Publikationen

Label-free proteomics by data-dependent acquisition enables the unbiased quantification of thousands of proteins, however it notoriously suffers from high rates of missing values, thus prohibiting consistent protein quantification across large sample cohorts. To solve this, we here present IceR (Ion current extraction Re-quantification), an efficient and user-friendly quantification workflow that combines high identification rates of data-dependent acquisition with low missing value rates similar to data-independent acquisition. Specifically, IceR uses ion current information for a hybrid peptide identification propagation approach with superior quantification precision, accuracy, reliability and data completeness compared to other quantitative workflows. Applied to plasma and single-cell proteomics data, IceR enhanced the number of reliably quantified proteins, improved discriminability between single-cell populations, and allowed reconstruction of a developmental trajectory. IceR will be useful to improve performance of large scale global as well as low-input proteomics applications, facilitated by its availability as an easy-to-use R-package.

High-throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples from a variety of sources, including fresh-frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phase-enhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96-well format. AutoSP3 performs unbiased protein purification and digestion, and delivers peptides that can be directly analyzed by LCMS, thereby significantly reducing hands-on time, reducing variability in protein quantification, and improving longitudinal reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low-input samples, reproducibly quantifying 500–1,000 proteins from 100 to 1,000 cells. Furthermore, we applied this approach to a cohort of clinical FFPE pulmonary adenocarcinoma (ADC) samples and recapitulated their separation into known histological growth patterns. Finally, we integrated autoSP3 with AFA ultrasonication for the automated end-to-end sample preparation and LCMS analysis of 96 intact tissue samples. Collectively, this constitutes a generic, scalable, and cost-effective workflow with minimal manual intervention, enabling reproducible tissue proteomics in a broad range of clinical and non-clinical applications.

Computational and mathematical models are key to obtain a system-level understanding of biological processes, but their limitations have to be clearly defined to allow their proper application and interpretation. Crowdsourced benchmarks in the form of challenges provide an unbiased assessment of methods, and for the past decade, the Dialogue for Reverse Engineering Assessment and Methods (DREAM) organized more than 15 systems biology challenges. From transcription factor binding to dynamical network models, from signaling networks to gene regulation, from whole-cell models to cell-lineage reconstruction, and from single-cell positioning in a tissue to drug combinations and cell survival, the breadth is broad. To celebrate the 5-year anniversary of Cell Systems, we review the genesis of these systems biology challenges and discuss how interlocking the forward- and reverse-modeling paradigms allows to push the rim of systems biology. This approach will persist for systems levels approaches in biology and medicine.