12-Plex DiLeu Isobaric Labeling Enabled High-Throughput Investigation of Citrullination Alterations in the DNA Damage Response

Li, Zihui; Wang, Bin; Yu, Qinying; Shi, Yuping; Li, Lingjun

doi:10.1021/acs.analchem.1c04073

Cited by 6 publications

(16 citation statements)

References 67 publications

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“…Despite these advances, high‐throughput characterization of protein modifications remains a challenging task. 1465 , 1466 , 1467 First, there is a lack of effective antibodies and reagents to enrich the modified peptides of some modification types, such as methylation. 1468 Second, large‐scale PTMomics analysis of clinical samples can be difficult due to the limited availability of samples from clinical cohorts.…”

Section: Perspectivementioning

confidence: 99%

“…Deciphering the complex biological functions of proteins requires a deep understanding of protein modification, referred to as the “PTM code.” 1462–1464 In recent years, the rapid development of MS‐based proteomics technology has greatly advanced the research progress of protein PTMs. Despite these advances, high‐throughput characterization of protein modifications remains a challenging task 1465–1467 . First, there is a lack of effective antibodies and reagents to enrich the modified peptides of some modification types, such as methylation 1468 .…”

Section: Perspectivementioning

confidence: 99%

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Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications

Zhong

Xiao

Xie

et al. 2023

MedComm

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Protein posttranslational modifications (PTMs) refer to the breaking or generation of covalent bonds on the backbones or amino acid side chains of proteins and expand the diversity of proteins, which provides the basis for the emergence of organismal complexity. To date, more than 650 types of protein modifications, such as the most well‐known phosphorylation, ubiquitination, glycosylation, methylation, SUMOylation, short‐chain and long‐chain acylation modifications, redox modifications, and irreversible modifications, have been described, and the inventory is still increasing. By changing the protein conformation, localization, activity, stability, charges, and interactions with other biomolecules, PTMs ultimately alter the phenotypes and biological processes of cells. The homeostasis of protein modifications is important to human health. Abnormal PTMs may cause changes in protein properties and loss of protein functions, which are closely related to the occurrence and development of various diseases. In this review, we systematically introduce the characteristics, regulatory mechanisms, and functions of various PTMs in health and diseases. In addition, the therapeutic prospects in various diseases by targeting PTMs and associated regulatory enzymes are also summarized. This work will deepen the understanding of protein modifications in health and diseases and promote the discovery of diagnostic and prognostic markers and drug targets for diseases.

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Section: Perspectivementioning

confidence: 99%

Section: Perspectivementioning

confidence: 99%

Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications

Zhong

Xiao

Xie

et al. 2023

MedComm

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“…This was the first study to introduce a mass probe for easier detection of citrullinated residues and was also a milestone study, as it inspired further citrullination derivatization design [51,52,72,73].…”

Section: Probe Design and Development For Citrullination Identificationmentioning

confidence: 99%

“…Recent work has combined the aforementioned novel derivatization methods with DiLeu isobaric tags to achieve multiplexed quantitative analysis of citrullination from up to 12 samples [72]. This approach was applied to investigate citrullination alterations in response to DNA damage-induced stress using human cell lines.…”

Section: Probe Design and Development For Citrullination Identificationmentioning

confidence: 99%

“…This approach was applied to investigate citrullination alterations in response to DNA damage-induced stress using human cell lines. In total, they identified and quantified 78 citrullination sites from 63 citrullinated proteins in three DNA damage treatment groups and one control group [72]. This strategy combined the pipeline for isobaric labeling with previous methods for derivatization and enrichment of citrullinated peptides, enabling high-throughput quantitation of citrullinated proteins from complex biological samples with greatly improved accuracy.…”

Section: Probe Design and Development For Citrullination Identificationmentioning

confidence: 99%

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Recent advances in characterization of citrullination and its implication in human disease research: From method development to network integration

Wang

Fields

2023

Proteomics

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Post‐translational modifications (PTM) of proteins increase the functional diversity of the proteome and have been implicated in the pathogenesis of numerous diseases. The most widely understood modifications include phosphorylation, methylation, acetylation, O‐linked/N‐linked glycosylation, and ubiquitination, all of which have been extensively studied and documented. Citrullination is a historically less explored, yet increasingly studied, protein PTM which has profound effects on protein conformation and protein‐protein interactions. Dysregulation of protein citrullination has been associated with disease development and progression. Identification and characterization of citrullinated proteins is highly challenging, complicated by the low cellular abundance of citrullinated proteins, making it difficult to identify and quantify the extent of citrullination in samples, coupled with challenges associated with development of mass spectrometry (MS)‐based methods, as the corresponding mass shift is relatively small, +0.984 Da, and identical to the mass shift of deamidation. The focus of this review is to discuss recent advancements of citrullination‐specific MS approaches and integration of the potential methodology for improved citrullination identification and characterization. In addition, the association of citrullination in disease networks is also highlighted.

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Mass Spectrometry-Based Precise Identification of Citrullinated Histones via Limited Digestion and Biotin Derivative Tag Enrichment

Wang,

Li,

Shi

et al. 2024

Anal. Chem.

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Histone citrullination is an essential epigenetic post-translational modification (PTM) that affects many important physiological and pathological processes, but effective tools to study histone citrullination are greatly limited due to several challenges, including the small mass shift caused by this PTM and its low abundance in biological systems. Although previous studies have reported frequent occurrences of histone citrullination, these methods failed to provide a high-throughput and site-specific strategy to detect histone citrullination. Recently, we developed a biotin thiol tag that enabled precise identification of protein citrullination coupled with mass spectrometry. However, very few histone citrullination sites were identified, likely due to the highly basic nature of these proteins. In this study, we develop a novel method utilizing limited digestion and biotin derivative tag enrichment to facilitate direct in vivo identification of citrullination sites on histones. We achieve improved coverage of histone identification via partial enzymatic digestion and lysine block by dimethylation. With biotin tag-assisted chemical derivatization and enrichment, we also achieve precise annotation of histone citrullination sites with high confidence. We further compare different fragmentation methods and find that the electron-transfer-dissociation-based approach enables the most in-depth analysis and characterization. In total, we unambiguously identify 18 unique citrullination sites on histones in human astrocytoma U87 cells, including 15 citrullinated sites being detected for the first time. Some of these citrullination sites are observed to exhibit noticeable alterations in response to DNA damage, which demonstrates the superiority of our strategy in understanding the roles of histone citrullination in critical biological processes.

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12-Plex DiLeu Isobaric Labeling Enabled High-Throughput Investigation of Citrullination Alterations in the DNA Damage Response

Cited by 6 publications

References 67 publications

Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications

Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications

Recent advances in characterization of citrullination and its implication in human disease research: From method development to network integration

Mass Spectrometry-Based Precise Identification of Citrullinated Histones via Limited Digestion and Biotin Derivative Tag Enrichment

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