing entire proteomes for relatively simple organisms (e.g. yeast) while covering decent portions of more complex proteomes (e.g. mammals). Our lab is equipped with state-of-the-art mass spectrometric technology (Thermo Orbitrap Velos, Bruker Maxis Qq-Tof) that we use for the development of quantitative proteomic techniques using stable-isotope labelling (e.g. SILAC and chemical approaches), and their application to study protein dynamics and interactions.
Our biological interest focuses on three main topics. The first is in developmental biology, with an emphasis on stem cell biology. We apply quantitative proteomics to the initial steps in haematopoiesis, studying FACS-sorted haematopoietic stem cells and multipotent progenitors isolated from mice. This should provide molecular and mechanistic clues as to how stem cells progress from a quiescent to an activated state. Knowing that proteins change in expression does not necessarily explain how this change is regulated, and therefore, secondly, we are interested in the underlying phenomenon of protein turnover, defined by protein synthesis and degradation. Using chemical biological tools, we can selectively capture proteins that are newly synthesised upon cellular stimulation, isolating them from the background of ‘old’ (pre-existing) proteins. Profiling these newly synthesised proteins quantitatively over time provides a valuable link between genome regulation and protein output. A third research topic is in the area of transcriptional regulation, where we are interested in identifying proteins that interact with DNA in a sequence-specific manner. This is complementary to the concept of chromatin IP, where we don’t ask the question where a particular protein binds to the genome, but rather what proteins bind to a defined genomic region. We have implemented the tools to identify proteins interacting with enhancer elements in fruit flies, identifying candidate regulatory proteins that are now being tested for their functionality in embryonic development.
Future projects and goals
Our future work can be divided into three major areas:
- Studying the changing proteome during gain and loss of pluripotency. We will focus on differentiation of haematopoietic stem cells and on reprogramming of fibroblasts to iPS cells.
- investigating protein turnover in mammalian cells and yeast, using various perturbations and growth conditions;
- studying protein-DNA and protein-RNA interactions to identify proteins modulating transcription and translation.
10 years of the human genome
EMBL scientists mark a decade since the first draft human genome sequence. Click on the image to play.
Duration: 6 mins.