Earticle

The aim of this study was to evaluate the changes of protein patterns in granulosa cells and corpus luteum during the estrus cycle in bovine ovary by proteomics ^techniques. Our study was devided into five steps for follicular, ovulatory, early-lteal, midluteal and late-luteal. The protein was extracted from glanulosa cell and corpus luteum proteins by using M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was 700 μg. Immobilized pH gradient (IPG) strip was used 18 cm and 3 11 NL. SDS-PAGE was used 10% acrylamide gel. The protein spots were visualized by Coomassie Brilliant Blue (CBB) staining, analyzed by MALDI mass spectrometry and searched on NCIBlnr. As the result, 61 spots of total 85 spots were repeated on follicular stage and 51 spots of total 114 spots were repeated on ovulatory stage. 40 spots of total 129 were repeated on early-luteal and 49 spots of total 104 spots were repeated on mid-luteal stage. Also 41 spots of total 60 spots were repeated on last-luteal stage. There were differences in the ovulation (follicular∼ovultory stage) in which the spots of follicular stage 19 was only and in ovulation stage was 10 spots. The difference between the luteinization (ovultory∼mid-luteal stage) was the spots counted in each stage. The spots of ovulatory stage was 1, early-luteal stage was 1 and in mid-luteal stage was 2. Eleven spots were found in mid-luteal stage and 2 spots were found in last-luteal stage. In conclusion, we confirmed that there were 7 spots in ovulation, 4 spots in luteinization and 2 spots in luteolysis. Spot No. 89-93 from ovulation were transferrin, and spot No.94 and 95 were HSP60. Spot No. 103 were Dusty PK, spot No. 135 were OGDC-E2, and spot No. 175, 176 were Rab GDI beta from luteinization. Spot No. 178 and 179 from luteolysis were vimentin.

목적: 본 연구는 미용업의 위생수준에 관하여 연구하고, 클리퍼의 모발 분진에 오염된 미생물을 신속하게 동정하여 환경위생인식 개선과 소독제 개발의 자료를 제공할 목적으로 수행하였다. 방법: 경기지역 미용실 근무자 248명을 대상으로 설문조사를 하였으며, 50개의 미용실에서 클리퍼 모발 분진을 수거하여 MALDI-TOF 동정법으로 미생물을 동정하였다. 통계처리는SPSSWIN (Ver. 19.0K, SPSS Inc., USA)을 사용하였다. 결과: 미용실내에서의 위생교육은 ‘있다’ 50.4%, 기기 종류별 소독교육은 ‘있다’ 64.9%로 나타났다. 위생교육의 필요성은 ‘대학원이상’ 93.3%로 가장 높게 나타났으며, 손에 피부질환 상태에서계속 근무하는 경우는 51.6%로 나타났다. 클리퍼의 소독은 ‘매일 1회’ 35.9%로 나타났으며, 소독을 하지 않는 이유로는 ‘수시사용의 번거로움’ 35.0%로 가장 높게 나타났다. 클리퍼 분진에서 MALDI-TOF MS 방법으로 동정된 미생물은 Escherichia coli AKD00698이며, score는 208.0으로 나타났다. 결론: 상기 결과를 바탕으로, 미용실 종사자들의 위생관리 인식 교육의필요성을 확인할 수 있었고, 동정된 미생물을 통해 분변 및 다른 균의 오염 가능성을 예측할 수 있어 위생학적으로 매우 중요한 의의를 지닌다.

