Mitochondrial redox systems as central hubs in plant metabolism and signaling

Plant mitochondria are indispensable for plant metabolism and are tightly integrated into cellular homeostasis. This review provides an update on the latest research concerning the organization and operation of plant mitochondrial redox systems, and how they affect cellular metabolism and signaling, plant development, and stress responses. New insights into the organization and operation of mitochondrial energy systems such as the tricarboxylic acid cycle and mitochondrial electron transport chain (mtETC) are discussed. The mtETC produces reactive oxygen and nitrogen species, which can act as signals or lead to cellular damage, and are thus efficiently removed by mitochondrial antioxidant systems, including Mn-superoxide dismutase, ascorbate–glutathione cycle, and thioredoxin-dependent peroxidases. Plant mitochondria are tightly connected with photosynthesis, photorespiration, and cytosolic metabolism, thereby providing redox-balancing. Mitochondrial proteins are targets of extensive post-translational modifications, but their functional significance and how they are added or removed remains unclear. To operate in sync with the whole cell, mitochondria can communicate their functional status via mitochondrial retrograde signaling to change nuclear gene expression, and several recent breakthroughs here are discussed. At a whole organism level, plant mitochondria thus play crucial roles from the first minutes after seed imbibition, supporting meristem activity, growth, and fertility, until senescence of darkened and aged tissue. Finally, plant mitochondria are tightly integrated with cellular and organismal responses to environmental challenges such as drought, salinity, heat, and submergence, but also threats posed by pathogens. Both the major recent advances and outstanding questions are reviewed, which may help future research efforts on plant mitochondria.

Plant mitochondria are key components of redox homeostasis and play vital roles in regulating cellular metabolism, thereby affecting development and stress tolerance at the whole plant level.

Advances

• Improved quantitative MS-based approaches have accelerated the study of mitochondrial protein abundance, turnover and PTMs.
• Mitochondrial enzymes and cellular compartments operate interactively and efficiently exchange substrates.
• Roles for mitochondrial retrograde signaling in plant growth, during physiologically relevant stress conditions and in interaction with other organelles such as the chloroplasts, have been clarified.
• Further insights into mitochondrial antioxidant and peroxidase systems and how they affect other redox systems, enzymes, and whole plant growth have been generated.
• Our understanding of how mitochondria help plants power development and cope with adversity has improved.

     

Oxygen and reactive oxygen species-dependent regulation of plant growth and development

Oxygen and reactive oxygen species (ROS) have been co-opted during evolution into the regulation of plant growth, development, and differentiation. ROS and oxidative signals arising from metabolism or phytohormone-mediated processes control almost every aspect of plant development from seed and bud dormancy, liberation of meristematic cells from the quiescent state, root and shoot growth, and architecture, to flowering and seed production. Moreover, the phytochrome and phytohormone-dependent transmissions of ROS waves are central to the systemic whole plant signaling pathways that integrate root and shoot growth. The sensing of oxygen availability through the PROTEOLYSIS 6 (PRT6) N-degron pathway functions alongside ROS production and signaling but how these pathways interact in developing organs remains poorly understood. Considerable progress has been made in our understanding of the nature of hydrogen peroxide sensors and the role of thiol-dependent signaling networks in the transmission of ROS signals. Reduction/oxidation (redox) changes in the glutathione (GSH) pool, glutaredoxins (GRXs), and thioredoxins (TRXs) are important in the control of growth mediated by phytohormone pathways. Although, it is clear that the redox states of proteins involved in plant growth and development are controlled by the NAD(P)H thioredoxin reductase (NTR)/TRX and reduced GSH/GRX systems of the cytosol, chloroplasts, mitochondria, and nucleus, we have only scratched the surface of this multilayered control and how redox-regulated processes interact with other cell signaling systems.

Oxygen and reactive oxygen species regulate plant growth, development, and differentiation through multiple interlinked signaling pathways.

