Data-independent acquisition method for ubiquitinome analysis reveals regulation of circadian biology

Hansen, Fynn M.; Tanzer, Maria C.; Bruening, F.; Bludau, Isabell; Schulman, Brenda A.; Robles, Morgane; Karayel, Özge; Mann, Matthias

doi:10.1101/2020.07.24.219055

Cited by 6 publications

(6 citation statements)

References 76 publications

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“…For example, the effect of each of the ∼80 yeast E3 ligases on the global proteome could be ascertained in just 5 days, or each of the ∼117 yeast kinases in about 1 week. In addition, the DIA-based workflow can be easily adapted to identify changes in posttranslational modifications including phosphorylation, ubiquitination, and acetylation, when coupled with an enrichment step ( 44 , 82 – 84 ).…”

Section: Discussionmentioning

confidence: 99%

DIA-based systems biology approach unveils E3 ubiquitin ligase-dependent responses to a metabolic shift

Karayel

Michaelis

Mann

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The yeastSaccharomyces cerevisiaeis a powerful model system for systems-wide biology screens and large-scale proteomics methods. Nearly complete proteomics coverage has been achieved owing to advances in mass spectrometry. However, it remains challenging to scale this technology for rapid and high-throughput analysis of the yeast proteome to investigate biological pathways on a global scale. Here we describe a systems biology workflow employing plate-based sample preparation and rapid, single-run, data-independent mass spectrometry analysis (DIA). Our approach is straightforward, easy to implement, and enables quantitative profiling and comparisons of hundreds of nearly complete yeast proteomes in only a few days. We evaluate its capability by characterizing changes in the yeast proteome in response to environmental perturbations, identifying distinct responses to each of them and providing a comprehensive resource of these responses. Apart from rapidly recapitulating previously observed responses, we characterized carbon source-dependent regulation of the GID E3 ligase, an important regulator of cellular metabolism during the switch between gluconeogenic and glycolytic growth conditions. This unveiled regulatory targets of the GID ligase during a metabolic switch. Our comprehensive yeast system readout pinpointed effects of a single deletion or point mutation in the GID complex on the global proteome, allowing the identification and validation of targets of the GID E3 ligase. Moreover, this approach allowed the identification of targets from multiple cellular pathways that display distinct patterns of regulation. Although developed in yeast, rapid whole-proteome–based readouts can serve as comprehensive systems-level assays in all cellular systems.

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Section: Discussionmentioning

confidence: 99%

DIA-based systems biology approach unveils E3 ubiquitin ligase-dependent responses to a metabolic shift

Karayel

Michaelis

Mann

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…The TIMS ion mobility separation is slower than the fast tandem-MS scans of Scanning SWATH, and dia-PASEF is hence less suited for being used in conjunction with ultra-fast chromatographic gradients generated with analytical flow LC systems (Messner et al, 2020a(Messner et al, , 2020b. However, the ion mobility separation and the resulting boost in sensitivity can maximize proteomic depth when only limited sample amounts are available, such as in single cell proteomics (Brunner et al, 2020) or in the quantitative analyses of posttranslational modifications (Bekker-Jensen et al, 2020;Hansen et al, 2021;Steger et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

High sensitivity dia-PASEF proteomics with DIA-NN and FragPipe

Demichev

Yuan

et al. 2021

Preprint

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The dia-PASEF technology exploits ion mobility separation for high-sensitivity analysis of complex proteomes. Here, we demonstrate neural network-based processing of the ion mobility data, which we implement in the DIA-NN software suite. Using spectral libraries generated with the MSFragger-based FragPipe computational platform, the DIA-NN analysis of dia-PASEF raw data increases the proteomic depth by up to 69% compared to the originally published dia-PASEF workflow. For example, we quantify over 5200 proteins from 10ng of HeLa peptides separated with a 95-minute nanoflow gradient, and over 5000 proteins from 200ng using a 4.8-minute separation with an Evosep One system. In complex samples, featuring a mix of human and yeast lysates, the workflow detects over 11700 proteins in single runs acquired with a 100-minute nanoflow gradient, while demonstrating quantitative precision. Hence, the combination of FragPipe and DIA-NN provides a simple-to-use software platform for dia-PASEF data analysis, yielding significant gains in high-sensitivity proteomics.

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“…The antibody is coupled to the beads using chemistry that does not affect the epitope binding regions. Since this HSmag anti-K-ε-GG formulation does not require an initial chemical cross-linking step to covalently couple the antibody to affinity beads, 1-2 days of procedural time are saved 7,21,22 . The magnetic bead formulation also provides the means to transfer ubiquitin enrichment protocols to a magnetic particle processor for automating and increasing the throughput of sample handling steps.…”

Section: Resultsmentioning

confidence: 99%

Automating UbiFast for High-throughput and Multiplexed Ubiquitin Enrichment

Rivera

Olive

Bergstrom

et al. 2021

Preprint

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Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites is necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-ε-GG peptides are enriched with anti-K-ε-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-ε-GG antibody (mK-ε-GG) and a magnetic particle processor. We report the identification of ~20,000 ubiquitylation sites from a TMT10-plex with 500 μg input per sample processed in ~2 hours. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility and significantly reduced processing time. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of this method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples.

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Data-independent acquisition method for ubiquitinome analysis reveals regulation of circadian biology

Cited by 6 publications

References 76 publications

DIA-based systems biology approach unveils E3 ubiquitin ligase-dependent responses to a metabolic shift

DIA-based systems biology approach unveils E3 ubiquitin ligase-dependent responses to a metabolic shift

High sensitivity dia-PASEF proteomics with DIA-NN and FragPipe

Automating UbiFast for High-throughput and Multiplexed Ubiquitin Enrichment

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