Mass spectrometric analysis compatible with digital microfluidics (DMF) technique.
- 주제(키워드) Digital microfluidics (DMF) , nano-DESI , Protein digestion , peptide mass fingerprint (PMF)
- 발행기관 서강대학교 일반대학원
- 지도교수 오한빈
- 발행년도 2019
- 학위수여년월 2019. 2
- 학위명 석사
- 학과 및 전공 일반대학원 화학과
- 실제URI http://www.dcollection.net/handler/sogang/000000063937
- UCI I804:11029-000000063937
- 본문언어 한국어
- 저작권 서강대학교 논문은 저작권보호를 받습니다.
초록/요약
Phosphorylation is one of the most important post-translational modifications (PTMs) of proteins, which modulates a wide range of biological functions and activities of proteins. The analysis of phosphopeptides is still one of the most challenging tasks in proteomics research by mass spectrometry. In this study, phosphopeptide enrichment carried out on a digital microfluidic (DMF) chip was demonstrated by analyzing phosphopeptides in the tryptic digested β-casein (bovine, 25.1kDa) and ovalbumin (chicken, 44.3kDa). This approach was made using a magnetic bead (MB)-based titanium dioxide (TiO2)-solid phase extraction (SPE) procedure, which selectively enriches phosphopeptides from tryptic digested protetins. And perfluorinated solvents are used as an surfactant to facilitate droplet movement and limit protein adsorption onto the device surface. The enriched phos phosphopeptides were detected using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). To evaluate this method efficiency, we analyzed the enriched phosphopeptides on DMF chip using Nano-LC(ESI source). This study shows that the phosphopeptide enrichment analysis can be automated and analyzed with a small sample volume on the DMF chip.
more초록/요약
Digital microfluidics(DMF) offers benefits of automatic reaction times, reduced sample volume, and the ability to integrate various analysis platform. In recent years, methodologies have been indicated that can prepare various chemical and biochemical sample using a digital microfluidic technique and finally analyze them using mass spectrometry. However, there have been a restriction that DMF and mass spectrometry have to be coupled in time and space. In this report, new platform, on-chip nano-DESI-MS(Nanospray Desorption Electrospray Ionization Mass Spectrometry)was established to overcome the coupling limitation. This is a method with which sample can be prepared independently using DMF before being analyzed by mass spectrometry. We presented a method for on-chip digestion of proteins and nano-DESI MS of digested protein. It was experimentally proved that in the case the solvent was vaporized and sample remained in solid phase, it can be analyzed independently using nano-DESI MS. Presently, Cytochrome c(bovine, MW 12.1kDa) and Beat-casein(bovine, MW 25.1kDa) proteins were analyzed by nano-DESI MS treated from on-chip digestion and MS/MS data was obtained from each of proteins.
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