Prognosis and diagnosis of male fertility is a most important for animal breeding system and human reproduction. Conventional semen analysis generally provides information on the quantitative parameters of spermatozoa, but yields no information concerning its functional competence. Thus, new methods for diagnosis and prognosis of male fertility will need to be developed to ensure more accurate assessments. Proteomics have used to find candidate biomarkers for male fertility, but the relationship between the proteome and fertility was not fully understood. Therefore, we performed a comprehensive proteomic approach to investigate small and large litter size boar spermatozoa and identify proteins related to negative male fertility. In present study, 20 proteins showed differential expression levels in small and large litter size groups. Nineteen of these proteins were abundantly expressed in the small litter group. Interestingly, only one protein was highly expressed in the large litter size spermatozoa. We then identified signaling pathways associated with the differentially expressed protein markers. Glutathione S-transferase Mu3 and glutathione peroxidase 4 were related to the glutathione metabolic pathway and arginine vasopressin receptor 2 was linked to vasopressin R2/STAT. Taken together, our results suggest that identified negative fertility-related biomarkers may be used as negative biomarkers for the detection of inferior male fertility such as sub-fertility or infertility.

The roots of Platycodon grandiflorum species either dried or fresh, are used as an ingredient in salads and traditional cuisine in Korea. To interpret the root proteins, a systematical and targeting analysis were carried out from diploid and tetraploid roots. Two dimensional gels stained with CBB, a total of 39 differential expressed proteins were identified from the diploid root under in vivo condition using image analysis by Progenesis Same Spot software. Out of total differential expressed spots, 39 differential expressed protein spots (≥ 1.5-fold) were analyzed using LTQ-FTICR mass spectrometry. Except two proteins, the rest of the identified proteins were confirmed as down-regulated such as Isocitrate dehydrogenase, Proteasome subunit alpha type-2-B. However, the most of the identified proteins from the explants were mainly associated with the oxidoreductase activity, nucleic acid binding, transferase activity and catalytic activity. The exclusive protein profile may provide insight clues for better understanding the characteristics of proteins and metabolic activity in various explants of the economically important medicinal plant Platycodon grandiflorum.

In rice, the stage of the meiosis in the pollen is sensitive stage resulted in the pollen sterility to reduce yield. Dianxi4 is a cold tolerant line. To monitoring the proteome expression patterns in the pollen of Dianxi4 under the cold stress, shotgun proteomic analysis was conducted to the anther of Dianxi4. The rice plant was grown in the peedy rice field then in the 10 DBH(days before heading), one individual rice plant was moved in the growth chamber under the condition of12℃/RH70%(12h day/12h night). Also the plant used as control was moved in the growth chamber unde the condition of 28℃/RH70%(12h day/12h night). after 4 days treatment, the plant were moved in a greenhouse. The treated rice anther were collected in the one day before heading. From the shotgun proteomic analysis, total of 3,855 non-redundant proteins were identified. Among them, 2,360 proteins were reproducibly identified through the treatment and replications. By the T-test, 1,181 differentially expressed proteins were detected. Through the GO analysis, proteins related in gene expression, cellular process, cellular biosynthetic process were enriched.

Although it is known that the composition of HMW-GSs and LMW-GSs are important factor for end-product quality as bread, noodle and cookie, it is still not clear which HMW-GSs and LMW-GSs confer specific processing properties. In this study, to investigate distinctive glutenin proteins and expression level for characteristic processing properties, we carried out qualitative and quantitative analysis of gluetenin protein in noodle and bread wheat cultivars by two-dimensional electrophoresis. Unexpectedly, five LMW-GS spots were found to be expressed at a common position in all cultivars and these spots may play something in glutenin biosynthesis. Also we found LMS-GS spots to distinguish Korean wheat cultivars mostly used as noodle and western bread wheat cultivars. These spots may contribute to characteristic processing properties. The 2DE results for each cultivar will be used as reference map or protein marker discriminating wheat cultivars, wheat and rice, imported and Korean flour. For quantitative analysis of gluetenin, we calculated relative expression level of the HMW-GS, LMW-GS and HMW-GS/ LMW-GS ratio in each cultivar by 2DE. The results presented in this study provide new insight into relation of specific glutenin proteins and end-use quality and will be useful to choose elite breeding line for improvement of wheat flour quality.