Advances

• Developmentally regulated hypoxia and reactive oxygen species (ROS) production are key features of the stem cell niches, providing information about stem cell position, the environment, and metabolic state.
• Protein cysteine oxidation is central to oxygen and ROS signaling. However, S-nitrosylation, S-glutathionylation, S-sulfhydration, and S-sulfenylation modifications can occur on the same cysteine. The influence of each modification on stability, localization, and function remains unknown.
• Numerous intersecting ROS signaling pathways are probable and likely depend on the site of ROS production and the nature of the oxidized receptor protein. ROS sensors such as the hydrogen peroxide (H₂O₂)-INDUCED Ca²⁺ INCREASES 1 (HPCA1) leucine rich receptor kinase translate redox signals into protein modifications to regulate signaling cascades. H₂O₂ perception/transduction is dependent on thiol-dependent mechanisms policed by the ferredoxin/thioredoxin (TRX), NAD(P)H TRX reductase C (NTRC), reduced glutathione (GSH), and glutaredoxin (GRX) systems.
• ROS waves transmit redox signals from cell to cell in the apoplast, and probably through plasmodesmata. Long-distance transport of H₂O₂ and other ROS, therefore, appears to be unnecessary. Similarly, contact sites between organelles allow ROS transfer.
• Convergence points for oxygen and ROS signaling occur on proteins such as ROH OF PLANT 2 (ROP2) GTPase,RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), and TRX-h to regulate meristematic activity via TARGET OF RAPAMYCIN (TOR) kinase activity.

     

Live monitoring of plant redox and energy physiology with genetically encoded biosensors

Genetically encoded biosensors pave the way for understanding plant redox dynamics and energy metabolism on cellular and subcellular levels.

ADVANCES

• Methodological advances in fluorescent protein-based in vivo biosensing have been instrumental for several paradigm shifts in our understanding of cell physiology, metabolism and signaling.
• An increasing number of genetically encoded biosensors has been used to dissect the dynamics of several distinct redox couples and energy physiology in plants.
• In vivo monitoring using biosensors has pioneered the simultaneous read-out of different physiological parameters in different subcellular locations by parallelized plate reader-based, multiwell fluorimetry, or expression strategies for multiple sensors in parallel.
• Sensing dynamic changes in hydrogen peroxide levels is possible with sensors of the HyPer family, or roGFP fusion variants with a thiol peroxidase.
• Peredox and SoNar family sensors enable direct visualization of NADH/NAD⁺, while iNAP family sensors respond to NADPH concentration in plants.
• Sensor variants with different sensitivity ranges enable use of the most appropriate variant for the specific in vivo environment or experimental scope.

     

Motif-based endomembrane trafficking

Endomembrane trafficking, which allows proteins and lipids to flow between the different endomembrane compartments, largely occurs by vesicle-mediated transport. Transmembrane proteins intended for transport are concentrated into a vesicle or carrier by undulation of a donor membrane. This is followed by vesicle scission, uncoating, and finally, fusion at the target membrane. Three major trafficking pathways operate inside eukaryotic cells: anterograde, retrograde, and endocytic. Each pathway involves a unique set of machinery and coat proteins that pack the transmembrane proteins, along with their associated lipids, into specific carriers. Adaptor and coatomer complexes are major facilitators that function in anterograde transport and in endocytosis. These complexes recognize the transmembrane cargoes destined for transport and recruit the coat proteins that help form the carriers. These complexes use either linear motifs or posttranslational modifications to recognize the cargoes, which are then packaged and delivered along the trafficking pathways. In this review, we focus on the different trafficking complexes that share a common evolutionary branch in Arabidopsis (Arabidopsis thaliana), and we discuss up-to-date knowledge about the cargo recognition motifs they use.

Trafficking protein complexes recognize specific linear motifs or modifications on integral membrane proteins and this recognition guides their transport between the different cellular compartments.

ADVANCED

• Plant research is slowly gaining insight into the linear trafficking motifs used by the various AP complexes.Recent observations point out that steady-state accumulation of cargo proteins at the plasma membrane is not necessarily caused by to impaired internalization.
• TSET/TPC, the most recently identified member of the heterotetrameric adaptor complex-containing coat (HTAC-CC) family, and the identification of an endocytic-autophagosomal degradation pathway operating between the contact sites of the endoplasmic reticulum with the plasma membrane and the vacuole provide previously undiscovered additional layers of complexity to endomembrane trafficking in plants.

