Cell-type-specific metabolic labeling of nascent proteomes in vivo

Álvarez-Castelao, Beatriz; Schanzenbächer, Christoph T.; Hanus, Cyril; Glock, Caspar; Dieck, Susanne tom; Dörrbaum, Aline Ricarda; Bartnik, Ina; Nassim-Assir, Belquis; Ciirdaeva, Elena; Mueller, Anke; Dieterich, Daniela C.; Tirrell, David A.; Langer, Julian D.

doi:10.1038/nbt.4016

Cited by 183 publications

(214 citation statements)

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“…Mass spectra were acquired over the mass range 350–1600 m /z, and sequence information was acquired by computer‐controlled, data‐dependent automated switching to MS/MS mode using collision energies based on mass and charge state of candidate ions (TOP15, “FT‐IT”‐mode, MS resolution 120k, MS 2 injection time: 50 ms). All samples were measured in technical triplicate LC‐MS/MS runs (three “technical replicates” of each condition and each of the two biological replicates for each strain) …”

Section: Methodsmentioning

confidence: 99%

Proteome Analysis of Enriched Heterocysts from Two Hydrogenase Mutants from Anabaena sp. PCC 7120

et al. 2019

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Cyanobacteria are oxygenic photosynthetic prokaryotes and play a crucial role in the Earth's carbon and nitrogen cycles. The photoautotrophic cyanobacterium Anabaena sp. PCC 7120 has the ability to fix atmospheric nitrogen in heterocysts and produce hydrogen as a byproduct through a nitrogenase. In order to improve hydrogen production, mutants from Anabaena sp. PCC 7120 are constructed by inactivation of the uptake hydrogenase (ΔhupL) and the bidirectional hydrogenase (ΔhoxH) in previous studies. Here the proteomic differences of enriched heterocysts between these mutants cultured in N2‐fixing conditions are investigated. Using a label‐free quantitative proteomics approach, a total of 2728 proteins are identified and it is found that 79 proteins are differentially expressed in the ΔhupL and 117 proteins in the ΔhoxH variant. The results provide for the first time comprehensive information on proteome regulation of the uptake hydrogenase and the bidirectional hydrogenase, as well as systematic data on the hydrogen related metabolism in Anabaena sp. PCC 7120.

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Section: Methodsmentioning

confidence: 99%

Proteome Analysis of Enriched Heterocysts from Two Hydrogenase Mutants from Anabaena sp. PCC 7120

et al. 2019

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“…Chemical labeling enables the enrichment of cell-surface proteins (Nunomura et al, 2005;Wollscheid et al, 2009;Zhang et al, 2003) but does not provide cell-type specificity. Alternative technologies for cell-type-specific proteomics, such as bio-orthogonal metabolic labeling (Alvarez-Castelao et al, 2017;Liu et al, 2018) or organelle tagging (Fecher et al, 2019), are not amenable to the analysis of cell-surface proteomes.…”

Section: Profiling Cell-surface Proteomes and Their Dynamics In Intacmentioning

confidence: 99%

Cell-Surface Proteomic Profiling in the Fly Brain Uncovers New Wiring Regulators

Han

et al. 2019

Preprint

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Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues, and thus govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally-resolved approach to profile cellsurface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed a global downregulation of wiring molecules and an up-regulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 new cellsurface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally-resolved in situ cell-surface proteomic profiling in discovering new regulators of brain wiring.

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“…The temporal resolution of QUAD can identify alterations in protein stability prior to development of disease phenotypes, thus identifying potential targets to ameliorate or prevent pathogenesis. With the development of noncanonical amino acids with cell-type specificity [72], AHA can be replaced to allow QUAD analysis to quantitate cell-specific protein stability in animal models of disease.…”

Section: Overall This Demonstrates How the Quantitation Of Protein Smentioning

confidence: 99%

Quantitative Analysis of Global Protein Stability Rates in Tissues

McClatchy

Gao

Lavalle-Adam

et al. 2019

Preprint

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Protein degradation is an important component of the proteostasis network, but no techniques are available to globally quantitate degradation rates in tissues. In this study, we demonstrate a new method QUAD (Quantification of Azidohomoalanine Degradation) that can accurately quantitate degradation rates in tissues. QUAD analysis of mouse tissues reveal that unique degradation trends can define different tissue proteomes. Within a tissue, specific protein characteristics are correlated with different levels of protein stability. Further investigation of the TRIC chaperonin with strikingly different subunit stabilities suggests a non-canonical chaperonin in brain tissue. Consistent with the theory that the proteostasis network is compromised with age, we discovered that protein stability is globally enhanced in brains of old mice compared to young mice. AbstractProtein degradation is an essential mechanism for maintaining homeostasis in response to internal and external perturbations. Disruption of this process is implicated in many human diseases, but quantitation of global stability rates has not yet been achieved in tissues. We have developed QUAD (Quantification of Azidohomoalanine Degradation), a technique to quantitate global protein degradation using mass spectrometry. Azidohomoalanine (AHA) is pulsed into mouse tissues through their diet. The mice are then returned to a normal diet and the decrease of AHA abundance can be quantitated in the proteome. QUAD analysis reveals that protein stability varied within tissues, but discernible trends in the data suggest that cellular environment is a major factor dictating stability. Within a tissue, different organelles, post-translation modifications, and protein functions were enriched with different stability patterns. Surprisingly, subunits of the TRIC molecular chaperonin possessed markedly distinct stability trajectories in the brain. Further investigation revealed that these subunits also possessed different subcellular localization and expression patterns that were uniquely altered with age and in Alzheimer's disease transgenic mice, indicating a potential non-canonical chaperonin. Finally, QUAD analysis demonstrated that protein stability is enhanced with age in the brain but not in the liver. Overall, QUAD allows the first global quantitation of protein stability rates in tissues, which may lead to new insights and hypotheses in basic and translational research.

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Cell-type-specific metabolic labeling of nascent proteomes in vivo

Cited by 183 publications

References 50 publications

Proteome Analysis of Enriched Heterocysts from Two Hydrogenase Mutants from Anabaena sp. PCC 7120

Proteome Analysis of Enriched Heterocysts from Two Hydrogenase Mutants from Anabaena sp. PCC 7120

Cell-Surface Proteomic Profiling in the Fly Brain Uncovers New Wiring Regulators

Quantitative Analysis of Global Protein Stability Rates in Tissues

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