Minimal sample requirement for highly multiplexed protein quantification in cell lines and tissues by PCT‐SWATH mass spectrometry

The amount of sample available for clinical and biological proteomic research is often limited and thus significantly restricts clinical and translational research. Recently, we have integrated pressure cycling technology (PCT) assisted sample preparation and SWATH‐MS to perform reproducible proteomic quantification of biopsy‐level tissue samples. Here, we further evaluated the minimal sample requirement of the PCT‐SWATH method using various types of samples, including cultured cells (HeLa, K562, and U251, 500 000 to 50 000 cells) and tissue samples (mouse liver, heart, brain, and human kidney, 3–0.2 mg). The data show that as few as 50 000 human cells and 0.2–0.5 mg of wet mouse and human tissues produced peptide samples sufficient for multiple SWATH‐MS analyses at optimal sample load applied to the system. Generally, the reproducibility of the method increased with decreasing tissue sample amounts. The SWATH maps acquired from peptides derived from samples of varying sizes were essentially identical based on the number, type, and quantity of identified peptides. In conclusion, we determined the minimal sample required for optimal PCT‐SWATH analyses, and found smaller sample size achieved higher quantitative accuracy.     

Analysis of the protein expression profiling during rice callus differentiation under different plant hormone conditions

Plant hormones function to coordinate plant growth and development. While the plant hormones, mainly auxin and cytokinin, are exogenously added to various plant tissue cultures, their effects on the organogenesis are apparent, but little is known concerning the molecular mechanisms by which they function in cultured cells. Rice, as a model plant in monocots, is also suitable to tissue culture studies. Here, we used four types of regeneration mediums with different relative concentrations of cytokinin and auxin for rice callus differentiation, the calli at different differentiation stages were collected for proteomic analysis. 2-dimensional electrophoresis revealed that 213 protein spots significantly differentially expressed during callus differentiation under different hormone conditions. By using mass spectrometry, 183 differentially expressed protein spots were identified to match 157 unique proteins. Most of these differential proteins were cellular/metabolic process-related proteins, whose different expression patterns may be correlated with the cytokinin and auxin regulation. Several hormone-related proteins were prominently featured in differentiated calli as compared with the initiated calli, such as alpha-amylase isoforms, mannose-binding rice lectin, putative dehydration stress-induced protein, cysteine endopeptidase and cystatin. All these results provide a novel insight into how the two plant hormones effect the callus differentiation in rice on the proteomic level.     

Protein interactome analysis of 12 mitogen‐activated protein kinase kinase kinase in rice using a yeast two‐hybrid system

The mitogen‐activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K‐interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two‐hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K‐interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full‐length cDNA in the rice KOME ( http://cdna01.dna.affrc.go.jp/cDNA/ ) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead‐associated domain‐containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K‐interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors.     

Proteomic analysis of changes in mitochondrial protein expression during fruit senescence

Fruit senescence has been reported to be an oxidative phenomenon, but the detailed mechanisms by which ROS regulate this process remain largely unknown. Here we show that senescence process of apple fruit was concomitant with the dynamic alterations in the mitochondrial proteome. Mitochondrial proteins involved in tricarboxylic acid cycle, electron transport chain, carbon metabolism, and stress response were found to be differentially expressed during fruit senescence. Alleviating oxidative stress by lowering the ambient oxygen concentration noticeably decreased the number of changed proteins and delayed fruit senescence, indicating the involvement of ROS in this process. To further investigate the regulatory effect of ROS on senescence process, we analyzed the mitochondrial proteome variations upon exposure to high oxygen (100%), which induces oxidative stress and accelerates fruit senescence. High oxygen treatment led to a further identification of differentially expressed proteins such as mitochondrial manganese superoxide dismutase, an antioxidant scavenging superoxide radicals produced in the mitochondria. Activity of manganese superoxide dismutase was reduced after high oxygen exposure, accompanied by an increase in oxidative protein carbonylation (damaged proteins). These data suggest that ROS may regulate fruit senescence by changing expression profiles of specific mitochondrial proteins and impairing the biological function of these proteins.     