     

Redox regulation of chloroplast metabolism

Regulation of enzyme activity based on thiol-disulfide exchange is a regulatory mechanism in which the protein disulfide reductase activity of thioredoxins (TRXs) plays a central role. Plant chloroplasts are equipped with a complex set of up to 20 TRXs and TRX-like proteins, the activity of which is supported by reducing power provided by photosynthetically reduced ferredoxin (FDX) with the participation of a FDX-dependent TRX reductase (FTR). Therefore, the FDX–FTR–TRXs pathway allows the regulation of redox-sensitive chloroplast enzymes in response to light. In addition, chloroplasts contain an NADPH-dependent redox system, termed NTRC, which allows the use of NADPH in the redox network of these organelles. Genetic approaches using mutants of Arabidopsis (Arabidopsis thaliana) in combination with biochemical and physiological studies have shown that both redox systems, NTRC and FDX-FTR-TRXs, participate in fine-tuning chloroplast performance in response to changes in light intensity. Moreover, these studies revealed the participation of 2-Cys peroxiredoxin (2-Cys PRX), a thiol-dependent peroxidase, in the control of the reducing activity of chloroplast TRXs as well as in the rapid oxidation of stromal enzymes upon darkness. In this review, we provide an update on recent findings regarding the redox regulatory network of plant chloroplasts, focusing on the functional relationship of 2-Cys PRXs with NTRC and the FDX–FTR–TRXs redox systems for fine-tuning chloroplast performance in response to changes in light intensity and darkness. Finally, we consider redox regulation as an additional layer of control of the signaling function of the chloroplast.

Thiol-dependent redox regulatory and antioxidant systems act concertedly to modulate chloroplast metabolism and signaling function.

Advances

• Plant chloroplasts harbor a complex redox network composed of the FDX–FTR–TRXs pathway, linking redox regulation to light, and NTRC, an NADPH-dependent system required for the activity of TRXs. Both systems adjust chloroplast performance to environmental cues.
• A relevant function of NTRC is redox control of 2-Cys PRXs, which maintains the reductive activity of chloroplast TRXs in the light. The NTRC–2-Cys PRXs redox system helps fine-tune the redox state of chloroplast enzymes thereby adjusting photosynthetic performance to changes in light.
• 2-Cys PRXs participate in the rapid oxidative inactivation of chloroplast enzymes in the dark, mediating the transfer of reducing equivalents from reduced enzymes, via TRXs, to hydrogen peroxide.
• Involvement of redox regulation in chloroplast retrograde signaling modulates early stages of plant development and response to environmental stress.

     

The redoxome: Proteomic analysis of cellular redox networks

Redox-regulated proteins play fundamentally important roles not only during the defense of organisms against oxidative stress conditions but also as targets of cellular signaling events. This realization has spurred the development of proteomic techniques geared towards characterizing the redoxome; proteins with highly reactive cysteine residues, whose thiol oxidation state controls the function of the proteins, and by extension, the pathways they are part of. We will here summarize the most recent advances made in the field of redox proteomic analysis, aimed to elucidate the cellular redox networks that appear to control prokaryotic and eukaryotic organisms.     

Adaptation of the parasitic plant lifecycle: germination is controlled by essential host signaling molecules

Parasitic plants are plants that connect with a haustorium to the vasculature of another, host, plant from which they absorb water, assimilates, and nutrients. Because of this parasitic lifestyle, parasitic plants need to coordinate their lifecycle with that of their host. Parasitic plants have evolved a number of host detection/host response mechanisms of which the germination in response to chemical host signals in one of the major families of parasitic plants, the Orobanchaceae, is a striking example. In this update review, we discuss these germination stimulants. We review the different compound classes that function as germination stimulants, how they are produced, and in which host plants. We discuss why they are reliable signals, how parasitic plants have evolved mechanisms that detect and respond to them, and whether they play a role in host specificity. The advances in the knowledge underlying this signaling relationship between host and parasitic plant have greatly improved our understanding of the evolution of plant parasitism and are facilitating the development of more effective control measures in cases where these parasitic plants have developed into weeds.

