Quantitative, Time-Resolved Proteomic Analysis by Combining Bioorthogonal Noncanonical Amino Acid Tagging and Pulsed Stable Isotope Labeling by Amino Acids in Cell Culture

Bagert, John D.; Xie, Yushu Joy; Sweredoski, Michael J.; Qi, Yuhong; Hess, Sonja; Schuman, Erin M.; Tirrell, David A.

doi:10.1074/mcp.m113.031914

Cited by 80 publications

(78 citation statements)

References 31 publications

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“…Excess systemic ncAAs and their metabolites have the potential to induce pathological states similar to what has been shown with naturally occurring amino acids such as hyperhomocysteinemia (excess Met)27. Nevertheless, data from numerous in vitro and in vivo reports910131718, in addition to our in vivo demonstration that AHA does not disrupt mammalian development (Fig. 3), indicate that short term administration of ncAAs has minimal adverse effects.…”

Section: Discussionsupporting

confidence: 70%

“…A limitation of these methods is that protein synthesis stops upon puromycin incorporation, reducing the biological activity of the truncated proteins and preventing their utility for long term labeling. In contrast to these methods, BONCAT enables the enrichment of newly synthesized proteins within complex mixtures91034 and does not overtly perturb protein synthesis levels as demonstrated in vitro 10, in vivo 13 and in this study.…”

Section: Discussionmentioning

confidence: 86%

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Incorporation of non-canonical amino acids into the developing murine proteome

Calve

Witten

Ocken

et al. 2016

Sci Rep

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Analysis of the developing proteome has been complicated by a lack of tools that can be easily employed to label and identify newly synthesized proteins within complex biological mixtures. Here, we demonstrate that the methionine analogs azidohomoalanine and homopropargylglycine can be globally incorporated into the proteome of mice through facile intraperitoneal injections. These analogs contain bio-orthogonal chemical handles to which fluorescent tags can be conjugated to identify newly synthesized proteins. We show these non-canonical amino acids are incorporated into various tissues in juvenile mice and in a concentration dependent manner. Furthermore, administration of these methionine analogs to pregnant dams during a critical stage of murine development, E10.5–12.5 when many tissues are assembling, does not overtly disrupt development as assessed by proteomic analysis and normal parturition and growth of pups. This successful demonstration that non-canonical amino acids can be directly administered in vivo will enable future studies that seek to characterize the murine proteome during growth, disease and repair.

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Section: Discussionsupporting

confidence: 70%

Section: Discussionmentioning

confidence: 86%

Incorporation of non-canonical amino acids into the developing murine proteome

Calve

Witten

Ocken

et al. 2016

Sci Rep

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“…BONCAT has been used to study proteome-wide responses in mammalian and microbial culture (Dieterich et al, 2006;Zhang et al, 2014;Bagert et al, 2014) and to analyze subpopulations of cells in complex multicellular eukaryotes (Erdmann et al, 2015;Genheden et al, 2015;Yuet et al, 2015), but has yet to be employed in plant systems. Here, we applied BONCAT to Arabidopsis (Arabidopsis thaliana) to visualize proteins synthesized within 3 h after imposition of four significant abiotic perturbations: light stress (shift to darkness), high temperature, salt, and osmotic stress.…”

mentioning

confidence: 99%

Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue

Glenn

Stone

et al. 2017

Plant Physiol.

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Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal noncanonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis (Arabidopsis thaliana) seedlings. BONCAT relies on the translational incorporation of a noncanonical amino acid probe into cellular proteins. In this study, the probe is the Met surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesized proteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min interval defined by an Aha pulse and that the method can be used to detect proteins made under conditions of light stress, osmotic shock, salt stress, heat stress, and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquid chromatography-mass spectrometry to identify and quantify proteins synthesized during heat stress and recovery from heat stress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed to enhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out with commercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stress responses as well as translational and posttranslational regulation in plants.

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“…Another study that examined the potential artifacts in protein quantification that might be caused by AHA labeling was published recently [78]. In this paper, it was shown that although complete replacement of Met by AHA during labeling of HeLa cells after 24 hours had no significant effect on protein identification, it did alter protein abundances.…”

Section: Strategy and Methods To Identify Newly Synthetized Proteins mentioning

confidence: 94%

Exploring ribosome composition and newly synthesized proteins through proteomics and potential biomedical applications

Št́astná

Gottlieb

Eyk

2017

Expert Review of Proteomics

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Introduction: Protein synthesis is the outcome of tightly regulated gene expression which is responsive to a variety of conditions. Efforts are ongoing to monitor individual stages of protein synthesis to ensure maximum efficiency and accuracy. Due to post-transcriptional regulation mechanisms, the correlation between translatome and proteome is higher than between transcriptome and proteome. However, the most accurate approach to assess the key modulators and final protein expression is directly by using proteomics. Areas covered: This review covers various proteomic strategies that were used to better understand post-transcriptional regulation, specifically during and early after translation. The methods that identify both regulatory proteins associated with translational components and newly synthesized proteins are discussed. Expert commentary: Emerging proteomic approaches make it possible to monitor protein dynamics in cells, tissues and whole animals. The ability to detect alteration in protein abundance soon after their synthesis enables earlier recognition of disease causing factors and candidates to prevent/rectify disease phenotype.

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Quantitative, Time-Resolved Proteomic Analysis by Combining Bioorthogonal Noncanonical Amino Acid Tagging and Pulsed Stable Isotope Labeling by Amino Acids in Cell Culture

Cited by 80 publications

References 31 publications

Incorporation of non-canonical amino acids into the developing murine proteome

Incorporation of non-canonical amino acids into the developing murine proteome

Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue

Exploring ribosome composition and newly synthesized proteins through proteomics and potential biomedical applications

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