Analysis of protein carbonylation — pitfalls and promise in commonly used methods

Abstract

Oxidation of proteins has received a lot of attention in the last decades due to the fact that they have been shown to accumulate and to be implicated in the progression and the pathophysiology of several diseases such as Alzheimer, coronary heart diseases, etc. This has also resulted in the fact that research scientists are becoming more eager to be able to measure accurately the level of oxidized protein in biological materials, and to determine the precise site of the oxidative attack on the protein, in order to get insights into the molecular mechanisms involved in the progression of diseases. Several methods for measuring protein carbonylation have been implemented in different laboratories around the world. However, to date no methods prevail as the most accurate, reliable, and robust. The present paper aims at giving an overview of the common methods used to determine protein carbonylation in biological material as well as to highlight the limitations and the potential. The ultimate goal is to give quick tips for a rapid decision making when a method has to be selected and taking into consideration the advantage and drawback of the methods.     

SWATH‐based quantitative proteomics reveals the mechanism of enhanced Bombyx mori nucleopolyhedrovirus‐resistance in silkworm reared on UV‐B treated mulberry leaves

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most acute infectious diseases in silkworm, which has led to great economic loss in sericulture. Previous study showed that the content of secondary metabolites in mulberry leaves, particularly for moracin N, was increased after UV‐B irradiation. In this study, the BmNPV resistance of silkworms reared on UV‐B treated and moracin N spread mulberry leaves was improved. To uncover the mechanism of enhanced BmNPV resistance, silkworm midguts from UV‐B treated mulberry leaves (BUM) and moracin N (BNM) groups were analyzed by SWATH‐based proteomic technique. Of note, the abundance of ribosomal proteins in BUM and BNM groups was significantly changed to maintain the synthesis of total protein levels and cell survival. While, cytochrome c oxidase subunit II, calcium ATPase and programmed cell death 4 involved in apoptotic process were up‐regulated in BNM group. Expressions of lipase‐1, serine protease precursor, Rab1 protein, and histone genes were increased significantly in BNM group. These results suggest that moracin N might be the main active component in UV‐B treated mulberry leaves which could improve the BmNPV‐resistance of silkworm through promoting apoptotic cell death, enhancing the organism immunity, and regulating the intercellular environment of cells in silkworm. It also presents an innovative process to reduce the mortality rate of silkworms infected with BmNPV.     

Shotgun proteomic analysis of soybean embryonic axes during germination under salt stress

Seed imbibition and radicle emergence are generally less affected by salinity in soybean than in other crop plants. In order to unveil the mechanisms underlying this remarkable salt tolerance of soybean at seed germination, a comparative label‐free shotgun proteomic analysis of embryonic axes exposed to salinity during germination sensu stricto (GSS) was conducted. The results revealed that the application of 100 and 200 mmol/L NaCl stress was accompanied by significant changes (>2‐fold, P<0.05) of 97 and 75 proteins, respectively. Most of these salt‐responsive proteins (70%) were classified into three major functional categories: disease/defense response, protein destination and storage and primary metabolism. The involvement of these proteins in salt tolerance of soybean was discussed, and some of them were suggested to be potential salt‐tolerant proteins. Furthermore, our results suggest that the cross‐protection against aldehydes, oxidative as well as osmotic stress, is the major adaptive response to salinity in soybean.     

Comparative proteomic analysis of methyl jasmonate‐induced defense responses in different rice cultivars

Jasmonate is an important endogenous chemical signal that plays a role in modulation of plant defense responses. To understand its mechanisms in regulation of rice resistance against the fungal pathogen Magnaporthe oryzae, comparative phenotype and proteomic analyses were undertaken using two near‐isogenic cultivars with different levels of disease resistance. Methyl‐jasmonate (MeJA) treatment significantly enhanced the resistance against M. oryzae in both cultivars but the treated resistant cultivar maintained a higher level of resistance than the same treated susceptible cultivars. Proteomic analysis revealed 26 and 16 MeJA‐modulated proteins in resistant and susceptible cultivars, respectively, and both cultivars shared a common set of 13 proteins. Cumulatively, a total of 29 unique MeJA‐influenced proteins were identified with many of them known to be associated with plant defense response and ROS accumulation. Consistent with the findings of proteomic analysis, MeJA treatment increased ROS accumulation in both cultivars with the resistant cultivar showing higher levels of ROS production and cell membrane damage than the susceptible cultivar. Taken together, our data add a new insight into the mechanisms of overall MeJA‐induced rice defense response and provide a molecular basis of using MeJA to enhance fungal disease resistance in resistant and susceptible rice cultivars.     

