• Journal of Plant Biotechnology
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• v.48 no.3
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• pp.165-172
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• 2021

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 protein 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 efficacies of the Azo and SDS surfactants using rice leave proteins. This approach led to identification of 3,365 proteins of which 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 substitute of SDS and promising for bottom-up proteomics as it facilitates robust protein extraction, rapid washing step during enzymatic digestion, and MS analysis.

• Parasites, Hosts and Diseases
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• v.58 no.2
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• pp.161-171
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• 2020

The ticks feed large amount of blood from their hosts and transmit pathogens to the victims. The salivary gland plays an important role in the blood feeding. When the female ticks are near engorgement, the salivary gland gradually loses its functions and begins to rapidly degenerate. In this study, data-independent acquisition quantitative proteomics was used to study changes in the phosphorylation modification of proteins during salivary gland degeneration in Haemaphysalis longicornis. In this quantitative study, 400 phosphorylated proteins and 850 phosphorylation modification sites were identified. Trough RNA interference experiments, we found that among the proteins with changes in phosphorylation, apoptosis-promoting Hippo protein played a role in salivary gland degeneration.

• Mass Spectrometry Letters
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• v.4 no.1
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• pp.1-9
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• 2013

Fungal biotechnology has been well established in food and healthcare sector, and now being explored for lignocellulosic biorefinery due to their great potential to produce a wide array of extracellular enzymes for nutrient recycling. Due to global warming, environmental pollution, green house gases emission and depleting fossil fuel, fungal enzymes for lignocellulosic biomass refinery become a major focus for utilizing renewal bioresources. Proteomic technologies tender better biological understanding and exposition of cellular mechanism of cell or microbes under particular physiological condition and are very useful in characterizing fungal secretome. Hence, in addition to traditional colorimetric enzyme assay, mass-spectrometry-based quantification methods for profiling lignocellulolytic enzymes have gained increasing popularity over the past five years. Majority of these methods include two dimensional gel electrophoresis coupled to mass spectrometry, differential stable isotope labeling and label free quantitation. Therefore, in this review, we reviewed more commonly used different proteomic techniques for profiling fungal secretome with a major focus on two dimensional gel electrophoresis, liquid chromatography-based quantitative mass spectrometry for global protein identification and quantification. We also discussed weaknesses and strengths of these methodologies for comprehensive identification and quantification of extracellular proteome.

• Environmental Analysis Health and Toxicology
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• v.22 no.4
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• pp.305-312
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• 2007

환경오염을 신속하게 모니터링하기 위한 생물지표의 개발은 증가하고 있는 오염의 심각성에 비추어 매우 중요한 과제로 여겨지고 있다. 본 연구에서는 독성물질처리에 의하여 선택적으로 발현이 조절되는 단백질의 동정을 통하여 독성물질에 대한 단백질 생물지표를 발굴하고자 시도하였다. 즉, 송사리(Oryzias latipes)를 유기인계 살충제인 다이아지논(diazinon)에 0, 0.1, 1, 5 mg/L 농도로 24시간 노출시킨 후, 머리와 몸통부분으로 나누어 단백질 발현패턴을 분석하였다. 본 시스템에서 다이아지논 처리에 의하여 유의적으로 발현이 증가된 단백질로서 alpha-tubulin, ribonuclease pancreatic precursor, protein hfq 등을 동정하였으며, 이 가운데 alpha-tubulin과 $hsp90{\beta}$의 발현이 다이아지논 농도에 의존적으로 증가하는 것을 semi-quantitative RT-PCR방법으로 확인하였다. 이와 같이 다이아지논 처리에 특이적으로 발현이 증가된 송사리 단백질들은 노출평가를 위한 생물지표로서 개발에 응용될 수 있을 것으로 평가된다.

• BMB Reports
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• v.47 no.5
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• pp.292-297
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• 2014

Age-related macular degeneration (AMD) is the leading cause of blindness in the world. Evidence indicates that the suppression of the ubiquitin-proteasome system (UPS) contributes to the accumulation of toxic proteins and inflammation in retinal pigment epithelium (RPE), the functional abnormalities and/or the degeneration of which are believed to be the initiators and major pathologies of AMD. To identify new protein associations with the altered UPS in AMD, we used LC-ESI-MS/MS to perform a proteomic analysis of the aqueous humor (AH) of AMD patients and matched control subjects. Six UPS-related proteins were present in the AH of the patients and control subjects. Four of the proteins, including 26S proteasome non-ATPase regulatory subunit 1 (Rpn2), were increased in patients, according to semi-quantitative proteomic profiling. An LC-MRM assay revealed a significant increase of Rpn2 in 15 AMD patients compared to the control subjects, suggesting that this protein could be a biomarker for AMD.

• BMB Reports
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• v.47 no.2
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• pp.80-85
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• 2014

Altered acetylcholine (Ach) homeostasis is associated with loss of viability in flies, developmental defects in mice, and cognitive deficits in human. Here, we assessed the importance of Ach in Caenorhabditis elegans development, focusing on the role of Ach during dauer formation. We found that dauer formation was disturbed in choline acetyltransferase (cha-1) and acetylcholinesterase (ace) mutants defective in Ach biosynthesis and degradation, respectively. When examined the potential role of G-proteins in dauer formation, goa-1 and egl-30 mutant worms, expressing mutated versions of mammalian $G_o$ and $G_q$ homolog, respectively, showed some abnormalities in dauer formation. Using quantitative mass spectrometry, we also found that dauer larvae had lower Ach content than did reproductively grown larvae. In addition, a proteomic analysis of acetylcholinesterase mutant worms, which have excessive levels of Ach, showed differential expression of metabolic genes. Collectively, these results indicate that alterations in Ach release may influence dauer formation in C. elegans.

