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Measuring yeast kinase activity

Title Measuring yeast kinase activity: Computational tools for generating SRM-assays to quantify phosphorylation states of proteins
Student Hannes Roest
Type MSc
Completion Date 2010-04-26
Awards ETH Medaille and Willi-Studer Prize

Protein phosphorylation is a post-translational modification that regulates many of the dynamic changes in the cell. In order to understand the cells reponse to internal and external stimuli, the quantitatie study of phosphorylation states in temporal resolution is necessary. Here we describe an approach using computational tools to generate quantitative SRM-based assays to study such phosphorylation events using tandem mass spectrometry. For this study, we focused on inferring the activity of eukaryotic protein kinases (ePKs) in yeast via their phosphorylation state.

We combined publicly available MS-generated phosphoidentification data from Phos- phoPep with sequencing data and annotations from kinase.com to generate multiple sequence alignments of the yeast kinase domains. We used evolutionary conservation together with aligned phosphosite identifications to find potential regulatory phospho- rylation sites in the activation segment of the kinase domains. The corresponding phos- phopeptides were synthesized externally and then analyzed by tandem MS in order to generate reference spectra for SRM-assay development.

In the course of this preliminary study, we developed several computational tools that were used to address the challenges encountered:

A database framework termed compep was developed in order to analyze tryptic pep- tidomes and select proteotypic peptides efficiently. It was used to generate lists of pep- tides that were synthesized based on predictions of external tools (MS1 signal intensity and retention time predictions) and criteria such as occurrence in another peptidome, occurrence in other gene loci or other splice variants from the same locus.

An interface between MySQL and the spectral search program SpectraST was imple- mented which allows the use of powerful SQL queries to create custom spectral libraries. We show the feasibility and efficacy of this approach by creating chimeric spectral li- braries that contain spectra from five different species and classifying 15 000.mzXML files by species using this library. To address the problem of the uniqueness of transitions in an SRM-assay in a com- 4 plex background, the SRMCollider was developed. This tool compares all transitions of a given peptide to all potential transitions in the selected background. It reports an interference when another transition is in proximity in the RT-Q1-Q3 space which might distort the result of the assay.

A web interface is available that allows the user to supply the query peptide with a web browser and retrieve the results directly or in an OpenOffice.org compatible CSV file.

We implemented the workflow described above, describe a theoretical analysis of each individual step and provide a computational toolkit that facilitates its execution on any set of target proteins. We developed a program that can select proteotypic peptides according to several criteria. We map the RT-Q1-Q3 space of a human peptidome and provide insights into SRM-assay development. We used the developed MySQL- SpectraST interface to create an organism-specific classifier-library. In conclusion, we provide computational framework whose potential applications extend beyond the scope of the work presented here.