Increasing the Throughput and Reproducibility of Activity-Based Proteome Profiling Studies with Hyperplexing and Intelligent Data Acquisition

Journal of Proteome Research, Journal Year: 2024, Volume and Issue: 23(8), P. 2934 - 2947

Published: Jan. 22, 2024

Intelligent data acquisition (IDA) strategies, such as a real-time database search (RTS), have improved the depth of proteome coverage for experiments that utilize isobaric labels and gas phase purification techniques (i.e., SPS-MS3). In this work, we introduce inSeqAPI, an instrument application programing interface (iAPI) program enables construction novel algorithms. First, analyze biotinylated cysteine peptides from ABPP to demonstrate method within inSeqAPI performs similarly equivalent vendor method. Then, describe PairQuant, designed hyperplexing approach utilizes protein-level isotopic labeling peptide-level TMT labeling. PairQuant allows analysis 36 conditions in single sample achieves ∼98% both peptide pair partners hyperplexed experiment well 40% improvement number quantified sites compared with non-RTS acquisition. We applied study ligandable nucleus leading identification additional druggable on protein- DNA-interaction domains transcription regulators nuclear ubiquitin ligases.

Language: Английский

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Structural and mechanistic basis for nucleosomal H2AK119 deubiquitination by single-subunit deubiquitinase USP16

Nature Structural & Molecular Biology, Journal Year: 2024, Volume and Issue: 31(11), P. 1745 - 1755

Published: June 25, 2024

Language: Английский

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USP16 is an ISG15 cross-reactive deubiquitinase that targets pro-ISG15 and ISGylated proteins involved in metabolism

Proceedings of the National Academy of Sciences, Journal Year: 2023, Volume and Issue: 120(50)

Published: Dec. 6, 2023

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for existence of other cross-reactive proteases, these remain be identified. Here, we identify deubiquitinase USP16 as an means activity-based profiling. Recombinant cleaved pro-ISG15 isopeptide-linked model substrates in vitro, well ISGylated from cell lysates. Moreover, interferon-induced stimulation ISGylation was increased depletion USP16. The USP16-dependent interactome indicated that deISGylating may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, glutamic-oxaloacetic transaminase 1. thus contribute regulation a subset metabolism-related during type-I interferon responses.

Language: Английский

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Increasing the Throughput and Reproducibility of Activity-Based Proteome Profiling Studies with Hyperplexing and Intelligent Data Acquisition

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Sept. 13, 2023

Abstract Intelligent data acquisition (IDA) strategies, such as real-time database search (RTS), have improved the depth of proteome coverage for experiments that utilize isobaric labels and gas phase purification techniques (i.e., SPS-MS3). While most applications IDA been focused on analysis protein abundance, these approaches recently applied to activity-based profiling (ABPP) studies aimed at characterization site engagement by small molecules. In this work, we extend capabilities offered vendor software through a program called InSeqAPI. First, demonstrate robust performance InSeqAPI in biotinylated cysteine peptides from ABPP experiments. Then, describe PairQuant, method within designed hyperplexing approach utilizes protein-level isotopic labeling peptide-level TMT labeling. PairQuant allows 36 conditions single sample achieves ∼98% both peptide pair partners hyperplexed experiment well 40% improvement number quantified sites compared non-RTS acquisition. We study ligandable nucleus leading an identification additional druggable protein-and DNA-interaction domains transcription regulators nuclear ubiquitin ligases.

Language: Английский

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Research progress of the Otubains subfamily in hepatocellular carcinoma

Biomedicine & Pharmacotherapy, Journal Year: 2024, Volume and Issue: 179, P. 117348 - 117348

Published: Aug. 28, 2024

In cancer research, oncogenesis can be affected by modulating the deubiquitination pathway. Ubiquitination regulates proteins post-translationally in variety of physiological processes. The Otubain Subfamily includes OTUB1 (ovarian tumor-associated proteinase B1) and OTUB2(ovarian B2). They are deubiquitinating enzymes, which research hotspots tumor immunotherapy, with their implications extending across spectrum development. Understanding important role tumorigenesis, includ-ing hepatocellular carcinoma (HCC) is crucial. HCC has alarming global incidence rates mortality statistics, ranking among top five prevalent cancers Malaysia

Language: Английский

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Roles of deubiquitinases in urologic cancers (Review)

Oncology Letters, Journal Year: 2024, Volume and Issue: 28(6)

Published: Oct. 14, 2024

Human health is endangered by the occurrence and progression of urological cancers, including renal cell carcinoma, prostate cancer bladder cancer, which are usually associated with activation oncogenic factors inhibition suppressors. The primary mechanism for protein breakdown in cells ubiquitin‑proteasome system, whilst deubiquitinases contribute to reversal this process. However, both important homeostasis. Deubiquitination may also be involved control cycle, proliferation apoptosis, dysregulated deubiquitination malignant transformation, invasion metastasis urologic malignancies. Therefore, a comprehensive summary mechanisms underlying cancers provide novel strategies insights diagnosis treatment. present review aimed methodically clarify role deubiquitinating enzymes urinary system as well their prospective application prospects clinical

Language: Английский

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