• Mass Spectrometry Letters
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• v.12 no.4
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• pp.126-130
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• 2021

The mass spectrometry (MS) provides the mass-to-charge ratios of atoms, molecules, stable/metastable complexes, and their fragments. I have taken a long journey with MS to address outstanding issues and problems by experiments and theory and gain insights into underlying principles in chemistry. By looking through the mass-to-charge ratio, I have studied thermochemical problems in silicon chemistry, the infrared multiphoton dissociation spectroscopy of organometallic intermediates, unimolecular dissociations of halotoluene radical cations, and the kinetics of association/dissociation of alkali halide triple ions with Lewis bases. Various MS platforms have been used to characterize non-covalent interactions between porphyrins and fullerenes and those between the group IIB ions and trioctylchalcogenides, and to examine the binding of the group IA, IIA and porphyrin ions to G-quadruplex DNA. Recently, I have focused on mass-balanced H/D isotope dipeptide tags for MS-based quantitative proteomics, a simple chemical modification method for MS-based lipase assay, and the kinetics and dynamics of energy-variable collision-induced dissociation of chemically modified peptides. Now, I see an important role of MS in global issues in the post-COVID era, as the society demands high standards for indoor air quality to contain the airborne-pathogen transmission as well as in-situ monitoring and tracking of carbon emissions to reduce global warming.

• Radiation Oncology Journal
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• v.22 no.4
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• pp.298-306
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• 2004

Purpose: The purpose of this study was to identify Radiosensitivity of proteins in tissues with different radiosensitivity. Materials and Methods: C3H/HeJ mice were exposed to 10 Gy. The mice were sacrifiud 8 hrs after radiation. Their spleen and liver tissues were collected and analyzed histologicaly for apoptosis. The expressions of radiosusceptibillty protein were analyzed by 2-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Resilts: The Peak of apoptosis levels were $35.3{\pm}1.7{\%}$ in spleen and $0.6{\pm}0.2{\%}$ in liver at 8 hrs after radiation. Liver, radioresistant tissues, showed that the levels of ROS metabolism related to proteins such as cytochromm c, glutathione S transferase, NADH dehydrogenase, riken cDNA and peroxiredoxin Vl increased after radiation. The expression of cytochrome c increased significantly in spleen and liver tissues after radiation. In spleen, radiosensitivity tissue, the identified proteins showed a significantly quantitative alteration following radiation. It was categorized as signal transduction, apoptosis, cytokine, Ca signal related protein, stress-related protein, cytoskeletal regulation, ROS metabolism, and others. Conclusion: Differences of radiation-induced apoptosis by tissues specifted were coupled with the induction of related radiosensitivity and radioresistant proteins. The result suggests that apoptosis relate protein and redox proteins play important roles in this radiosusceptibility.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5409-5413
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• 2012

At present, multiple myeloma (MM) remains an incurable disease and cologenic cells may be responsible for disease relapse. It has been proposed that CD20+/CD138- NCI-H929 cells could be hallmarks of MM clonogenic cells. Here, the immunology phenotype of NCI-H929 cells is described. Only a small population of CD20+/CD138- cells (<1%) was found in the NCI-H929 cell line, but CD20+/CD138- cells were not detected. We found that CD20+/CD138+ cells were able to exhibit cologenic capacity by colony formation assay and continuous passage culture. Proteins were analyzed by 1D-SDS-PAGE and TMT based quantitative differential liquid chromatography tandem mass spectrometry (LC-MS/MS). 1,082 non-redundant proteins were identified, 658 of which were differentially expressed with at least a 1.5-fold difference. 205 proteins in CD20+ cells were expressed at higher levels and 453 proteins were at lower levels compared with CD20- cells. Most proteins had catalytic and binding activity and mainly participated in metabolic processes, cell communication and molecular transport. These results proved that there are different biological features and protein expression profile between CD20+ and CD20- cells in the NCI-H929 cell line.

• Mass Spectrometry Letters
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• v.8 no.4
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• pp.114-118
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• 2017

While saliva can be considered as good biological fluid for monitoring biomarkers due to many advantages including its communication with blood and the non-invasive nature during its sampling, its applications to that purpose is still limited. As a part of efforts to expand the applications of saliva to the protein biomarker research, we carried out global absolute quantitation of proteins in human whole saliva (WS) by bottom-up proteomics techniques mainly based on nLC-Q-IMS-TOF employing $MS^E$. From the analyses of a pooled WS sample collected from 22 healthy Korean volunteers, 93 proteins ranging from $5.89{\times}10^1ng/mL$ (immunoglobulin heavy chain) to $1.59{\times}10^4ng/mL$ (${\alpha}-amylase$ 1) were confirmed. For the validation of the present results, human serum albumin in the same sample was quantitated by ELISA and its result was compared with that from the nLC-Q-IMS-TOF study. As a result, there was no significant difference between two results from individual approaches ($1.18{\times}10^4{\pm}0.03{\times}10^4 ng/mL$ from nLC-Q-IMS-TOF experiments vs. $1.23{\times}10^4{\pm}0.07{\times}10^4ng/mL$ from ELISA experiments, n=3, p=0.309). To our knowledge, this is the first global absolute quantitation of proteins in human whole saliva and information from the present study can be widely used as the first level reference for the discovery of new protein biomarkers from human whole saliva as well as for quantitative applications of human whole saliva proteins.