Theme leaders: Shabaz Mohammed (UU) and Rainer Bischoff (RUG)

Background

In high-throughput proteomics often protein fractionation is omitted and the entire sample is digested to create a pool containing hundreds of thousands of peptides which is getting close to being directly accessible to mass spectrometry for peptide and then consequently protein identifications. The complexity of the analytes demands selective, fast and sensitive instrumentation alongside constant method development in sample preparation, fractionation, pre-concentration, chromatographic separation and detection. In the last few years a constant growth in proteome coverage has been achieved but maintaining a dynamic growth will require not only further optimization of current technologies but will also require developing separation/enrichment strategies which incorporate and exploit the physicochemical nature of peptides such as those that are phosphorylated, acetylated or modified in any other form.

In this theme there will be a concerted effort to address three main areas; performance enhancement, new separation/enrichment technologies and implementation of chip based methodologies.  The research can be segmented further into 6 number of discrete projects that will be overseen by 3 participating partners (UU, RUG and NVI) in collaboration with leading proteomics manufacturers. Certain projects represent continuation and further application of successful strategies developed in NPC1 (e.g. the TiO2 phosphopeptide enrichment, protein LC) while others represent projects with both academic and industrial manufacturers input. All maintain aspects that require close co-operation and collaboration thus maintaining an internal cohesion and perspective.

Approach

Regarding enhancing the performance of current technologies what is required is down-scaling the HPLC components to allow separations to true nanolitre flow rates, an order lower than current state of the art.  Such progress will allow sensitivity and dynamic range of detectable components to be increased via near-suppression free electrospray ionization which is only possible at those flow-rates (projects E1.2 and E1.5).  Increase of separation power will be provided column elongatation and smaller particles and by continuing improvements in SCX through tighter integration with targets.  For example, interest in vivo proteolytic events requires detection of protein termini and so applying amine blocking chemistry combined with appropriate SCX conditions enrichment and fractionation can be achieved. Such enhancements will be applied to immunoproteomics or more specifically B and T-cell epitope mapping as it craves increased powers of peptide detection and will represent an ideal and highly relevant subject.

Successful strategies from NPC1 included HILIC separations and TiO₂ based phosphopeptide enrichment.  However, these technologies still represent fledgling ideas that require much more exploration and so are the launch pad for projects E1.1 and E1.2.  Part of the proteomics wide acceptance of TiO₂ and HILIC was related to its ability to produce results from low amounts of material.  However, an essential part of most experiments require quantitation, a technique that still places limitations on sample amounts.  This project will also develop automated on-column labeling for quantiation strategies that will reduce sample handling, improve ease of use and also allow material levels to be reduced. These advances will amongst others be directly applied to projects on the quantification of proteasome sub-units in different mice-tissues, which is part of the theme Chemical Approaches to Proteome Biology.

A central aspect of all enabling technologies is accessibility to a wide community and thus an entire project will focus on incorporate our techniques into chip form, with the participation of leading manufacturers, allowing non-experts to benefit from our novel enabling technologies.

In this theme is also incorporated another continuation of a successful strategy developed in Groningen by Veenhoff to enrich for specific organelles prior to proteomic analysis. In particular the high-quality isolation of the yeast nucleus as well as the nuclear envelopes and pore complexes thereof, will be further optimized, explored and applied in NPC2. This E1.4 project is part of this theme as it requires extensive and high-expertise separation and enrichment strategies. The developed tools are directly beneficial to yeast projects in the Proteome Biology of Micro-organisms theme and other yeast proteome projects within and outside/related to the NPC program.

In E1.3 affinity enrichment techniques will be further developed. Notably our approach to the activity-based profiling of proteases will form part of this project. Up to now we have focused our attention on metalloproteases (MMPs and ADAMs) as part of the STW-funded projects GPC 6150 and 8008 (link to the Chemical Proteomics theme of NPC2; collaboration with Hermen Overkleeft; Leiden University). Enrichment will be automated and coupled on-line to a flow-through trypsin reactor combined with chip-LC-MS/MS. This prototype of an instrumental set-up will be used to implement other affinity enrichment strategies, for example, using lectins or metaloxides (see project E1.2). The system will be applied to the analysis of body fluids (e.g. epithelial lining fluid from COPD patients) to investigate the relation between active proteases, disease progression and response to therapy (TIP project T1-108). Finally in E1.6 analytical tools are developed to investigate the the human erythrocyte proteome (i.e. red blood cells). Using a successful depletion technique introduced in NPCI, we will set-up a protein fractionation technique based on multidimensional chromatography. In combination with quantification methods such as fluorescent- and/or in vitro isobaric mass labeling, we will enable the differential analysis of protein levels in RBCs from healthy individuals and patients with erythrocyte disorders. This work is performed in collaboration with the department of Clinical Chemistry and Haematology at the UMCU that has a long standing track record in hereditary red cell research and is an international reference site for red cell diagnostics.

Deliverables

• Efficient selective proteasome sub-unit inhibitors and ABPs
• True nanolitre flow LC-MS for near-suppression free attomole sensitivity proteomics
• Novel separation methods for targeted sub-peptidome fractionation e.g. efficient selective purification of Protein Termini
• Efficient facile on-line chemical labeling strategies for quantitation
• Novel microfluidic devices (chips) allowing multidimensional separations or enrichment, labeling and quantitation.
• An integrated total analysis system for affinity enrichment, trypsin digestion and nano(chip)LC-MS/MS for the high-sensitivity, functional profiling of proteases
• Improved methods for organelle separation that allow time-dependent quantitative proteomics, e.g. cell cycle-dependent analysis of nuclear envelope composition.
• Multidimensional Protein Separation technologies
• Efficient depletion of high abundant proteins in Blood and Tissue