Root parasitic plants grow on the roots of other plants and germinate only in the presence of that host, on which they completely depend, through the perception of host presence signaling molecules called germination stimulants.

Outstanding questions

• Have we overlooked the role of germination stimulants in facultative parasites?
• What is the biological relevance of the observation that many plant species produce and secrete a range of different strigolactones?
• Have parasitic plants evolved mechanisms to compensate for low phosphorus availability, a condition that stimulates their germination?
• What is the contribution of the HTL strigolactone receptors to host specificity in parasitic plants or does downstream signaling play a role?
• What other, nonstrigolactone, germination stimulants can parasitic plants respond to and does this require adaptation in the HTL receptors?
• What is the role of germination and underlying mechanism in the rapid adaptation of (orobanchaceous) parasitic plants to a new host?

     

The mechanism of host-induced germination in root parasitic plants

Chemical signals known as strigolactones (SLs) were discovered more than 50 years ago as host-derived germination stimulants of parasitic plants in the Orobanchaceae. Strigolactone-responsive germination is an essential adaptation of obligate parasites in this family, which depend upon a host for survival. Several species of obligate parasites, including witchweeds (Striga, Alectra spp.) and broomrapes (Orobanche, Phelipanche spp.), are highly destructive agricultural weeds that pose a significant threat to global food security. Understanding how parasites sense SLs and other host-derived stimulants will catalyze the development of innovative chemical and biological control methods. This review synthesizes the recent discoveries of strigolactone receptors in parasitic Orobanchaceae, their signaling mechanism, and key steps in their evolution.

A family of receptors that evolved in the Orobanchaceae family enable seeds of parasitic plants to sense strigolactones from a nearby host root and germinate.

Advances

• Strigolactone perception by parasite seed is mediated by a clade of neofunctionalized KAI2d proteins that evolved from a receptor that mediates karrikin responses in other plants.
• KAI2d proteins use a similar mechanism to perceive SLs as D14, which mediates growth responses to SLs in nonparasites, but activate different signaling pathways.
• Crystal structure analyses and chemical probes reveal features of KAI2d ligand-binding pockets that contribute to their specificity.

     

Peptide-based Interaction Proteomics

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Highlights

• •Peptide-based screens provide a scalable approach to study protein-protein interactions.
• •These screens help to characterize the function of structurally disordered regions.
• •The impact of posttranslational modifications can be directly investigated.

     

Hormonal impact on photosynthesis and photoprotection in plants

Photosynthesis is not only essential for plants, but it also sustains life on Earth. Phytohormones play crucial roles in developmental processes, from organ initiation to senescence, due to their role as growth and developmental regulators, as well as their central role in the regulation of photosynthesis. Furthermore, phytohormones play a major role in photoprotection of the photosynthetic apparatus under stress conditions. Here, in addition to discussing our current knowledge on the role of the phytohormones auxin, cytokinins, gibberellins, and strigolactones in promoting photosynthesis, we will also highlight the role of abscisic acid beyond stomatal closure in modulating photosynthesis and photoprotection under various stress conditions through crosstalk with ethylene, salicylates, jasmonates, and brassinosteroids. Furthermore, the role of phytohormones in controlling the production and scavenging of photosynthesis-derived reactive oxygen species, the duration and extent of photo-oxidative stress and redox signaling under stress conditions will be discussed in detail. Hormones have a significant impact on the regulation of photosynthetic processes in plants under both optimal and stress conditions, with hormonal interactions, complementation, and crosstalk being important in the spatiotemporal and integrative regulation of photosynthetic processes during organ development at the whole-plant level.

In addition to mediating stoma-induced reductions in photosynthesis during stress, phytohormones modulate the spatiotemporal and integrative regulation of photosynthetic and photoprotection processes.

Advances

• Hormones strongly impact photosynthesis, both indirectly and directly.
• Not only CKs, but also auxin, GAs, and SLs are essential to modulate photosynthetic rates under optimal conditions at the whole-plant level.
• ABA, JAs, SA, and ethylene play a major role in the regulation of photosynthesis under various stress conditions.
• An integrated hormonal response at the whole-plant level allows the most adequate photosynthetic response to every developmental and stress situation.

