A TMT-based quantitative proteomic analysis provides insights into the protein changes in the seeds of high- and low- protein content soybean cultivars

민철우,GUPTARAVI,NGUYEN VAN TRUONG,배진우,고종민,이병원,김선태 한국식물생명공학회 2020 JOURNAL OF PLANT BIOTECHNOLOGY Vol.47 No.3

The presence of high amounts of seed storage proteins (SSPs) improves the overall quality of soybean seeds. However, these SSPs pose a major limitation due to their high abundance in soybean seeds. Although various technical advancements including mass-spectrometry and bioinformatics resources were reported, only limited information has been derived to date on soybean seeds at proteome level. Here, we applied a tandem mass tags (TMT)-based quantitative proteomic analysis to identify the significantly modulated proteins in the seeds of two soybean cultivars showing varying protein contents. This approach led to the identification of 5,678 proteins of which 13 and 1,133 proteins showed significant changes in Daewon (low-protein content cultivar) and Saedanbaek (high-protein content cultivar) respectively. Functional annotation revealed that proteins with increased abundance in Saedanbaek were mainly associated with the amino acid and protein metabolism involved in protein synthesis, folding, targeting, and degradation. Taken together, the results presented here provide a pipeline for soybean seed proteome analysis and contribute a better understanding of proteomic changes that may lead to alteration in the protein contents in soybean seeds.

High-throughput proteome analysis reveals changes of primary metabolism and energy production under artificial aging treatment in Glycine max seeds

민철우,김유지,GUPTARAVI,김소운,한원영,고종민,강항원,윤원병,정명근,김용철,김선태 한국응용생명화학회 2016 Applied Biological Chemistry (Appl Biol Chem) Vol.59 No.6

This study was conducted to obtain basic information on protein profile changes by artificial aging in soybean seeds. Seed proteins were extracted using the protamine sulfate precipitation method, which improves the detection of low-abundance proteins (LAPs) by depleting the major seed storage proteins . Isolated proteins were separated by high-resolution two-dimensional gel electrophoresis (2-DE), and differentially modulated protein spots were identified by MALDI-TOF/TOF. A total of 33 differential proteins were identified of which 31 and 2 showed decreased and increased abundances, respectively. Functional annotation of the identified proteins revealed that proteins were mainly associated with primary metabolism (55%) and response to stimulus (20.9%). Proteins with increased abundance were associated with nutrient reservoir activity (spots 5, 10), while the decreased abundance proteins were mainly involved in the primary metabolism such as carbohydrate metabolic process (spots 1–3, 11), protein folding (spots 6–9, 33), glucose metabolic process (spot 25) oxidoreductase activity (spots 19–24), UDP-glucose pyrophosphorylase activity (spots 12, 13). These results provide information about proteome changes, especially, LAPs during artificial seed aging treatment.

Label-free quantitative proteomic analysis determines changes in amino acid and carbohydrate metabolism in three cultivars of Jerusalem artichoke tubers

민철우,정원용,박지현,문기범,고현준,손정훈,전재흥,김현순,GUPTARAVI,김선태,조혜선 한국식물생명공학회 2019 Plant biotechnology reports Vol.13 No.2

Jerusalem artichoke (JA) tubers are an important bio-economy developing crop because of its invaluable bioproducts in both food and biofuel aspects. However, the molecular mechanism of its tuberization, and the differences among different cultivars have been little studied to date. Therefore, here we selected PJA, DJA, and HJA cultivars of JA tubers, showing variations in their tuber epidermal pigmentation, underground tuberization, and inulin content. A comparative proteome analysis led to the identification of 402 proteins in the tubers of which 114 were significantly modulated among different cultivars. Gene Ontology (GO) analysis showed proteins related to the biosynthesis of amino acids and carbohydrate metabolism were differentially modulated in the tubers of three cultivars. Results from the inulin content measurement and proteome analysis suggest that Sucrose:sucrose 1-fructosyltransferase (1-SST) prioritizes inulin biosynthesis rather than rate-limiting enzyme fructan:fructan 1-fructosyltransferases (1-FFT). Furthermore, we confirmed the relationship between transcript-protein expression levels was in discord within inulin biosynthesis enzymes 1-SST and 1-FFT with the terms in previous RT-qPCR results using the same tubers. Our data represent the first report of comparative tuber proteome profiling of different JA and provide the metabolic and molecular basis for understanding carbohydrate metabolism in the tuber tissue.

