Biology subjects

Wallmann, G.

Publications and source records attributed to Wallmann, G..

3 recordsLinked to original sources

bioRxiv2023

SPARCS, a platform for genome-scale CRISPR screening for spatial cellular phenotypes

Forward genetic screening associates phenotypes with genotypes by randomly inducing mutations and then identifying those that result in phenotypic changes of interest. Here we present spatially resolved CRISPR screening (SPARCS), a platform for microscopy-based genetic screening for spatial cellular phenotypes. SPARCS uses automated high-speed laser microdissection to physically isolate phenotypic variants in situ from virtually unlimited library sizes. We demonstrate the potential of SPARCS in a genome-wide CRISPR-KO screen on autophagosome formation in 40 million cells. Coupled to deep learning image analysis, SPARCS recovered almost all known macroautophagy genes in a single experiment and discovered a role for the ER-resident protein EI24 in autophagosome biogenesis. Harnessing the full power of advanced imaging technologies, SPARCS enables genome-wide forward genetic screening for diverse spatial phenotypes in situ.

systems biology↗

bioRxiv2023

Data-Driven Optimization of DIA Mass-Spectrometry by DO-MS

Mass spectrometry (MS) enables specific and accurate quantification of proteins with ever increasing throughput and sensitivity. Maximizing this potential of MS requires optimizing data acquisition parameters and performing efficient quality control for large datasets. To facilitate these objectives for data independent acquisition (DIA), we developed a second version of our framework for data-driven optimization of mass spectrometry methods (DO-MS). The DO-MS app v2.0 (do-ms.slavovlab.net) allows to optimize and evaluate results from both label free and multiplexed DIA (plexDIA) and supports optimizations particularly relevant for single-cell proteomics. We demonstrate multiple use cases, including optimization of duty cycle methods, peptide separation, number of survey scans per duty cycle, and quality control of single-cell plexDIA data. DO-MS allows for interactive data display and generation of extensive reports, including publication quality figures, that can be easily shared. The source code is available at: github.com/SlavovLab/DO-MS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/526809v3_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1334corg.highwire.dtl.DTLVardef@cec8bcorg.highwire.dtl.DTLVardef@1dcc07eorg.highwire.dtl.DTLVardef@1e1abd2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Synchro-PASEF allows precursor-specific fragment ion extraction and interference removal in data-independent acquisition

Data-independent acquisition (DIA) methods have become increasingly popular in mass spectrometry (MS)-based proteomics because they enable continuous acquisition of fragment spectra for all precursors simultaneously. However, these advantages come with the challenge of correctly reconstructing the precursor-fragment relationships in these highly convoluted spectra for reliable identification and quantification. Here we introduce a scan mode for the combination of trapped ion mobility spectrometry (TIMS) with parallel accumulation - serial fragmentation (PASEF) that seamlessly and continuously follows the natural shape of the ion cloud in ion mobility and peptide precursor mass dimensions. Termed synchro-PASEF, it increases the detected fragment ion current several-fold at sub-second cycle times. Consecutive quadrupole selection windows move synchronously through the mass and ion mobility range, defining precursor-quadrupole relationships. In this process, the quadrupole slices through the peptide precursors, which separates fragment ion signals of each precursor into adjacent synchro-PASEF scans. This precisely defines precursor - fragment relationships in ion mobility and mass dimensions and effectively deconvolutes the DIA fragment space. Importantly, the partitioned parts of the fragment ion transitions provide a further dimension of specificity via a lock and key mechanism. This is also advantageous for quantification, where signals from interfering precursors in the DIA selection window do not affect all partitions of the fragment ion, allowing to retain only the specific parts for quantification. Overall, we establish the defining features of synchro-PASEF and explore its potential for proteomic analyses.

biochemistry↗