Purpose: This research was conducted to study the hygiene level of beauty shop industry and to provide data for improving the environmental hygiene of beauty shops and developing sanitizers by identifying the contaminated microorganisms from hair dust in the clippers promptly. Methods: This survey targeting 248 beauty shop workers in Gyeonggi province was carried out, and hair dust in the clippers from 50 beauty shops was collected. Additionally, microorganisms were identified by using the MALDI-TOF identification method. The SPSSWIN (Ver. 19.0K, SPSS Inc., USA) was used for statistical processing. Results: For the hygiene education in the beauty shop, 50.4% answered ‘Yes’ and for the sterilization education for each apparatus type, 64.9% answered ‘Yes’. For the necessity for hygiene education, 93.3% of respondents with education level of graduate school graduation or higher answered ‘It is necessary’ and for continuous working with skin disease, 51.6% of the respondents answered ‘Yes’. For clipper sterilization, 35.9% answered ‘Once a day’, and for the reason of no clipper sterilization, 35.0% answered ‘Inconvenience due to frequent usage’. The microorganism identified from the dust in the clipper with the MALDI-TOF MS method was Escherichia coli AKD00698 of which the score was 208.0. Conclusion: The results of this research showed that it is necessary to educate beauty shop workers about the hygiene management. Moreover, it is very important significance in the view of hygiene. Because we are able to predict the contamination possibility of other bacteria.

目的: 通过研究美容店业的卫生水平, 鉴定理发店的剪发灰尘中的微生物，并为改善美容店的环境卫生意识和消毒剂的 开发提供研究资料。方法: 这项调查对京畿道地区248名美容店人员进行问卷调查，并在50个美容店中，收集剪发灰尘， 利用MALDI-TOF方法鉴定微生物，以及利用SPSSWIN (Ver. 19.0K, SPSS Inc., USA)进行统计处理。结果: 在问卷调查中， 在美容店中是否有卫生教育，其中50.4% 回答“是”； 是否有不同类型设备的灭菌教育，其中64.9% 回答“是”；对于卫生 教育的必要性，研究生以上学历的受访者中，93.3%的受访者回答“这是必要的”；手上具有皮肤病的情况下，是否会 连续工作，51.6%的受访者回答“是”； 剪刀的消毒方面，“每日一次消毒”占35.9%，不进行消毒的原因中“频繁使用造 成的不便”占35.0%，显示最高；在剪发灰尘污染的微生物中，利用MALDI-TOF MS方法鉴定的微生物是Escherichia coli AKD00698，其得分为208.0。结论: 这项研究的结果表明，有必要对美容店从事者的卫生管理认识进行教育。此外，通 过鉴定的微生物，我们能够预测其他细菌的污染可能性，因此卫生教育具有非常重要的意义。

생체에서의 황화수소는 의약 분야와 발효 산업에 있어서 중요한 역할을 하고 있다. 본 연구에서는 Saccharomyces cerevisiae을 이용하여 기질인 L-cysteine과 β-mercaptoethanol로 부터 β-replacement 반응에 인한 황화수소를 발생시킬 수 있는 효소를 간편하고 신속하게 동정하는 방법의 확립을 목적으로 하였다. 효소에 의해 발생된 황화수소와 Pb-acetate의 반응으로 생성된 Pb-S를 gel상에서 간편하게 확인한 후, 확인된 단백질들을 이온 교환컬럼를 수행한 후 gel에서 추출하는 방법으로 MALDI-TOF MS의 시료를 간단히 얻을 수 있었다. PMF 방법과 MS/MS ion search 분석을 통해 간편하게 효모에서 황화수소를 형성할 가능성이 있는 세가지 단백질의 동정에 성공하였다. 이 세 가지 단백질은 CYS3, CYS4, MET17 유전자의 단백질로서 cystathionine γ-lyase, CBS, OASS 임이 밝혀졌다. 그리고 이 세 단백질들은 L-cysteine과 β-mercaptoethanol의 존재하에서 황화수소를 실제로 생성함을 확인하였다. 본 연구에서 표적의 물질을 생산하는 단백질들을 젤상에서의 활성측정과 MALDI-TOF MS를 이용하여 간단히 그리고 정확하게 동정하는 방법을 확립하였다.

Hydrogen sulfide (H2S) is a by-product of metabolism of amino acids including sulfur and alcoholic fermentation, it is generally thought of in terms of a poisonous gas. Though H2S can have a negative impact on the perceived quality of fermented drinks due to an undesirable aroma, it plays prominent roles as a neuromodulator in the mammalian brain as well as a smooth muscle relaxant. Nowadays studies on the proteins which produce H2S are carried out in various fields such as structure, function, and metabolism. Here we propose to develop a simple and rapid H2S forming assay method, which will lead to speed up preparing the H2S forming proteins for identification by MALDI-TOF MS analysis. We detected three kinds of proteins which produce H2S in the crude extract of Saccharomyces cerevisiae. Those proteins were cystathionie β-synthase, O-acetylserine sulfhydrylase, and cystathionine γ-lyase.

