2012

2011

2010

2009

Bobby currently supervises the following students:

• Bo-Tao Xin
• Guillem Paniagua Sorriano
• Nan Li

Chemical tools for chemical biology/functional proteomics

Schematic overview of the functional proteomics pipeline

The fascinating world of proteins is a 4-dimensional space defined by protein sequence, 3D structure, expression level and function. From the available draft genomes protein sequence can be predicted, however, the link between protein ID and function can only be determined empirically. This tedious process of annotation can be greatly accelerated by combining the power of organic chemistry, bio-chemistry and LC-MS based proteomics analysis as functional proteomics.

Design and synthesis of chemical entities that can target specifically and bind covalently to the active site of enzymes is one of the core business of our department. In a 2003 key publication [1], our functional proteomics concept of chemistry in living cells and 2-step labeling strategy for enzyme profiling was presented. The proteasome [2] was chosen as model enzyme because it is essential for life, it is present in the cytoplasm and nucleus and it is involved in the regulation of many cellular pathways. 2-step molecular probes that target the proteasome in living cells contain a war head (electrophilic trap that binds to the active site peptide), an oligo-peptide proteasome homing sequence, a spacer and a ligation handle. The ligation handle is a small bio-orthogonal group (azide or acetylene) that doesn’t interact with any bio-molecule. This construct is fed to life cells, proteasomes are targeted, cells are lysed and an affinity tag, usually biotin, is installed via a water compatible chemical ligation [3,4].

In 2004, my first job was to set up the biochemistry and the proteomics facility that will enrich, analyse and identify the proteins captured by our molecular probes. As MSc and PhD in bio-pharmaceutical sciences and postdoc in cell biology and genetics it was fun to set up cell culture models and introduce SDS-PAGE and western blot analysis protocols in the department. Recently we acquired a state-of-the-art LTQ-Orbitrap mass spectrometer that accurately determines the exact mass of peptides with femtomolar (10-15 M) sensitivity and produces a staggering 6 MS/MS fragmentation scans per second. Sequest and Mascot database search engines are used to identify the enriched proteins.

We have several ambitious future plans with both synthetic and proteomics challenges. Synthesis of new warheads, new molecular probes and synthesis of heavy, stable isotope (13C, 15N) labeled, probes will facilitate the relative quantification of enzymatic activities in a ICAT-like fashion [5]. For example, quantifying the proteasome activity in cancer metastasis cells can be achieved by treating cancer cells with heavy labeled probes and normal cells with light probes. Cancer and normal cells are mixed, lysed and run through the functional proteomics pipe line. Because heavy and light labeled peptides elute simultaneously, they are measured in the same MS scan and their relative abundance can be determined simply from the peak-height. If the heavy labeled peptides are more abundant that the light peptides this will strongly indicate that cancer cells express a higher proteasome activity than normal cells.

Another challenge is to find mass spectrometry protocols to determine the peptide sequence of the active site with our molecular probe construct attached to it. This exotic mix of biological peptide and synthetic probe shows aberrant MS/MS fragmentation compared to fragmentation of a peptide only. Finally, we aim to expand the functional proteomics pipe-line to a robot driven high-throughput platform for routine analysis of many samples and enzymatic activities.