단백질체학적 통합접근을 통한 인삼의 생육단계별 염 스트레스 반응 단백질 동정

김소운,민철우,라비굽타,조익현,방경환,김선태 한국약용작물학회 2015 한국약용작물학술대회 발표집 Vol.2015 No.10

초기 염류 스트레스 반응 인삼 잎 단백질체 분석

김소운,민철우,GUPTARAVI,조익현,방경환,김영창,김기홍,김선태 한국약용작물학회 2014 한국약용작물학회지 Vol.22 No.5

Salt stress is one of the major abiotic stresses affecting the yield of ginseng (Panax ginseng C. A. Meyer). Theobjective of this study was to identify bio-marker, which is early responsive in salt stress in ginseng, using proteomicsapproach. Ginseng plants were exposed to 5 ds/m salt concentration and samples were harvested at 0, 6, 12 and 18 hoursafter exposure. Total proteins were extracted from ginseng leaves treated with salt stress using Mg/NP-40 buffer and wereseparated on high resolution 2-DE. Approximately 1003 ± 240 (0 h), 992 ± 166 (6 h), 1051 ± 51 (12 h) and 990 ± 160 (18 h)spots were detected in colloidal CBB stained 2D maps. Among these, 8 spots were differentially expressed and were identifiedby using MALDI-TOF/TOF MS or/and LC-MS/MS. Ethylene response sensor-1 (spot GL 1), nucleotide binding protein(spot GL 2), carbonic anhydrase-1 (spot GL 3), thylakoid lumenal 17.9 kDa protein (spot GL 4) and Chlorophyll a/b bindingprotein (spot GL 5, GL 6) were up-regulated at the 12 and 18 hour, while RuBisCO activase B (spot GL 7) and DNA helicase(spot GL 8) were down-

Ginseng (Panax sp.) proteomics: an update

김수원,이서현,민철우,조익현,방경환,현동윤,Ganesh Kumar Agrawal,Randeep Rakwal,Sajad Majeed Zargar,GUPTARAVI,김선태 한국응용생명화학회 2017 Applied Biological Chemistry (Appl Biol Chem) Vol.60 No.3

Panax ginseng, commonly known as ginseng, is a well-known medicinal plant that has been used as traditional medicine in China and Korea. Research in the past few decades supports the pharmacological effects of ginseng. For example, ginseng roots (extracts) exhibit multiple medicinal effects, such as anticancer, antiaging, and protection against circulatory shock, in humans. In this review, we summarize the progress made so far in the ginseng proteomics, starting from sample preparation to establishments of proteomes and databases. Both gel-based (1-DE and 2-DE in combination with LC–MS/MS) and gel-free proteomics technologies have been applied on wide range of samples, collected during different growth and developmental stages and under normal or adverse stress conditions. In particular, comparative proteome analysis has been carried out to investigate the protein profiles of Oriental, American and Indian ginsengs using majorly root and leaf tissues. Moreover, identification of stress-responsive proteins was a key focus that led to the detection of some of the common proteins such as heat shock protein (HSP), ATPase, enolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and ribonuclease/ ginseng major protein (GMP). Acquired proteomicsbased knowledge has been very fruitful in providing better insight into the ginseng biology, opening a door for comparative and translation research of other important medicinal plants. However, due to the fact that proteins undergo various post-transcriptional and post-translational modifications, obtained proteomics data do not always complement the transcriptomics data perfectly; therefore, future efforts would require the utilization of an integrated/holistic molecular-genetic (or omics) approach to explore the biology of this golden plant.

