From Data to Discovery: Maximizing Insights into Molecular Mechanisms with Spectronaut® - Biognosys

From Data to Discovery: Maximizing Insights into Molecular Mechanisms with Spectronaut®


Probing Protein Interactome Dynamics Using an Experimental Library of Protein Complex Interfaces

Enhancing Single-cell Proteomics through Tailored Data-Independent Acquisition and Advanced Data Processing

Cathy Marulli (Picotti Lab, ETH Zurich)

Methods to systematically monitor the dynamics of protein complexes are needed. We introduce FiLiP-MS, a structural proteomics workflow for global profiling of protein binding interfaces directly in cell lysates. We applied serial ultrafiltration and limited proteolysis-coupled mass spectrometry (LiP-MS) to generate a library of peptides at protein binding interfaces, based on protease accessibility differences between complex-bound and monomeric forms. We used the generated yeast library (6441 candidate interface markers from 1086 proteins at 5% FDR) to probe interactome changes in yeast grown under DNA-replication stress. We captured known and novel protein complex rearrangements, identified several rearrangements that depend directly or indirectly on activity of the SAGA acetyltransferase Gcn5 and discovered a link between Gcn5 activity and the regulation of P-bodies. FiLiP-MS enables protein complex dynamics to be monitored proteome-wide upon any perturbation, at high throughput and with peptide-level resolution, providing both global and molecular views of a system under study.

Erwin Schoof (DTU)

Single-cell proteomics by Mass Spectrometry (scp-MS provides valuable insight into distinct cell-states and signaling patterns present across cell populations. However, carrying out proteome profiling from the limited amount of material encapsulated in a single cell presents significant challenges. Although great strides forward have been facilitated by extensive efforts to minimize sample losses during sample preparation and clever data acquisition approaches, further development is needed to increase the quantifiable proteome depth. Accordingly, we carried out a comprehensive analysis of orbitrap-based data-independent acquisition (DIA) for limited material proteomics. We found a fundamental difference between optimal DIA methods for high- and low-load samples. To build further upon our findings, we adopted a high-resolution MS1 quantification approach, accommodating long injection times and high resolution, while keeping scan cycle times low for robust quantification. Additionally, the improvements made in recent Spectronaut updates have major impact on the quantifiable proteins from single cells. In conclusion, we present a complete experimental scp-MS workflow, combining DIA with simple single-cell sample preparation and the latest chromatographic and computational advances to showcase our developments by profiling real single cells.

Short Bio Cathy Marulli:

Cathy Marulli is a third-year PhD student in Prof. Paola Picotti’s lab at ETH Zurich, where she is developing novel structural proteomics methods. She did her Bachelor’s in “Interdisciplinary Sciences” and received her Master’s in “Systems Biology” at ETH Zurich in 2021. She received the ETH medal 2021 for the best master thesis which laid the foundation for the project she is going to present at EuPA.

Short Bio Erwin Schoof:

Erwin is an Associate Professor at the Technical University of Denmark, where he heads the Cell Diversity Lab. His group focuses on developing and applying single-cell proteomics and multi-omics approaches to complex biological systems in order to get a thorough understanding of cell differentiation. He was one of the very early adopters of single-cell proteomics by MS and continuously seeks new improvements. Today, he will tell us about the most recent iteration of his experimental workflow enhancing single-cell proteome coverage and reproducibility, also relying on Spectronaut innovations.

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