Stored cysteine proteinases start globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch (Vicia sativa L.) seed germination

Inhibition of protein synthesis by cycloheximide during vetch seed germination, did not prevent globulin breakdown as indicated by a decrease in vicilin- and legumin-specific immunosignals on Western blots. Protein bodies isolated from embryo axes and cotyledons of dry vetch (Vicia sativa L.) seeds using a non-aqueous method were found to be free of cytoplasmic and organellar contaminations. Lysates of these purified protein bodies were capable of degrading globulins; this process was blocked by the cysteine proteinase (CPR) inhibitor iodoacetic acid. Protein bodies contained the papain-like CPR2 and CPR4, and the legumain-like CPR VsPB2. In vitro assays showed that albumin extracts from protein bodies degraded oligopeptide substrates in the PepTag-Assay and degraded the legumain substrate N-benzoyl-asparaginyl-p-nitroanilide. We conclude that, during germination, globulin mobilization is initiated by stored CPRs in protein bodies of embryonic axes as well as cotyledons, and that de-novo-formed proteolytic enzymes mainly mediate bulk degradation of stored globulin in cotyledons after germination. Received: 14 February 2000 / Accepted: 16 August 2000     

Identification of centromeric and telomeric DNA‐binding proteins in rice

Centromeres and telomeres are DNA/protein complexes and essential functional components of eukaryotic chromosomes. Previous studies have shown that rice centromeres and telomeres are occupied by CentO (rice centromere satellite DNA) satellite and G‐rich telomere repeats, respectively. However, the protein components are not fully understood. DNA‐binding proteins associated with centromeric or telomeric DNAs will most likely be important for the understanding of centromere and telomere structure and functions. To capture DNA‐specific binding proteins, affinity pull‐down technique was applied in this study to isolate rice centromeric and telomeric DNA‐binding proteins. Fifty‐five proteins were identified for their binding affinity to rice CentO repeat, and 80 proteins were identified for their binding to telomere repeat. One CentO‐binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this protein, and proven to be responsible for the specific binding to CentO in vitro. Four proteins identified as telomere DNA‐binding proteins in this study were reported by different groups previously. These results demonstrate that DNA affinity pull‐down technique is effective in the isolation of sequence‐specific binding proteins and will be applicable in future studies of centromere and telomere proteins.     

Patterns of protein carbonylation following oxidative stress in wild-type and<Emphasis Type="Italic"> sigB Bacillus subtilis</Emphasis> cells

Oxidative stress causes damage to nucleic acids, membrane lipids and proteins. One striking effect is the metal-catalyzed, site-specific carbonylation of proteins. In the gram-positive soil bacterium Bacillus subtilis, the PerR-dependent specific stress response and the ^B-dependent general stress response act together to make cells more resistant to oxidative stress. In this study, we analyzed the carbonylation of cytoplasmic proteins in response to hydrogen peroxide stress in B. subtilis. Furthermore, we asked whether the ^B-dependent response to oxidative stress also confers protection against protein carbonylation. To monitor the amount and specificity of protein damage, carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and the resulting stable hydrazones were detected by immunoanalysis of proteins separated by one- or two-dimensional gel electrophoresis. The overall level of protein carbonylation increased strongly in cells treated with hydrogen peroxide. Several proteins, including the elongation factors EF-G, TufA and EF-Ts, were found to be highly carbonylated. Induction of the peroxide specific stress response by treatment with sub-lethal peroxide concentrations, prior to exposure to otherwise lethal levels of peroxide, markedly reduced the degree of protein carbonylation. Cells starved for glucose also showed only minor amounts of peroxide-mediated protein carbonylation compared to exponentially growing cells. We could not detect any differences between wild-type and sigB cells starved for glucose or preadapted by heat treatment with respect to the amount or specificity of protein damage incurred upon subsequent exposure to peroxide stress. However, artificial preloading with proteins that are normally induced by ^B-dependent mechanisms resulted in a lower level of protein carbonylation when cells were later subjected to oxidative stress.Communicated by W. Goebel     