고려인삼의 건조 스트레스 처리에 따른 단백질체 비표지 정량분석

정주용(Ju Young Jung),민철우(Cheol Woo Min),신혜원(Hye Won Shin),Truong Van Nguyen,김지현(Ji hyun Kim),김의정(Eui Jung Kim),조익현(Ick Hyun Jo),김유진(Yu Jin Kim),김선태(Sun Tae Kim) 한국약용작물학회 2021 한국약용작물학회지 Vol.29 No.6

Background: Korean ginseng (Panax ginseng Meyer) has long been cultivated as an important medicinal plant. Moderate water loss resulting form drought stress can impairs the growth of ginseng and lead to yield reduction. However, the mechanisms by which drought stress affects ginseng at the proteome level remain pooly undersood. Methods and Results: We carried out label-free quantitative proteomic analysis of ginseng roots subjected to drought stress (grown at less than 10% soil moisture for 2 weeks) and, compared the results with a control samples of ginseng grown at 25% soil moisture. The analysis was carried out using liquid chromatography with tandem mass spectrometry. A total of 2,471 proteins were identified, and 195 of which showed significant modulation. Functional classification revealed that proteins involved in calcium signaling, and photosynthesis and, production of secondary metabolites were enriched in the control sample (cluster_1), whereas proteins associated with stress response redox reaction, electron transport, and protein synthesis were enriched in the drought-stressed root (cluster_2). Conclusions: Our results provide an overview of drought-induced proteomic changes in ginseng root, and illuminate their correlation with physiological changes, revealing potential proteomic markers of drought stress in ginseng.

Sound wave affects the expression of ethylene biosynthesis-related genes through control of transcription factors RIN and HB-1

Joo Yeol Kim,Hye Ryun Ahn,김선태,민철우,이수인,김진아,박수철,정미정 한국식물생명공학회 2016 Plant biotechnology reports Vol.10 No.6

We previously reported that sound wave treatment (1 kHz) delays fruit ripening in tomato (Solanum lycopersicum), affecting the expression of ethylene biosynthesis-related genes encoding 1-aminocyclopropane1-carboxylic acid (ACC) synthases (ACS) and ACC oxidases (ACO). In this study, we investigated the activity of the transcription factors RIN and HB-1, which function in the ethylene biosynthetic pathway, in response to sound treatment. To investigate whether RIN and HB-1 directly activate the transcription of ACS and ACO, we performed transcriptional activation analysis in Arabidopsis thaliana leaf protoplasts, transiently expressing RIN or HB-1 and using reporter constructs with promoters of the tomato ACS and ACO genes. Activation of the endogenous AtACS and AtACO genes was also measured by qPCR. The RIN- and HB-1-induced expression of these genes decreased, but the HB-1-induced expression of some genes increased after sound treatment. To confirm these results, we performed transient assays in Nicotiana tabacum, which produced results similar to those observed in Arabidopsis. The major ethylene biosynthesis-related genes harbor a CArG-box as a RIN-binding motif. These findings indicate that RIN and HB-1 affect the expression of ethylene biosynthesis-related genes in response to sound treatment, and they suggest that RIN may regulate the ethylene biosynthesis-related genes by binding to their CArG-boxes.

Development of a Simple and Reproducible Method for Removal of Contaminants from Ginseng Protein Samples Prior to Proteomics Analysis

Ravi Gupta(굽타 라비),So Wun Kim(김소운),Chul Woo Min(민철우),Gi-Ho Sung(성기호),Ganesh Kumar Agrawal(아그라왈 가네시 쿠마르),Randeep Rakwal(락왈 랜딥),Ick Hyun Jo(조익현),Kyong Hwan Bang(방경환),Young-Chang Kim(김영창),Kee-Hong Kim(김기홍 한국생명과학회 2015 생명과학회지 Vol.25 No.7

