Theme Leaders: Ron Heeren (AMOLF) and Albert Heck (UU)

Background

Mass spectrometry is rapidly maturing as a powerful tool for proteomics applications as new sample-preparation approaches, instrumentation developments and bioinformatics tools for analyzing data become increasingly available. No one other technology can now match both the throughput and the molecular information content that mass spectrometry provides. Instrument manufacturers have made a tremendous effort in the last decade and have come up with innovative commercially available instrumentation which continuously allow more speed, sensitivity, specificity for standard high-throughput proteomics. In this part of the NPC II program we aim to make progress beyond the standard mass spectrometry based analysis. We seek to further widen the scope of mass spectrometry based analysis in proteomics. Therefore, at AMOLF Heeren et al. have developed a platform for spatially resolved mass spectrometric imaging, which in this program will be further developed and implemented in NPC II. These efforts are linked with two european programs on nanoparticle based imaging on cells and tissue (EU-Meditrans) and the development of innovative imaging MS technology, and the manufacturers Bruker, Kratos/Shimadzu and ULVAC-PHI. At Utrecht Heck and Altelaar will further explore the possibilities of the emerging novel ion activation technique ETD, in collaboration with Bruker, Agilent and Thermo. In this part of the program the Bioinformatics theme will have a strong input as well, as novel algorithms need to be implemented. Finally, ongoing efforts in the direct analysis of intact protein complexes by the Heck group will be linked to NPC II via this program.

Approach

E3.1 Mass Spectrometry Imaging: Imaging mass spectrometry is developed and combined with new proteomics tools to improve local peptide and protein identification and visualization directly from unstained and unlabelled thin tissue sections. The coupling of ion mobility separation with imaging MS will be implemented realizing innovative possibilities to separate nominally isobaric species prior to mass analysis. This will result in the visualization of new spatial features directly from biomedical tissue. In addition new protocols for local tissue digestion will be developed and combined with tandem MS to improve the protein identification capabilities during imaging MS. The strategies will be combined into a new approach for 3-dimensional MS based imaging of small xenografted tumors and organs. The rendered 3D molecular images will be combined and compared with high resolution MRI 3D images rendering a new tool for the visualization of molecular anatomy. New bioinformatrics based visualization software tools will be developed for this purpose. Strong existing collaborations with Bruker and the BSIK-project Virtual Laboratory for e-sciences will be utilized and strengthened in this project.

E3.2 Electron transfer dissociation (ETD): To improve protein identification and mapping of post-translational modifications of we will develop and use ETD. Therefore, novel platforms developed by Thermo-Fisher, Bruker and/or Agilent given to us via -test-site agreements, will be used. ETD is a novel dissociation technology that may be used to fragment peptides. It is an alternative for more standard collision induced dissociation (CID). ETD requires optimization of sample preparation and data-analysis compared to the present used technique. Analysis of PTMs, such as phosphorylation and glycosylation, is difficult with standard CID since the modification is often labile, resulting in little to no peptide sequence information. ETD may be helpful in these areas. A benefit of ETD is its ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another. As in general the peptides/proteins delivered to the ETD ion trap will be bigger, we need to optimize in this project not only the digestion (with different proteases), but also the LC chromatography, which may be RP C4 or C8, or alternatively SCX, HILIC, TiO2 etc.

E3.3 Macromolecular Mass Spectrometry: The function of proteins largely depends on their interactions, which depends on their structure. Mass spectrometry based proteomics has created a plethora of information about protein-protein interaction networks. However, such data does not generate information on exact protein complex composition, structure and dynamics. New mass spectrometry based technologies especially targeted at the analysis of these latter features are surfacing allowing applications for investigating intact supramolecular protein (sub)-complexes. The analysis of intact non-covalently bound proteins assemblies has been termed native mass spectrometry. In this part of the program native mass spectrometry will be further developed and applied to a number of systems with high molecular weights (i.e. a few million Da). Direct links with NPC II projects focused on for instance the proteasome and transcription complexes are foreseen.

Deliverables

• Top-down analysis of protein complexes, revealing stoichiometry and topology
• Implementation of ion mobility for analysis of protein conformation
• Novel ETD based tools for de novo sequencing
• RP-TiO2-ETD and HILIC-TiO2-ETD platform for analysis of PTMs
• Software algorithms for ETD analysis of PTMs
• Coupling of ETD and Orbitrap analysis as advanced proteomics platform
• Implementation of Ion mobility for MALDI based direct tissue analysis
• Innovative high speed (3D)MALDI MS imaging tools
• New statistical analysis, validation and visualization algorithms for imaging MS
• Combined proteomics and image analysis for unlabelled tissue sections