A terminal alkyne and disulfide functionalized agarose resin specifically enriches azidohomoalanine labeled nascent proteins

Hou, Zhanwu; Han, Xiao; Wang, Zhen; Ghazanfar, Sana; Yang, Jeffy; Liu, Huadong

doi:10.1016/j.jchromb.2021.122527

Cited by 5 publications

(3 citation statements)

References 29 publications

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“…We aimed to investigate the effects of PA-stimulated insulin resistance on nascent proteomics changes using an AHA labeling technique commonly used to probe changes in protein translation ( 37 , 38 ). Phosphorylation proteome analysis indicated that protein translation regulation was involved in PA treatment.…”

Section: Resultsmentioning

confidence: 99%

4EBP2-regulated protein translation has a critical role in high-fat diet–induced insulin resistance in hepatocytes

Han,

Yang,

Zhang

et al. 2023

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 99%

4EBP2-regulated protein translation has a critical role in high-fat diet–induced insulin resistance in hepatocytes

Han,

Yang,

Zhang

et al. 2023

Journal of Biological Chemistry

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“…When AHA is added to medium to culture cells without methionine, it is present in the newly synthesized protein. In our previous study, we developed a terminal alkyne-and disulfide-functionalized agarose (TAD) resin that efficiently enriches the AHA-labeled proteome by click reaction (24). Following MS analysis, we quantified the nascent proteome and verified these results by western blot and molecular biology experiments.…”

Section: Popular Scientific Summarymentioning

confidence: 99%

Resveratrol regulates Hsp60 in HEK 293T cells during activation of SIRT1 revealed by nascent protein labeling strategy

Wang

Zhang

et al. 2022

Food & Nutrition Research

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Background: Resveratrol, a well-known natural compound and nutrient, activates the deacetylation ability of SIRT1, demonstrating p53-dependent apoptosis functions in many diseases. However, the nascent proteomic fluctuation caused by resveratrol is still unclear. Objective: In this study, we investigated the effect of resveratrol on the nascent proteome and transcriptome initiated by SIRT1 activation, and we explored the mechanism of resveratrol in HEK 293T cells. Methods: Bioorthogonal noncanonical amino acid tagging (BONCAT) is a method used to metabolically label nascent proteins. In this strategy, L-azidohomoalanine (AHA) was used to replace methionine (Met) under different conditions. Taking advantage of the click reaction between AHA and terminal alkyne- and disulfide-functionalized agarose resin (TAD resin), we were able to efficiently separate stimulation responsive proteins from the pre-existing proteome. Resveratrol responsive proteins were identified by Liquid Chromatograph-Mass Spectrometer/Mass Spectrometer (LC-MS/MS). Furthermore, changes in mRNA levels were analyzed by transcriptome sequencing. Results: Integrational analysis revealed a resveratrol response in HEK 293T cells and showed that Hsp60 was downregulated at both the nascent protein and mRNA levels. Knockdown of SIRT1 and Hsp60 provides evidence that resveratrol downregulated Hsp60 through SIRT1 and that Hsp60 decreased p53 through the Akt pathway. Conclusions: This study revealed dynamic changes in the nascent proteome and transcriptome in response to resveratrol in HEK 293T cells and demonstrated that resveratrol downregulates Hsp60 by activating SIRT1, which may be a possible mechanism by which resveratrol prevents p53-dependent apoptosis by regulating Hsp60.

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“…[28] Inorganic supports, in the form of silica, [29] and cobalt [30] -based functionalized particles have also been used as solid supports for the selective enrichment of bioorthogonally tagged peptides and proteins. Examples of cleavable linkers are levulinoyl esters, [31] disulfides, [32][33] diazobenzenes, [34] acid-or photo-cleavable linkers. [35][36] However, in practice, the use of these approaches is hindered by the fact that the sensitivity of such approaches is often low due to poor chemical release of the linker.…”

Section: Introductionmentioning

confidence: 99%

Bioorthogonal Peptide Enrichment from Complex Samples Using a Rink‐Amide‐Based Catch‐and‐Release Strategy

et al. 2023

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Uptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of low abundant exogenous antigens from complex cell extracts is difficult. Mass‐spectrometry based proteomics – the ideal analysis tool in this case – requires methods to retrieve such molecules with high efficiency and low background. Here, we present a method for the selective and sensitive enrichment of antigenic peptides from APCs using click‐antigens; antigenic proteins expressed with azidohomoalanine (Aha) in place of methionine residues. We here describe the capture of such antigens using a new covalent method namely, alkynyl functionalized PEG‐based Rink amide resin, that enables capture of click‐antigens via copper‐catalyzed azide‐alkyne [2 + 3] cycloaddition (CuAAC). The covalent nature of the thus formed linkage allows stringent washing to remove a‐specific background material, prior to retrieval peptides by acid‐mediated release. We successfully identified peptides from a tryptic digest of the full APC proteome containing femtomole amounts of Aha‐labelled antigen, making this a promising approach for clean and selective enrichment of rare bioorthogonally modified peptides from complex mixtures.

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A terminal alkyne and disulfide functionalized agarose resin specifically enriches azidohomoalanine labeled nascent proteins

Cited by 5 publications

References 29 publications

4EBP2-regulated protein translation has a critical role in high-fat diet–induced insulin resistance in hepatocytes

4EBP2-regulated protein translation has a critical role in high-fat diet–induced insulin resistance in hepatocytes

Resveratrol regulates Hsp60 in HEK 293T cells during activation of SIRT1 revealed by nascent protein labeling strategy

Bioorthogonal Peptide Enrichment from Complex Samples Using a Rink‐Amide‐Based Catch‐and‐Release Strategy

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