본 연구는 인삼의 잎과 뿌리 단백질 추출물에서 활성탄을 이용하여 염, 계면활성제, 색소를 제거하여 단백질체 분석 연구에 미치는 잠재적인 효과에 대한 평가를 기술하고 있다. 5%(w/v) 활성탄(100-400 mesh)과 함께 단백질 추출물을 30분간 4°C에서 반응시켜 염과 계면활성제를 제거한 후 SDS-PAGE를 분석하여 단백질의 양상을 관찰하였다. 엽록소 함량의 분석은 활성탄 처리 후 엽록소의 상당한 양(~33%)이 제거되는 것을 보여주었고, 이 분석은 염, 계면활성제 제거만이 아닌 색소의 제거에서도 활성탄의 잠재적 효과가 있음을 보여 주고 있다. 활성탄을 처리한 단백질 시료를 이용하여 이차원 전기영동과 PCA 통계분석을 시행한 결과 단백질은 gel에서 더 나은 해상도를 보여주었으며 단백질 시료의 정제에서도 활성탄의 효과가 있음을 확인하였다. 종합적으로, 이 결과들은 활성탄을 이용한 간단한 방법으로 다양한 식물 조직의 단백질 추출물에서 염, 계면활성제, 색소를 제거함으로써 고해상도의 단백질체 분석에 적용될 수 있을 것으로 기대된다. This study describes the effects of activated charcoal on the removal of salts, detergents, and pigments from protein extracts of ginseng leaves and roots. Incubation of protein extracts with 5% (w/v) activated charcoal (100-400 mesh) for 30 min at 4°C almost removed the salts and detergents including NP-40 as can be observed on SDS-PAGE. In addition, analysis of chlorophyll content showed significant depletion of chlorophyll (~33%) after activated charcoal treatment, suggesting potential effect of activated charcoal on removal of pigments too along with the salts and detergents. 2-DE analysis of activated charcoal treated protein samples showed better resolution of proteins, further indicating the efficacy of activated charcoal in clearing of protein samples. In case of root proteins, although not major differences were observed on SDS-PAGE, 2-DE gels showed better resolution of spots after charcoal treatment. In addition, both Hierarchical clustering (HCL) and Principle component analysis (PCA) clearly separated acetone sample from rest of the samples. Phenol and AC-phenol samples almost overlapped each other suggesting no major differences between these samples. Overall, these results showed that activated charcoal can be used in a simple manner to remove the salts, detergents and pigments from the protein extracts of various plant tissues.

벼의 차세대 단백질체 분석을 위한 질량분석기 호환의 광분해성 계면활성제의 적용

신혜원(Hye Won Shin),응웬반쯔엉(Truong Van Nguyen),정주용(Ju Young Jung),이기현(Gi Hyun Lee),장정우(Jeong Woo Jang),윤진미(Jinmi Yoon),라비굽타(Ravi Gupta),김선태(Sun Tae Kim),민철우(Cheol Woo Min) 한국식물생명공학회 2021 JOURNAL OF PLANT BIOTECHNOLOGY Vol.48 No.3

The solubilization of isolated proteins into the adequate buffer containing of surfactants is primary step for proteomic analysis. Particularly, sodium dodecyl sulfate (SDS) is the most widely used surfactant, however, it is not compatible with mass spectrometry (MS). Therefore, it must be removed prior to MS analysis through rigorous washing, which eventually results in inevitable loss. Recently, photocleavable surfactant, 4-hexylphenylazosulfonate (Azo), was reported which can be easily degraded by UV irradiation and is compatible with MS during proteomic approach using animal tissues. In this study, we employed comparative label-free proteomic analysis for evaluating the solubilization efficacy of the Azo and SDS surfactants using rice leave samples. This approach led to identification of 3,365 proteins and out of 682 proteins were determined as significantly modulated. Further, according to the subcellular localization prediction in SDS and Azo, proteins localized in the chloroplast were the major organelle accounting for 64% of the total organelle in the SDS sample, while only 37.5% of organelle proteins solubilized in the Azo were predicted to be localized in chloroplast. Taken together, this study validates the efficient solubilization of total protein isolated from plant material for bottom-up proteomics. Azo surfactant is suitable as substituents of SDS and promising for bottomup proteomics as it facilitates robust protein extraction, rapid washing step during enzymatic digestion, and MS analysis.