Yeast, Saccharomyces cerevisiae, produces hydrogen sulfide (H2S) as an undesirable by-product during alcoholic fermentation. Very low levels of H2S can be detected, so it can have a profound effect on final product quality. It is highly desirable to have yeast strains available for various fermented production that will not produce and release H2S. To confirm H2S producing proteins in Saccharomyces cerevisiae, we assayed the beta-replacement reaction activity with beta-mercaptoethanol and L-cysteine, which will form S-hydroxyethyl-L-cysteine and H2S. Formed H2S can easily be detected by the incubation with Pb-acetate that will make Pb-sulfide precipitation. When the crude extract of S. cerevisiae was incubated with beta-mercaptoethanol, L-cysteine and Pb-acetate, two proteins which made H2S on the Native-PAGE staining were detected. Because CYS4 gene deleted crude extracts gave only one H2S producing protein in the Native gel, cystathionine beta-synthase is one of the H2S producing protein. The other protein was identified as O-acetylhomoserine sulfhydrylase by MALDI-TOF mass analysis. We could produce CYS4 and Met 17 deficient yeast strain.

Salmonella is one of the most common bacteria that causes heavy losses in swine industry and major causative pathogen of food poisoning in public health. Various methods for the identification of Salmonella such as Gram staining, agglutination test, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) have been used. Several studies have demonstrated that Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI TOF) Mass Spectrometry (MS) identification is an efficient and inexpensive method for the rapid and routine identification of isolated bacteria. In this study, MALDI TOF MS could provide rapid, accurate identification of Salmonella spp. from swine compared with end point PCR and real time PCR.

지난 10여 년에 걸쳐, 임상미생물분야는 미생물 동정에 있어 다소 느린 전통적인 동정 알고리즘에서 신속히 결과를 확인할 수 있는 분자진단학적 방법 또는 질량분석법으로의 혁신적인 변화를 겪고 있다. 일찍이, 질량분석법은 임상화학검사실에서의 단백기반 시료 분석법으로 적용되어 왔다. 최근, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) 원리를 이용한 질량분석법이 미생물검사실에 도입되면서 미생물 동정에 있어 신속, 정확한 미생물 동정결과를 제공할 수 있게 되었다. 널리 알려진 제조사의 제품으로 2가지 장비 플랫폼이 기존의 표현형을 통한 동정 자동화 장비를 대체하고 있으며, 일부 경우에서는 염기서열 기반 동정기법 대신으로도 사용되고 있다. 이 종설은 임상연구나 임상미생물검사에 있어 MALDI-TOF 질량분석법의 현재 상황을 요약하고, 시료 처리, 분석 알고리즘, 해석 및 한계를 분석한 최근 연구들을 소개하고자 한다. 또한, 현재 상용화된 MALDI-TOF 질량분석기와 그 분석 소프트웨어에 대해서도 다룰 것이다. 마지막으로, 임상검사실에서 이러한 신규 검사법들의 잠재능력을 개발하기 위해 끊임없이 노력하고 개발자들과의 협력관계를 공고히 한다면 MALDI-TOF 질량분석법을 비롯한 새로운 기술에 있어 향후 진단 분야에서의 용도를 새롭게 정의할 수 있을 것이다.