In the present study, different expression of protein from Taekwang was revealed by 2-DE, and expressions of protein on each week after flowering was investigated. After analysis of expression of protein, MALDI-TOF was executed to identify expected protein function. Results revealed that there were three patterns of expression of protein during the maturing. The first pattern was that proteins were gradually expressed as up-regulation from 1 week to 6 week. The second pattern was that proteins were expressed gradually from 1 week to 5 week and then it started down-regulation in 6 week. The last pattern was that proteins were gradually as up-regulation from 1 week to 3 week and then down-regulation until 6 week. This phenomenon suggests that young stage has more protein related to correspondence mechanism against disease and growth and then maturing stage has more expression of protein related to storage protein. In MALDI-TOF analysis, p24 oleosin isoform A protein was identified that relates oleosin which is synthetic product in oil body. This protein spot increased gradually until 5 week and then decreased after 5 week. It explained that the protein is active until maturing stage to protect oil in seed and then its activity has gradually degraded. This result may be expected that a protein, related to growth of a seed has increased until maturing and then a seed fills up with a storage protein

Hepatocytes derived from human embryonic stem cells (hESCs) may be a useful source for the treatment of diseased or injured liver. However, a low survival rate of grafted hepatocytes and immune rejection are still major obstacles to be overcome. We previously showed that secreted proteins (secretome) from hESC-derived hepatocytes had a potential therapeutic power in the tissue repair of injured liver without cell transplantation. The purpose of the present study was to discover key protein(s) in the secretome of hESC-derived hepatocytes using proteomic analysis and to study the tissue repair mechanism which may be operated by the secretomes. Purified indocyanine green+ hepatocytes derived from hESCs displayed multiple hepatic features, including expression of hepatic genes, production of albumin, and glycogen accumulation. The nano-LC/ESI-QTOF-MS analysis identified 365 proteins in the secretome of hESC-derived hepatocytes and the protein functional network analysis was conducted using the MetaCore TM from GeneGO. In addition, 20 tissue regeneration-related transcription factors (TFs) were extrapolated through further proteomic analysis. After intraperitoneal injection, the secretome significantly promoted the liver regeneration in a mouse model of acute liver injury. Protein functional network analysis on the secretome-induced regenerating liver confirmed 20 transcription factors (TFs) which were identified in the ICGhigh cells. The upreguation of these tissue repair-related TFs were validated by qPCR and western blotting on the regenerating liver tissues. These results demonstrate that application of the secretome analysis in combination with the protein functional network mapping would provide a reliable tool to discover new tissue-regenerating proteins as well as to expand our knowledge of the mechanisms of tissue regeneration.

Understanding the host defense mechanisms in response to brown leaf spot disease caused by Cochliobolus miyabeanus is very important for production of resistant plant. In this study, two-dimensional gel electrophoresis (2-DGE) in conjunction with mass spectrometry was utilized to unravel changes of stress inducible proteins in rice leaves infected with C. miyabeanus. For this purpose, we firstly observed disease developmental process of C. miyabeanus in rice using trypan blue, anilin blue, acid fuchsin staining, and DAB staining for ROS detection and expressional abundance of ROS related proteins in rice leaves inoculated was confirmed by Western blotting. Proteins were extracted by PEG fractionation and their expression patterns were analyzed by 2-DGE and subjected to image analysis using the ImageMaster 6.0 2D Platinum software, resulting in the identification of 86 differentially expressed protein spots with significantly changes (p<0.05) compared with control. MALDI-TOFTOF-MS analysis revealed that 69 proteins including 42 and 27 significantly up- and down-regulated proteins, respectively, were identified. Based on gene ontology analysis, identified proteins were classified according to their functional groups: metabolism (20%), oxygen-detoxifying (13%), protein stress/defense (24%). Thus, these results for the first time suggest that differentially induced proteins may play a key role for understanding host defense mechanisms during rice -C. miyabeanus interaction.

Although a great deal of rice proteomic research has been conducted, there are relatively few studies specifically addressing the rice grain proteome. The existing rice grain proteomic research has focused on the identification of deferentially expressed proteins. Here, we performed comparative shotgun proteomic analysis of rice grain development to construct an in-depth proteome reference map, to reveal the expression patterns of the identified proteins, and to detect proteins that are expressed deferentially during grain development. A Korean rice variety, Ilpumbyeo was used. Proteins were extracted from rice grains 10, 20, and 30 days after flowering, as well as from mature grains. The protein expression patterns were revealed by a quantitative shotgun proteoemic analysis. By merging all of the identified proteins in this study, we identified 4,172 non-redundant. A Genome Ontology category enrichment analysis for the 4,172 proteins revealed that 52 categories were enriched, including the carbohydrate metabolic process, transport, localization, lipid metabolic process, and secondary metabolic process. The relative abundances of the 1,784 reproducibly identified proteins were compared to detect 484 differentially expressed proteins during rice grain development. Clustering analysis and Genome Ontology category enrichment analysis revealed that proteins involved in the metabolic process were enriched through all stages of development, suggesting that proteome changes occurred even in the desiccation phase. Interestingly, enrichments of proteins involved in protein folding were detected in the desiccation phase and in fully mature grain.

