Clinical Proteomic Study and Diagnostic Application for Antibiotic Resistant Bacteria Using High-End to Low-End Mass Spectrometry
Abstract
The long-term antibiotic abuse and lack of systematic management have threatened the health of human beings worldwide. According to the recent report of GLASS (GLobal Antimicrobial-resistance Surveillance System) supervised by WHO, super-bacteria with multi-drug antibiotic resistance are rapidly increasing worldwide. Especially, the dissemination of antibiotic-resistant Enterobacteriaceae are one of great concerns in the clinical settings worldwide. Fast detection of antibiotics-resistant biomarkers is very crucial in the clinical field for next treatment and surveillance. In general, PCR-based molecular diagnostics or antibiotic susceptibility tests have been used to identify antibiotics resistance, but each process needs high cost or is often time-consuming (2 ~ 3 days). In odrder to effectively prevent the infection of antibiotic-resistant Enterobacteriaceae, it is urgent to introduce low-cost and faster diagnosis methods rather than current methods. In this presentation, I introduce useful proteomic approaches to identify the presence of antibiotic resistance genes, and simply applicable detection methods to the laboratory medicine. Proteomics and target-oriented detection approach using high and medium resolution mass spectrometry (MS) allowed to identify a protein related to antibiotic-resistance and also to determine the size and sequence of clinically detectable proteoform. Further research on the proteoform has enabled to develop a method for rapid detection even on low-resolution MS. Since an MALDI-TOF MS platform has been widely used for identification of pathogenic microorganisms in general hospital and commercial labs, this detection method will be easily applied and used on the pre-installed platform. The detection method enables rapid monitoring and appropriate treatment of certain antibiotic resistant bacteria and is expected to ultimately contribute to human health and well being.
Clinical proetomics, divergence and convergence of mass spectrometry
Abstract
High resolution mass-spectrometry coupled with effective protein/peptide sepatation technique have been applied on biology and translational area. And combination of other omics technology such as transctriptomics and genomics with proteomics have been extended the spectrum of its application to the precision medicine. This divergence phase of proteomics technology is playing an important and novel role as a name of proteo-genomics. At the same time, area of proteomics is reached to deep down of clincal umnet needs by not olny instrument development but also deep underatanding about the usefulness of proteomics application on translational research. Nowaday, proteomics also have been in convergence phase in clinical and translaitional area, especially in biomarker development and validation.
Herein, I would like to introduce some applications of proteo-genomics as exampled of divergence phase of proteomics. Also, application of clinical proteomics as a convergence aspect of proteomics will be presented focusing on discovery of biomarker candidates from the diseases which there have been any diagnostic/prognostic biomarkers. Vaccine candidate discovery for contengeous disease such as Severe fever with thrombocytopenia syndrome (SFTS) and methicillin-resistant Staphylococcus aureus (MRSA) using proteomics will be introduced. Possibility of mass spectrometry-based diagnosis kit development using rdatiation expose spefecific protein markers will be one of the parts of this presentation.
This research was suppoorted by a grant of the Korea Health technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea. (Grant Number: HI15C2918) and the goverment-wide R&D Fund project for infectious disease research (Grant Number: HG18C0037). And this work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (Grant Number: NRF-2017M2A2A7A02061306).
Konkuk University School of Medicine, Konkuk University Medical Center
Title
Quantitative proteomic analysis of aqueous humor from patients
Abstract
To identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential windowed acquisition of all theoretical fragment ion mass spectra, SWATH-MS) for dry AMD patients and controls. After uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen (RPD)) and 2 controls (3 groups). Increased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells and compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both drusen and RPD. In conclusion, differential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.
In-depth proteomic analysis of pleural effusion for lung cancer biomarker discovery
Abstract
Lung cancer is the leading cause of cancer-related death worldwide and adenocarcinoma (ADC) is the predominant histological type of lung cancer. Pleural effusion (PE), a tumor-proximal body fluid, is associated with lung malignancy and serves as a promising source for biomarker discovery. We herein established the differential PE proteome from six types of exudative PEs, including three malignant PEs (MPEs), one paramalignant PE, and two benign diseases using a label-free semiquantitative proteomics approach. To facilitate discovery of lung ADC biomarkers, we applied several integrated strategies to narrow down the potential biomarkers efficiently. The potential biomarkers were validated by ELISA. The area under the receiver-operator characteristic curve for three combined biomarkers in discriminating lung cancer MPE from benign diseases was 0.903. We also demonstrated that cytological examination combined with PE biomarkers would improve the overall clinical diagnostic efficacy of lung cancer MPE. In addition, we generated drug resistance-associated PE proteome from lung ADC patients who received epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) treatments with or without the following chemotherapy via iTRAQ-based quantitative proteomics technology. The PE and serum levels of biomarker candidates were successfully determined by ELISA. We found that the serum levels of CDHP were significantly correlated to treatment efficacy of EGFR-TKI. Importantly, the CDHP levels at baseline were associated with progression-free survival of ADC patients with EGFR mutations. We further show that the CDHP expression was up-regulated in EGFR-TKI-resistant compared to responsive lung ADC cells. Our results collectively provide the useful databases for malignancy biomarker research and also for prognosis in ADC patients with EGFR-TKI therapy.
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