Protein precipitation of diluted samples in SDS‐containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach

Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest, especially because of the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work, two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in very diluted samples solubilized in a strong buffer (containing SDS). The amount of protein recovered after acetone and TCA/acetone precipitation was assessed, as well as the protein identification and relative quantification by SWATH‐MS yields were compared with the results from the same sample without precipitation. From this study, it was possible to conclude that in the case of diluted samples in denaturing buffers, the use of cold acetone as precipitation protocol is more favourable than the use of TCA/acetone in terms of reproducibility in protein recovery and number of identified and quantified proteins. Furthermore, the reproducibility in relative quantification of the proteins is even higher in samples precipitated with acetone compared with the original sample.     

Analysis of the rat hypothalamus proteome by data‐independent label‐free LC‐MS/MS

Studies of neuronal, endocrine, and metabolic disorders would be facilitated by characterization of the hypothalamus proteome. Protein extracts prepared from 16 whole rat hypothalami were measured by data‐independent label‐free nano LC‐MS/MS. Peptide features were detected, aligned, and searched against a rat Swiss‐Prot database using ProteinLynx Global Server v.2.5. The final combined dataset comprised 21 455 peptides, corresponding to 622 unique proteins, each identified by a minimum of two distinct peptides. The majority of the proteins (69%) were cytosolic, and 16% were membrane proteins. Important proteins involved in neurological and synaptic function were identified including several members of the Ras‐related protein family and proteins involved in glutamate biosynthesis.     

Increased protein carbonylation in leaves of Arabidopsis and soybean in response to elevated [CO2]

While exposure of C3 plants to elevated [CO2] would be expected to reduce production of reactive oxygen species (ROS) in leaves because of reduced photorespiratory metabolism, results obtained in the present study suggest that exposure of plants to elevated [CO2] can result in increased oxidative stress. First, in Arabidopsis and soybean, leaf protein carbonylation, a marker of oxidative stress, was often increased when plants were exposed to elevated [CO2]. In soybean, increased carbonyl content was often associated with loss of leaf chlorophyll and reduced enhancement of leaf photosynthetic rate (Pn) by elevated [CO2]. Second, two-dimensional (2-DE) difference gel electrophoresis (DIGE) analysis of proteins extracted from leaves of soybean plants grown at elevated [CO2] or [O3] revealed that both treatments altered the abundance of a similar subset of proteins, consistent with the idea that both conditions may involve an oxidative stress. The 2-DE analysis of leaf proteins was facilitated by a novel and simple procedure to remove ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from soluble soybean leaf extracts. Collectively, these findings add a new dimension to our understanding of global change biology and raise the possibility that oxidative signals can be an unexpected component of plant response to elevated [CO2].     

Comparative proteomics of dehydration response in the rice nucleus: New insights into the molecular basis of genotype‐specific adaptation

Dehydration is the most crucial environmental factor that considerably reduces the crop harvest index, and thus has become a concern for global agriculture. To better understand the role of nuclear proteins in water‐deficit condition, a nuclear proteome was developed from a dehydration‐sensitive rice cultivar IR‐64 followed by its comparison with that of a dehydration‐tolerant c.v. Rasi. The 2DE protein profiling of c.v. IR‐64 coupled with MS/MS analysis led to the identification of 93 dehydration‐responsive proteins (DRPs). Among those identified proteins, 78 were predicted to be destined to the nucleus, accounting for more than 80% of the dataset. While the detected number of protein spots in c.v. IR‐64 was higher when compared with that of Rasi, the number of DRPs was found to be less. Fifty‐seven percent of the DRPs were found to be common to both sensitive and tolerant cultivars, indicating significant differences between the two nuclear proteomes. Further, we constructed a functional association network of the DRPs of c.v. IR‐64, which suggests that a significant number of the proteins are capable of interacting with each other. The combination of nuclear proteome and interactome analyses would elucidate stress‐responsive signaling and the molecular basis of dehydration tolerance in plants.