Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling

Mann, Matthias; Hornung, Veit; Stafford, Che A.; Bludau, Isabell; Tanzer, Maria C.

doi:10.1101/2020.11.04.368159

Cited by 10 publications

(11 citation statements)

References 66 publications

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“…4f). CDK12 is known to be involved not only in cell cycle progression but also in TNF 44 and noncanonical NF-κB 45 signaling. While these previous reports may suggest a pro-inflammatory role of CDK12, our findings suggest that the role of CDK12 may be more nuanced or context-dependent, and we designated it for further investigation (see below).…”

Section: Identification Of Modifiers Of Microglial Activation By Cris...mentioning

confidence: 99%

A CRISPRi/a platform in iPSC-derived microglia uncovers regulators of disease states

et al. 2021

Preprint

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Microglia are emerging as key drivers of neurological diseases. However, we lack a systematic understanding of the underlying mechanisms. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human iPSC-derived microglia. We developed an efficient eight-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the "druggable genome". These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. A screen with single-cell RNA sequencing as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains and identified regulators of these states. A disease-associated state characterized by SPP1 expression was selectively depleted by CSF1R inhibition. Thus, our platform can systematically uncover regulators of microglia states, enabling their functional characterization and therapeutic targeting.

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Section: Identification Of Modifiers Of Microglial Activation By Cris...mentioning

confidence: 99%

A CRISPRi/a platform in iPSC-derived microglia uncovers regulators of disease states

et al. 2021

Preprint

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“…These include EGF-induced phosphorylation of E3 ligases like Mindbomb Homolog 2 (MIB2) (S309) and members of the linear ubiquitin chain assembly complex Ring Finger Protein 31 (RNF31) (S466) and Sharpin (S165), which are most frequently studied in the context of TNF signaling (supplemental Fig. S14) (56)(57)(58)(59). Similarly, phosphorylation of Receptor Interacting Serine/Threonine Protein Kinase 1 (RIPK1) on S320, which prevents TNF-induced cell death, was also increased upon EGF signaling (supplemental Fig.…”

Section: In-depth Phosphoproteomics Analysis Of the Egf-signaling Pat...mentioning

confidence: 99%

Rapid and in-depth coverage of the (phospho-)proteome with deep libraries and optimal window design for dia-PASEF

Skowronek

Thielert

Voytik

et al. 2022

Preprint

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Data-independent acquisition (DIA) methods have become increasingly attractive in mass spectrometry (MS)-based proteomics, because they enable high data completeness and a wide dynamic range. Recently, we combined DIA with parallel accumulation - serial fragmentation (dia-PASEF) on a Bruker trapped ion mobility separated (TIMS) quadrupole time-of-flight (TOF) mass spectrometer. This requires alignment of the ion mobility separation with the downstream mass selective quadrupole, leading to a more complex scheme for dia-PASEF window placement compared to DIA. To achieve high data completeness and deep proteome coverage, here we employ variable isolation windows that are placed optimally depending on precursor density in the m/z and ion mobility plane. This Automatic Isolation Design procedure is implemented in the freely available py_diAID package. In combination with in-depth project-specific proteomics libraries and the Evosep LC system, we reproducibly identified over 7,700 proteins in a human cancer cell line in 44 minutes with quadruplicate single-shot injections at high sensitivity. Even at a throughput of 100 samples per day (11 minutes LC gradients), we consistently quantified more than 6,000 proteins in mammalian cell lysates by injecting four replicates. We found that optimal dia-PASEF window placement facilitates in-depth phosphoproteomics with very high sensitivity, quantifying more than 35,000 phosphosites in a human cancer cell line stimulated with an epidermal growth factor (EGF) in triplicate 21 minutes runs. This covers a substantial part of the regulated phosphoproteome with high sensitivity, opening up for extensive systems-biological studies.

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“…A second challenge are missing values, especially in PTM studies. Although this problem is much reduced in DIA compared to DDA data, it still occurs frequently especially as sample size grows (39,(46)(47)(48). This is due to the complexity of spectra, the low abundance of modified peptides and the technical variability.…”

Section: Data Quality Assessment and Discovery Of Egf Signaling Event...mentioning

confidence: 99%

“…In recent decades, MS has become the tool of choice for large-scale identification and quantitation of proteins and their post-translational modifications (PTMs) and the computational workflows for the analysis of DDA have matured. DIA analysis is comparatively newer and less established, especially when PTMs are being analyzed (28,(36)(37)(38)(39). Although modern proteomics workflows report the localization of the identified PTMs and the associated probabilities, in our was used to evaluate the overall quality of the selected sample (A5_1_2451) (Fig.…”

Section: Visual Validation Of Peptidoforms Of the Proteins Of Interestmentioning

confidence: 99%

AlphaViz: Visualization and validation of critical proteomics data directly at the raw data level

Voytik

Skowronek

Zeng

et al. 2022

Preprint

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Although current mass spectrometry (MS)-based proteomics identifies and quantifies thousands of proteins and (modified) peptides, only a minority of them are subjected to in-depth downstream analysis. With the advent of automated processing workflows, biologically or clinically important results within a study are rarely validated by visualization of the underlying raw information. Current tools are often not integrated into the overall analysis nor readily extendable with new approaches. To remedy this, we developed AlphaViz, an open-source Python package to superimpose output from common analysis workflows on the raw data for easy visualization and validation of protein and peptide identifications. AlphaViz takes advantage of recent breakthroughs in the deep learning-assisted prediction of experimental peptide properties to allow manual assessment of the expected versus measured peptide result. We focused on the visualization of the 4-dimensional data cuboid provided by Bruker TimsTOF instruments, where the ion mobility dimension, besides intensity and retention time, can be predicted and used for verification. We illustrate how AlphaViz can quickly validate or invalidate peptide identifications regardless of the score given to them by automated workflows. Furthermore, we provide a 'predict mode' that can locate peptides present in the raw data but not reported by the search engine. This is illustrated the recovery of missing values from experimental replicates. Applied to phosphoproteomics, we show how key signaling nodes can be validated to enhance confidence for downstream interpretation or follow-up experiments. AlphaViz follows standards for open-source software development and features an easy-to-install graphical user interface for end-users and a modular Python package for bioinformaticians. Validation of critical proteomics results should now become a standard feature in MS-based proteomics.

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Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling

Cited by 10 publications

References 66 publications

A CRISPRi/a platform in iPSC-derived microglia uncovers regulators of disease states

A CRISPRi/a platform in iPSC-derived microglia uncovers regulators of disease states

Rapid and in-depth coverage of the (phospho-)proteome with deep libraries and optimal window design for dia-PASEF

AlphaViz: Visualization and validation of critical proteomics data directly at the raw data level

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