In the past decade, clinical microbiology underwent revolutionary changes in methods used to identify microorganisms, a transition from slow and traditional microbial identification algorithms to rapid molecular methods and mass spectrometry (MS). Earlier, MS was clinically used as a highly complex method that was adapted for protein-centered analysis of samples in chemistry laboratories. Recently, a paradigm-shift happened when matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS was implemented to be used in microbiology laboratories for rapid and robust methods for accurate microbial identification. Two instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases even genetic sequence-based identification practices. This review summarizes the current role of MALDI-TOF MS in clinical research, in diagnostic clinical microbiology laboratories, and serves as an introduction to MALDI-TOF MS, highlighting research associated with sample preparation, algorithms, interpretations, and limitations. Currently available MALDI-TOF MS instruments as well as software platforms that support the use of MALDI-TOF with direct specimens have been discussed in this review. Finally, clinical laboratories are consistently striving to extend the potential of these new methods, often in partnership with developmental scientists, resulting in novel technologies, such as MALDI-TOF MS, which could shape and define the diagnostic landscape for years to come.

본 연구에서는 일개 대학병원에서 2년간 분리된 혈액배양 양성배지에서 MALDI-TOF MS system을 이용하여 Grampositive bacilli를 동정한 결과를 균종별, 분기별로 분석하였다. Corynebacterium striatum은 총 89균주 중 66균주(74.2%), Bacillus cereus는 60균주 중 44 균주 (73.3%), Listeria monocytogenes는 25균주 중 25균주 (100%)로 2.0 이상의 높은 스코어에서 동정되었다. 미 동정 된 균주는 303균주 중 293균주는 혈액배양에서 1회 분리 균주로 감염균으로서의 의의가 없는 오염 균주로 간주되었다. 감염균으로서 의의가있는 동일 환자 2회 이상 분리 균주 대상 16S-rRNA sequencing 비교결과 총 50균주 중 43균주가 일치해 86.0% 동정이 가능하였다. 일치하지 않은 7균주 중 5균주는 MALDI-TOF MS로도 동정이 되지 않았다. 결론적으로 혈액배양에서 Gram-positive bacilli가 동정되는 경우, 일차적으로 MALDI-TOF MS를 이용하여 동정해보고이를 활용한다면 어렵고 비용이 많이 들던 Gram-positive bacilli 동정이 저비용으로 더욱 간편하고 정확해지며, Grampositive bacilli에 의한 감염 진단에도 도움이 될 것으로 판단된다.

In this study, Gram-positive bacilli (GPB) were identified by MALDI-TOF MS and analyzed according to the quaternary and microbial strains in the blood culture medium over a two year period at a university hospital. The results were as follows. The overall positive rate of blood culture was 9.97%. In 713 isolated GPB, 410 strains (57.5%) were identified using a microflex MALDI Biotyper. The positive rate of GPB among the blood culture positive bacteria was 8.2%, and the quarterly isolation rate was 9.8% in the third quarter of 2015, 8.7% in the second quarter of 2016, 8.1% in the third quarter of 2016, 8.1% in the first quarter of 2015, 7.9% in the first quarter of 2015, 7.9% in the second quarter of 2015, 6.8% in the first quarter of 2016, and 6.7% in the fourth quarter of 2015. The isolates were Corynebacterium striatum 89 (12.4%), Bacillus cereus 60 (8.4%), Bacillus subtilis 30 (4.2%), Paenibacillus urinalis 29 (4.1%), and Listeria monocytogenes 25 (3.5%). The results of 16S rRNA sequencing of 43 isolates (86.0%) were consistent with those of the other 50 isolates. Five out of the seven unmatched weeks were not identified by MALDI-TOF MS.

Proteomic analyses of synaptic vesicle fraction from rat brain have been performed for the better understanding of vesicle regulation and signal transmission. Two different approaches were applied to identify proteins in synaptic vesicle fraction. First, the isolated synaptic vesicle proteins were treated with trypsin, and the resulting peptides were analyzed using a high-pressure capillary reversed phase liquid chromatography/tandem mass spectrometry (cRPLC/MS/MS). Alternatively, proteins were separated by two-dimensional gel electrophoresis (2DE) and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS). Total 18 and 52 proteins were identified from cRPLC/MS-MS and 2DE-MALDI-TOF-MS analysis, respectively. Among them only 2 proteins were identified by both methods. Of the proteins identified, 70% were soluble proteins and 30% were membrane proteins. They were categorized by their functions in vesicle trafficking and biogenesis, energy metabolism, signal transduction, transport and unknown functions. Among them, 27 proteins were not previously reported as synaptic proteins. The cellular functions of unknown proteins were estimated from the analysis of domain structure, expression profile and predicted interaction partners.

