Quantitative Profiling of Protein O-GlcNAcylation Sites by an Isotope-Tagged Cleavable Linker

Qin, Ke; Zhu, Yuntao; Qin, Wei; Gao, Jinjun; Shao, Xuan; Wang, Yanling; Zhou, Wen; Wang, Chu; Chen, Xing

doi:10.1021/acschembio.8b00414

Cited by 77 publications

(88 citation statements)

References 52 publications

(81 reference statements)

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“…Consistent with the high levels of modification we observed in our pulldown, recent proteomics experiments using the same chemoenzymatic strategy have identified many different endogenous MYPT1 O-GlcNAcylation sites [42][43][44] localized around two different regions of the protein (Figure 5d). The most N-terminal of these areas, with modification at Ser379 and Thr381, is located near the portion of MYPT1 responsible for binding the phosphatase catalytic subunit PP1cδ and its substrate, phosphorylated MLC [45][46][47] .…”

Section: Mypt1 Is a Heavily And Dynamically O-glcnacylated Proteinsupporting

confidence: 89%

MYPT1 O-GlcNAc modification controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

Pedowitz

Batt

Darabedian

et al. 2019

Preprint

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Many intracellular proteins can be modified by N-acetylglucosamine, a posttranslational modification known as O-GlcNAc.Because this modification is found on serine and threonine side-chains, O-GlcNAc has the potential to dynamically regulate cellular signaling pathways through interplay with phosphorylation. Here, we discover and characterize one such pathway.First, we find that O-GlcNAcylation levels control the sensitivity of fibroblasts to actin contraction induced by the signaling lipid sphingosine-1-phosphate (S1P). In subsequent mechanistic investigations, we find that S1P agonizes the S1PR2 receptor, resulting in activation of the Rho/Rho kinase signaling pathway. This pathway typically culminates in the phosphorylation of myosin light chain (MLC), resulting in myosin activation and cellular contraction. We find that O-GlcNAcylation of the phosphatase subunit MYPT1 inhibits this pathway by blocking MYPT1 phosphorylation, maintaining its activity and causing the dephosphorylation of MLC. Therefore, MYTP1 O-GlcNAcylation levels function as a rheostat to regulate the sensitivity of cells to S1P-mediated cellular contraction. Our findings have important implications for the role of in fibroblast motility and differentiation, as well as several other signaling pathways that use MLC/MYTP1 to control actin contraction in various tissues.

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Section: Mypt1 Is a Heavily And Dynamically O-glcnacylated Proteinsupporting

confidence: 89%

MYPT1 O-GlcNAc modification controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

Pedowitz

Batt

Darabedian

et al. 2019

Preprint

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“…Previous reports suggested that streptavidin-biotin enrichment conducted after proteolysis could result in a higher number of identifications than enrichment conducted prior to protein digestion [30][31][32] . We therefore decided to perform two enrichment approaches in parallel to understand how either method would affect the total number of identifications.…”

Section: Resultsmentioning

confidence: 99%

Evaluation and Optimization of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Protein Interactomes

Rabalski

Bogdan

Baranczak

2019

Preprint

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Numerous reagents have been developed to enable chemical proteomic analysis of small molecule-protein interactomes. However, the performance of these reagents has not been systematically evaluated and compared. Herein, we report our efforts to conduct a parallel assessment of two widely-used chemically-cleavable linkers equipped with dialkoxydiphenylsilane (DADPS linker) and azobenzene (AZO linker) moieties. Profiling a cellular cysteinome using iodoacetamide alkyne probe demonstrated a significant discrepancy between the experimental results obtained through the application of each of the reagents. To better understand the source of observed discrepancy, a mass tolerant database search strategy using MSFragger software was performed. This resulted in identifying a previously unreported artifactual modification on the residual mass of the azobenzene linker. Furthermore, we conducted a comparative analysis of enrichment modes using both cleavable linkers. This effort determined that enrichment of proteolytic digests yielded a far greater number of identified cysteine residues than the enrichment conducted prior to protein digest. Inspired by recent studies where multiplexed quantitative labeling strategies were applied to cleavable biotin linkers, we combined this further optimized protocol using the DADPS cleavable linker with tandem mass tag (TMT) labeling to profile the FDA-approved covalent EGFR kinase inhibitor dacomitinib against the cysteinome of an epidermoid cancer cell line. Our analysis resulted in the detection and quantification of over 10,000 unique cysteine residues, a nearly 3-fold increase over previous studies that used cleavable biotin linkers for enrichment. Critically, cysteine residues corresponding to proteins directly as well as indirectly modulated by dacomitinib treatment were identified. Overall, our study suggests that the dialkoxydiphenylsilane linker could be broadly applied wherever chemically cleavable linkers are required for chemical proteomic characterization of cellular proteomes.

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“…The combination of metabolic labeling and click chemistry has emerged as a powerful method to study proteins and protein modifications (Mahal et al, ; Hang et al, ; Bond and Kohler, ; Dieterich et al, ; Hanson et al, ; Hong et al, ; Smeekens et al, ; Chen et al, ; Cheng et al, ; Spiciarich et al, ; Xiao and Wu, ; Xiao et al, ). Metabolic labeling of glycans with unnatural sugar analogs is an excellent way to study glycans and protein glycosylation (Kayser et al, ; Saxon and Bertozzi, ; Feng et al, ; Aguilar et al, , ; Park et al, ; Qin et al, ). In the recent two decades, the Bertozzi group has performed pioneering work on using unnatural sugar analogs to label glycoproteins (Mahal et al, ; Saxon and Bertozzi, ; Hang et al, ; Breidenbach et al, ).…”

Section: Global Analysis Of Glycoproteinsmentioning

confidence: 99%

Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

Xiao

Sun

Suttapitugsakul

et al. 2019

Mass Spectrometry Reviews

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Protein glycosylation is ubiquitous in biological systems and plays essential roles in many cellular events. Global and site‐specific analysis of glycoproteins in complex biological samples can advance our understanding of glycoprotein functions and cellular activities. However, it is extraordinarily challenging because of the low abundance of many glycoproteins and the heterogeneity of glycan structures. The emergence of mass spectrometry (MS)‐based proteomics has provided us an excellent opportunity to comprehensively study proteins and their modifications, including glycosylation. In this review, we first summarize major methods for glycopeptide/glycoprotein enrichment, followed by the chemical and enzymatic methods to generate a mass tag for glycosylation site identification. We next discuss the systematic and quantitative analysis of glycoprotein dynamics. Reversible protein glycosylation is dynamic, and systematic study of glycoprotein dynamics helps us gain insight into glycoprotein functions. The last part of this review focuses on the applications of MS‐based proteomics to study glycoproteins in different biological systems, including yeasts, plants, mice, human cells, and clinical samples. Intact glycopeptide analysis is also included in this section. Because of the importance of glycoproteins in complex biological systems, the field of glycoproteomics will continue to grow in the next decade. Innovative and effective MS‐based methods will exponentially advance glycoscience, and enable us to identify glycoproteins as effective biomarkers for disease detection and drug targets for disease treatment. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 9999: XX–XX, 2019.

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Quantitative Profiling of Protein O-GlcNAcylation Sites by an Isotope-Tagged Cleavable Linker

Cited by 77 publications

References 52 publications

MYPT1 O-GlcNAc modification controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

MYPT1 O-GlcNAc modification controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

Evaluation and Optimization of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Protein Interactomes

Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

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