     

Mechanisms of resistance and virulence in parasitic plant–host interactions

Parasitic plants pose a major biotic threat to plant growth and development and lead to losses in crop productivity of billions of USD annually. By comparison with “normal” autotrophic plants, parasitic plants live a heterotrophic lifestyle and rely on water, solutes and to a greater (holoparasitic plants) or lesser extent (hemiparasitic plants) on sugars from other host plants. Most hosts are unable to detect an infestation by plant parasites or unable to fend off these parasitic invaders. However, a few hosts have evolved defense strategies to avoid infestation or protect themselves actively post-attack often leading to full or partial resistance. Here, we review the current state of our understanding of the defense strategies to plant parasitism used by host plants with emphasis on the active molecular resistance mechanisms. Furthermore, we outline the perspectives and the potential of future studies that will be indispensable to develop and breed resistant crops.

Some plants are able to recognize parasitic plants as attacking pathogens and can fend them off by inducing defense responses.

Advances

• Receptor proteins have been discovered in host plants (i.e. sunflower, tomato, or cowpea) that detect parasitic plants as an invading pathogen and further induce plant immunity and resistance responses in hosts leading to a parasite rejection.
• Molecular patterns exist in parasitic plants that can be specifically detected by host plant receptors.
• The host plant receptors require co-receptors and signaling components (i.e. BAK1, SOBIR1, etc.) also known from plant immunity against microbes.
• Parasitic plants evolved strategies to circumvent and to suppress host plant immunity, i.e. by manipulating host cells with siRNAs or proteins that act as effectors.
• Similar to the interaction of plants with microbial pathogens, elements of PTI and ETI can be both observed in plant–parasitic plant interactions.

     

Discovery of a Redox Thiol Switch: Implications for Cellular Energy Metabolism

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Highlights

• •Develop a TMT-based proteomics tool to profile cysteine persulfides in the cellular proteomes.
• •Discover a Redox Thiol Switch from protein S-glutathioinylation to S-persulfidation (RTS^GS) with implications in the regulation of cellular energy metabolism under oxidative stress.

     

Optimal transportation theory for species interaction networks

1. Observed biotic interactions between species, such as in pollination, predation, and competition, are determined by combinations of population densities, matching in functional traits and phenology among the organisms, and stochastic events (neutral effects).
2. We propose optimal transportation theory as a unified view for modeling species interaction networks with different intensities of interactions. We pose the coupling of two distributions as a constrained optimization problem, maximizing both the system''s average utility and its global entropy, that is, randomness. Our model follows naturally from applying the MaxEnt principle to this problem setting.
3. This approach allows for simulating changes in species relative densities as well as to disentangle the impact of trait matching and neutral forces.
4. We provide a framework for estimating the pairwise species utilities from data. Experimentally, we show how to use this framework to perform trait matching and predict the coupling in pollination and host–parasite networks.

The coupling between species in a species interaction network can be modeled using optimal transportation. As an application of the MaxEnt principle, it jointly maximizes interaction utility and entropy. This allows for anticipating how the interaction coupling can change when species abundances change.     

Current progress in Striga management

The Striga, particularly S. he rmonthica, problem has become a major threat to food security, exacerbating hunger and poverty in many African countries. A number of Striga control strategies have been proposed and tested during the past decade, however, further research efforts are still needed to provide sustainable and effective solutions to the Striga problem. In this paper, we provide an update on the recent progress and the approaches used in Striga management, and highlight emerging opportunities for developing new technologies to control this enigmatic parasite.

Advances

• The recently established Striga control technologies, such as push-pull, toothpick, and imidazolinone seed dressing have opened up new opportunities for smallholder farmers to overcome this parasite.
• The development of low-cost and efficient germination stimulants together with an application protocol for rain-fed agriculture has made the suicidal germination strategy a realistic approach.
• Molecular elucidation of strigolactone biosynthesis and perception has led to the development of new chemicals that disrupt the communication between Striga and its hosts.

