Chemical proteomics for subcellular proteome analysis

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Protein functions are tightly regulated by their subcellular localization and dynamic alteration. Chemical proteomics offers convenience and efficiency for profiling protein features in a native context. In this review, we summarize the recent progress of subcellular-compartment-focused chemical proteomics which do not rely on organelle fractionation. Organelle-specific activity-based protein profiling (ABPP) and engineered ascorbate peroxidase (APEX) have been developed for proteome analysis within organelles and even sub-organelles. In parallel, our lab designed organelle-localizable reactive molecules (ORMs) to selectively tag nuclear and mitochondrial proteins. ORMs-based proteomics is applicable to primary neurons and brain slices, as well as cultured cell lines. In addition, we invented a conditional proteomics approach to elucidate zinc homeostasis by labeling and identifying proteins localized in Zn2+-rich space of live cells.

Introduction

Protein functions are closely associated with its subcellular distribution in live cells. Each subcellular compartment, or organelle, contains varied protein compositions that underlie the diversity of biochemical reactions in a single cell. Protein localization and expression level are dynamically varied in response to environmental changes. The spatiotemporal changes of proteomes reflect the biological states of resided organelles. Organelle-focused proteomics relying on subcellular fractionation have been used to identify the components of cellular organelles [1, 2, 3]. However, the conventional methods [1] often suffer from the limited specificity and the low coverage. Moreover, they can neither accurately report temporal dynamics of proteins, because of the time-consuming biochemical purification processes, nor readily access the sub-organelle proteomes.

Chemical proteomics is now a powerful strategy for the focused protein profiling [4, 5, 6]. The proteome of interest is covalently tagged with chemical reagents in live cells, followed by standard enrichment and mass spectrometry (MS) analysis, which allows for fixing protein information before cell lysis and, thus is able to obtain a snapshot of dynamically altered subcellular proteomes that cannot be addressed by the organelle fractionation. Organelle-focused chemical proteomics exploits spatially limited reactions by directing labeling reagents or enzymes to specific subcellular compartments. These may provide organelle and even sub-organelle proteome mapping with high spatiotemporal resolutions.

In addition to subcellular localization, cellular microenvironments, such as pH, concentrations of metals, hypoxia/hyperoxia and redox states, tightly regulate local protein structures and activities. Such environmental conditions are spatially heterogeneous and dynamically fluctuated in live cells and tissues. Useful methods to precisely address the local proteomes are enormously desirable for comprehensive elucidation of proteome dynamics. We firstly proposed a strategy termed ‘conditional proteomics’ [7••] as a powerful approach to selectively label and identify the conditional proteomes and profile their dynamics.

In this review, we introduce the recent progress of chemical proteomics that focus on subcellular compartments, including organelle-focused proteomics and conditional proteomics (Figure 1).

Section snippets

Organelle-specific ABPP

Activity-based protein profiling (ABPP) represents one of the most successful examples of chemical proteomics, which was invented by Cravatt’s group [8,9]. Proteins exhibiting an enzymatic activity-of-interest are selectively labeled by activity-based probes (ABPs) through a bioorthogonal reaction. For organelle-specific ABPP, Wright and co-workers developed a lysosome-targeting ABP by conjugating a weakly basic amine (DAMP) to a cathepsin-reactive warhead [10]. Liquid chromatography

Conditional proteomics

As described above, organelle-focused chemoproteomic approaches enabled to profile protein localizations, the dynamically altered expression levels of organelle proteins and these activities. Recently, we reported a new method that can explore an ‘environment’ in which proteins exist in live cells [7••].

It is now discussed that Zn (II) ion (Zn2+) acts as a signaling molecule inside and outside cells, and the local concentration of Zn2+ dynamically changes. Zinc signaling and dynamics play

Conclusions and perspectives

Chemical proteomics has been proved powerful for subcellular proteome analysis. It does not rely on conventional organelle purification and can provide detailed insights into the microenvironments of diverse subcellular compartments. Proximity labeling, especially by APEX, have been successfully applied to high-resolution proteome mapping restricted in specific organelles and even organelle sub-compartments. The two types of chemical proteomics developed by our group, using ORMs or AIZin, do

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank Dr. Yuki Yasueda and Dr. Takayuki Miki for their contributions to the development of ORMs-based organelle focused proteomics and zinc conditional proteomics. This work was supported by Grant-in-Aid for JSPS fellows (17F17344) to Hao Zhu, Grant-in-Aid for Young Scientists (B) (18K14334) to Tomonori Tamura, and the Japan Science and Technology Agency (JST) Core Research for Evolutional Science and Technology (CREST) to Itaru Hamachi. This work was also supported by a Grant-in-Aid for

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