경제동물에 있어 정자의 수정능력의 예측은 매우 중요한 일이다. 그러나 현재까지 이용되고 있는 정자의 기능적 분석은 정자의 운동성, 활력, 농도 및 일반적인 형태적 분석 등과 같은 양적 형질의 평가만을 수행할 뿐 수정능력 예측을 위한 객관적이고 정확한 도구는 개발되지 못하였다. 즉, 인반적인 정자의 분석은 웅성의 수정능력을 진단하는데 있어 그 한계를 가지고 있는 실정이다. 우수 동물 및 농가의 생산력을 증진시키고 안정적인 생산체제를 구축하기 위하여 새로운 정자의 수정능력 예측법의 개발이 시급한 실정이다. 최근 보다 정확하고 객관적인 수정능력을 예측하기 위하여 정자의 hyperactivation, 수정능획득과 같은 intact-acrosome, 정자의 투명대 결합능력, 정상적 DNA의 상태 등과 같은 다양한 방법이 수행되고 있다. 그러나 이러한 방법들이 정자의 기능과 수정능력 사이의 상관관계가 매우 적기 때문에 정확한 수정능력의 예측에 있어 많은 의문이 제기되고 있다. Proteomics는 postgenome 시대에 새로운 연구로서, 생물학적 과정에 관련된 세포 mechanism을 식별하는 proteome의 질적, 양적 비교로 정의할 수 있다. 정자는 transcription이 불활성화 되어 있는 세포이므로 proteomics를 이용하여 정자의 분자적 기능을 보다 정확히 이해할 수 있다. 또한 정자 proteome의 비교 분석은 정자가 수정을 위한 수정능획득의 방법과 정자가 서로 다른 수정 능력을 가지는 원인을 이해하는데 있어 매우 중요하다. 본 연구의 목적은 돼지의 체내 수정능력을 예측하는 방법의 최적화를 통하여 새로운 예측방법을 개발하고, 나아가 정자의 수정능과 관련된 프로테옴을 동정하여 정자의 수정능력을 예측하는 바이오 마커로의 이용 가능성을 입증하고자 하였다. 본 실험의 분석 결과의 민감도를 높이기 위하여 모든 과정을 최적화 하는 과정을 거쳤으며, quality control을 실시하여 안정적인 결과를 도출하였다. 투명대를 제거한 햄스터 난자와 미성숙 돼지 난포란을 이용한 정자의 침투능력검사에 있어 heparin을 처리한 실험구에서 가장 높은 침투율을 나타냈다. 정자 침투능력검사에 있어 투명대를 제거한 햄스터 난자를 공시하였을 때 돼지의 미성숙 난포란을 이용한 결과보다 유의적으로 높은 결과를 나타냈다. 소의 동결정액을 이용하여 quality control을 실시하여 실험의 변이를 제거하였으며, 또한 암컷의 변이 요인 또한 가능한 모두 제거하였다. 최적화된 SPA (sperm penetration assay using zona-free hamster oocytes)는 돼지의 평균 산자수와 유의적인 상관관계를 나타냈으나, 수태율과는 어떠한 상관관계도 나타내지 않았다. 산자수 8마리 이하를 예측하기 위하여 Sperm Fertility Index (SFI)을 1.2로 cutoff 하였을 경우 정확성은 92%이며, 산자수 10마리 이상을 예측하기 위하여 SFI를 2.5로 정하였을 경우 96%의 정확성을 나타내어 수정능력 예측을 위한 방법으로 유용성이 입증되었다. 산자수가 낮은 group과 높은 group의 정자로부터 발현되는 단백질의 수준을 분석한 결과, 총 108개의 단백질 spot을 찾을 수 있었다. SigmaGel programe을 이용하여 spot의 농도를 분석한 결과, 25개의 단백질 spot이 서로 다르게 발현하였으며, 산자수가 낮은 정자에서 보다 높은 농도로 발현하고 있음을 알 수 있었다. 25개의 단백질 spot을 LC-MS/MS 방법을 이용하여 분석한 결과, 총 20개의 단백질을 동정할 수 있었으며, 이들 20개 단백질 중 mitochondrial trifunctional proteins이 산자수가 높은 group의 정자에서 높은 발현을 나타냈으며, PHGPx와 Arginine Vasopressin Receptor 2가 산자수가 낮은 group의 정자에서 높은 발현을 나타내었다. 이러한 결과들은 이들 방법들이 단지 정자의 수정능력을 분석하는데만 이용할 수 있는 것이 아니라 biomarker로서 정자의 수정능력을 조절하는 인자와 관련된 유전자를 발굴하는데도 유용할 것으로 사료된다.