     

Proteomic identification and quantification of S-glutathionylation in mouse macrophages using resin-assisted enrichment and isobaric labeling

S-Glutathionylation (SSG) is an important regulatory posttranslational modification on protein cysteine (Cys) thiols, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols, and covalent capture of reduced thiols using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was initially validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG modification compared to controls. This approach was extended to identify potential SSG-modified Cys sites in response to H₂O₂, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys sites from 265 proteins that were sensitive to S-glutathionylation in response to H₂O₂ treatment, thus providing a database of proteins and Cys sites susceptible to this modification under oxidative stress. Functional analysis revealed that the most significantly enriched molecular function categories for proteins sensitive to SSG modifications were free radical scavenging and cell death/survival. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of SSG-modified proteins. The analytical strategy also provides a unique approach to determining the major pathways and cellular processes most susceptible to S-glutathionylation under stress conditions.     

Reproducibility,Specificity and Accuracy of Relative Quantification Using Spectral Library-based Data-independent Acquisition

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Highlights

• •Provision of data generated on the basis of a gold standard spike-in sample set.
• •Choice of spectral library has great impact on identification and quantification.
• •DIA is superior to DDA in quantification reproducibility, specificity and accuracy.
• •DIA outperforms DDA in the quantification of low protein amounts.
• •Quantification on peptide level is generally preferable.

     

Oxidative stress activates NORAD expression by H3K27ac and promotes oxaliplatin resistance in gastric cancer by enhancing autophagy flux via targeting the miR-433-3p

Oxaliplatin resistance undermines its curative effects on cancer and usually leads to local recurrence. The oxidative stress induced DNA damage repair response is an important mechanism for inducing oxaliplatin resistance by activating autophagy. ELISA is used to detect target genes expression. TMT-based quantitative proteomic analysis was used to investigate the potential mechanisms involved in NORAD interactions based on GO analysis. Transwell assays and apoptosis flow cytometry were used for biological function analysis. CCK-8 was used to calculate IC50 and resistance index (RI) values. Dual-luciferase reporter gene assay, RIP and ChIP assays, and RNA pull-down were used to detect the interaction. Autophagy flux was evaluated using electron microscope and western blotting. Oxidative stress was enhanced by oxaliplatin; and oxaliplatin resistance gastric cancer cell showed lower oxidative stress. TMT labeling showed that NORAD may regulate autophagy flux. NORAD was highly expressed in oxaliplatin-resistant tissues. In vitro experiments indicate that NORAD knockdown decreases the RI (Resistance Index). Oxaliplatin induces oxidative stress and upregulates the expression of NORAD. SGC-7901 shows enhanced oxidative stress than oxaliplatin-resistant cells (SGC-7901-R). NORAD, activated by H3K27ac and CREBBP, enhanced the autophagy flux in SGC-7901-R to suppress the oxidative stress. NORAD binds to miR-433-3p and thereby stabilize the ATG5- ATG12 complex. Our findings illustrate that NORAD, activated by the oxidative stress, can positively regulate ATG5 and ATG12 and enhance the autophagy flux by sponging miR-433-3p. NORAD may be a potential biomarker for predicting oxaliplatin resistance and mediating oxidative stress, and provides therapeutic targets for reversing oxaliplatin resistance.Subject terms: Gastric cancer, Oncogenes

     

Organellar Maps Through Proteomic Profiling – A Conceptual Guide

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Highlights

• •Organelle profiling maps capture localizations of 1000s of proteins in one experiment.
• •Comparing maps +/− perturbation reveals disease mechanisms & cellular responses.
• •A conceptual guide to planning and interpreting organellar profiling experiments.
• •A cross-study consensus set of human organellar marker proteins.

     

Molecular Dynamics Simulation-assisted Ionic Liquid Screening for Deep Coverage Proteome Analysis

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Highlights

• •Mechanistic insights into ionic liquids and proteins at molecular level.
• •Extractants prescreen for proteome analysis with MD simulation system.
• •A loss-less sample preparation method developed for in-depth proteome profiling.
• •Over 3,300 proteins were confidently identified from 1,000 HeLa cells in a 1 h run.
• •Label-free quantitative proteome analysis of human liver cancer tissues.