Urosepsis due to Aerococcus urinae is rare in clinical settings with only a few of reported cases worldwide by 16S rRNA sequencing. Here we report a case of sepsis caused by A. urinae in a 86 year-old male with complicated urinary tract infection which was confirmed through peptide mass fingerprinting of matrix-assisted laser desorption ionization time of flight mass spectrometry.

Purpose: The genus Aeromonas is a pathogen that is well known to cause severe clinical illnesses, ranging from gastroenteritis to sepsis. Accurate identification of A. hydrophila, A. caviae, and A. veronii is important for the care of patients. However, species identification remains difficult using conventional methods. The aim of this study was to compare the accuracy of different methods of identifying Aeromonas at the species level: a biochemical method, matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF MS), 16S rRNA sequencing, and housekeeping gene sequencing (gyrB, rpoB). Materials and Methods: We analyzed65 Aeromonas isolates recovered from patients at a university hospital in Koreabetween 1996 and 2012. The isolates were recovered from frozen states and testedusing the following four methods: a conventional biochemical method, 16S rRNA sequencing, housekeeping gene sequencing with phylogenetic analysis, and MALDI-TOF MS. Results: The conventional biochemical method and 16S rRNA sequencing identified Aeromonas at the genus level very accurately, although species level identification was unsatisfactory. MALDI-TOF MS system correctly identified 60 (92.3%) isolates at the species level and an additional four (6.2%) at the genus level. Overall, housekeeping gene sequencing with phylogenetic analysis was found to be the most accurate in identifying Aeromonas at the species level. Conclusion: The most accurate method of identification of Aeromonas to species level is by housekeeping gene sequencing, although high cost and technical difficulty hinder its usage in clinical settings. An easy-to-use identification method is needed for clinical laboratories, for which MALDI-TOF MS could be a strong candidate.

Background: Diverse microbiota exist in the lower respiratory tract. Although next generation sequencing (NGS) is the most widely used microbiome analysis technique, it is difficult to implement NGS in clinical microbiology laboratories. Therefore, we evaluated the performance of conventional culture methods together with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying microbiota in bronchoalveolar lavage (BAL) fluid. Methods: BAL fluid samples (n=27) were obtained from patients undergoing diagnostic bronchoscopy for lung mass evaluation. Bacterial and fungal culture was performed with conventional media used in clinical microbiology laboratories. On an average, 20 isolated colonies were picked from each agar plate and identified by MALDI-TOF MS. Microbiome analysis using 16S rRNA NGS was conducted for comparison. Results: Streptococcus spp. and Neisseria spp. were most frequently cultured from the BAL fluid samples. In two samples, Enterobacteriaceae grew predominantly on MacConkey agar. Actinomyces and Veillonella spp. were commonly identified anaerobes; gut bacteria, such as Lactobacillus, Bifidobacterium, and Clostridium, and fungi were also isolated. NGS revealed more diverse bacterial communities than culture, and Prevotella spp. were mainly identified solely by NGS. Some bacteria, such as Staphylococcus spp., Clostridium spp., and Bifidobacterium spp., were identified solely by culture, indicating that culture may be more sensitive for detecting certain bacteria. Conclusions: Culture and NGS of BAL fluid samples revealed common bacteria with some different microbial communities. Despite some limitations, culture combined with MALDI-TOF MS might play a complementary role in microbiome analysis using 16S rRNA NGS.

The unique Br signature was utilized for C-terminal amino acid sequencing of model peptides. C-terminal carboxyl group was selectively derivatized in peptides, containing side chain carboxyl group, using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and Br was introduced using 4-bromophenylhydrazine hydrochloride (BPH) in a one pot reaction. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) tandem mass spectra were obtained carrying the Br signature in the y-series ions. The Br signature facilitated C-terminal sequencing and discrimination of C-terminal carboxyl groups in the free acid and amide forms.