Optimization of phosphoproteomic workflows for challenging sample types and limited sample availability

Abstract

In this work, novel strategies for phosphoproteomic analysis for sample preparation, data acquisition and statistical analysis were presented. The advantages achieved on three different biological questions were demonstrated: 1. Rapid evolutionary adaptation of osmoregulatory pathways in Magnaporthe oryzae 2. Phosphoproteomic response of a novel therapeutic strategy against human osteosarcoma 3. Phosphoproteomic response of treated murine T-Cells To elucidate rapid evolutionary adaptation in M.oryzae, a highly robust phosphoproteomics workflow suitable for high-throughput was successfully established, that involves heat inactivation of degrading enzymes in combination with harsh cell wall lysis conditions by high concentrations of denaturing agents. To improve data quality, we successfully develop and applied an ion-mobility enhanced, data independent acquisition strategy for phosphopeptidomics, which eliminates most of the issues resulting from a high number of missing values often observed in the commonly applied data dependent acquisition mode. The Bruker timsTOF Pro 2 integrates a trapped ion mobility separation (TIMS) before actual mass spectrometry measurement, which increases the accuracy of identification of phosphopeptides and separates co-eluting and isobaric phosphopeptide isomers. The developed method yields a data quality (such as phosphosite and peptide sequence confidence) and data completeness. For comprehensive data evaluation, a already widely applied statistical analysis from transcriptome analysis, i.e. linear models instead of t-test was successfully adopted. Here, we could prove the suitability of the analytical strategy with comparing osmoregulatory response of wild type samples with previously acquired knowledge from literature. The developed strategy was applied to a time-course experiment of osmoregulatory deficient sample set comprised from MoHog1 deleted mutants. In addition, a highly sensitive phosphopeptide enrichment method was developed to meet the needs of clinical proteomics and in-vivo animal experiments, that in most cases only yield very minute protein amounts which is not sufficient for classical protocols using TiO2 phosphopeptide enrichment in spin-tips. The workflow displayed high accuracy and reproducibility requiring only 25 µg total protein and was applied to characterize the phosphoproteome of human osteosarcoma and murine T-Cells.