Quantitative analysis of mass spectrometry proteomics data : Software for improved life science

The rapid advances in life science, including the sequencing of the human genome and numerous other techiques, has given an extraordinary ability to aquire data on biological systems and human disease. Even so, drug development costs are higher than ever, while the rate of new approved treatments is historically low. A potential explanation to this discrepancy might be the difficulty of understanding the biology underlying the acquired data; the difficulty to refine the data to useful knowledge through interpretation. In this thesis the refinement of the complex data from mass spectrometry proteomics is studied. A number of new algorithms and programs are presented and demonstrated to provide increased analytical ability over previously suggested alternatives. With the higher goal of increasing the mass spectrometry laboratory scientific output, pragmatic studies were also performed, to create new set on compression algorithms for reduced storage requirement of mass spectrometry data, and also to characterize instrument stability. The final components of this thesis are the discussion of the technical and instrumental weaknesses associated with the currently employed mass spectrometry proteomics methodology, and the discussion of current lacking academical software quality and the reasons thereof. As a whole, the primary algorithms, the enabling technology, and the weakness discussions all aim to improve the current capability to perform mass spectrometry proteomics. As this technology is crucial to understand the main functional components of biology, proteins, this quest should allow better and higher quality life science data, and ultimately increase the chances of developing new treatments or diagnostics

Development of a combined chemical biology tool using synthetic probes and tandem mass spectrometry to elucidate heat shock protein 90 KDA's C-terminal- domain binding sites

Heat shock protein 90 kDa (Hsp90) has long been an appealing drug target for cancer treatment due to its pivotal role in conformational maturation and refolding of many proteins directly related to malignant progression. Previously various Hsp90 N-terminal domain (NTD) inhibitors have progressed to clinical trials in patients, unfortunately these studies were suspended due to high toxicity-related issues. On the other hand, the lack of a high-resolution crystal structure of the C-terminal domain (CTD) of human Hsp90 hampers the discovery of next generations of CTD inhibitors and presents challenges for further structure-based drug design. This study attempts to provide validated evidence for the Novobiocin binding site in yeast Hsp90 CTD by combinatorial use of photochemistry-based affinity protein labelling and analytical tools including NMR and mass spectrometry. This research on completion will shed light on the development of Hsp90 CTD inhibitors, which provide an alternative route to effective development of cancer drug candidates based on inhibition/interruption of Hsp90. The use of ligands conjugated to photoaffinity labelling (PAL) reagents for chemical modification of active site residues can circumvent limitations such as lack of complete crystal structure in CTD region, allowing detailed information on the sites of protein-ligand interactions to be acquired through high resolution MS and MS/MS experiments. Bifunctional PAL compounds incorporated a diazirine group and a novobiocin component, based on a previously reported glucosyl-novobiocin scaffold, which has been reported to demonstrate 200-fold increase in CTD inhibitory effects towards Hsp90 activities compared to that of novobiocin. The synthesis of the diazirine group we developed alleviated the high temperature and highpressure conditions reported in literature. Also, in the functionalisation of the affinity providing novobiocin was based on the Williamson ether mechanism and resulted in regioisomers. An isomer due to rearrangement from coumarin to flavone in novobiocin core was identified by advanced NMR, and this phenomenon has recently been reported by our group. Further detailed studies of photoactivation revealed the physiochemical properties of these probes, and such information was very critical for the ease of data analysis in the protein labelling stage. These novobiocin derivatives were activated under UV irradiation in the presence of Hsp90 proteins and adducts were detected using detailed MS and MS/MS analysis. The protein-PAL complexes were identified using MALDITOF MS and related techniques to confirm the labelling of yeast Hsp90. Complexes were further subjected to enzymatic digestion and result products were analysed by tandem MS (Orbitrap MS). Currently, the peptide mapping of chemically labelled proteins and localisation of chemically labelled amino acid are frequently carried out by manual analysis, which is time-consuming and requires relatively high level of modified peptide in the enzymatic digestion mixture. In this project, by using PeptideShaker with custom modification of the pre-identified mass difference, the modified peptide (at W585) was identified at relatively low protein labelling yield. W585 in yeast Hsp90 has been recently reported to be related to client remodelling coincident with stabilizing yHsp90 in an open conformation. The peptide region in the vicinity of this residue was further analysed and the binding pocket was identified by correlating the experimental results to ICM Pro based molecular docking, providing a direct visualisation of the yeast Hsp90 CTD binding site. Even these probes demonstrated low affinity towards human Hsp90, given the close homology between Hsp90 tertiary structures from yeast and human origins, the combination of both experimental and computational results obtained yeast Hsp90 labelling is expected to cast light on the understanding of Hsp90-novobiocin binding rationale, and contribute to future structure based drug design (SBDD) in the search for more potent Hsp90 C-terminal domain inhibitors

specL - An R/Bioconductor package to prepare peptide spectrum matches for use in targeted proteomics

MOTIVATION Targeted data extraction methods are attractive ways to obtain quantitative peptide information from a proteomics experiment. SWATH (Sequential Window Acquisition of all Theoretical Spectra) and DIA (Data Independent Acquisition) methods increase reproducibility of acquired data because the classical precursor selection is omitted and all present precursors are fragmented. However, especially for targeted data extraction, MS coordinates (retention time information precursor and fragment masses) are required for the particular entities (peptide ions). These coordinates are usually generated in a so called discovery experiment earlier on in the project if not available in public spectral library repositories. The quality of the assay panel is crucial to ensure appropriate downstream analysis. For that, a method is needed to create spectral libraries and to export customisable assay panels. RESULTS Here, we present a versatile set of functions to generate assay panels from spectral libraries for use in targeted data extraction methods (SWATH/DIA) in the area of proteomics. Availability: specL is implemented in the R language and available under an open-source license (GPL-3) in Bioconductor since BioC 3.0 (R-3.1) http://www.bioconductor.org (Trachsel, Panse, and Grossmann, 2015). A vignette with a complete tutorial describing data import/export and analysis is included in the package and can also be found as supplement material of this paper. CONTACT [email protected],[email protected]