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제114회 대한화학회 학술발표회, 총회 및 기기전시회 안내 A new phosphopeptide enrichment strategy using phospho-specific antibody combined with online-mHFER-nLC-ESI-MS/MS

2014년 8월 28일 16시 43분 43초
ANAL.P-575 이곳을 클릭하시면 발표코드에 대한 설명을 보실 수 있습니다.
10월 15일 (수요일) 16:00~19:00
저자 및
이선영, 이슬기, 홍종기, 강덕진1,*
경희대학교 약학과, Korea
1한국표준과학연구원(KRISS) 삶의질측정표준본부, Korea
Phosphorylation, one of post-translational modifications, is well known as a key regulator implicating in cellular growth, differentiation, and signaling. However, mass spectrometry (MS)-based shotgun proteomics to identify and quantify phosphoproteome is not fully accessible yet, due to their low abundance in biological samples (e.g., sera, cells, and etc.) and low ionization efficiency during electrospray ionization (ESI). Herein, we newly introduced an enrichment strategy for phosphoproteomics by which phosphopeptides having an affinity with phospho-specific antibodies (pAbs) can be automatically isolated from ordinary peptides during online microbore hollow fiber enzyme reactor (mHFER)-nLC-ESI-MS/MS experiments. To examine the potential in phosphoproteomics, the tryptic peptides from MCF7 were mixed with pAbs, having a molecular weight of above 50 kDa, and followed by which the pAb-phosphopeptide complexs introduced into the mHFER (10 kDa in MW-cutoff) with continuous flow rate of 5 uL/min. During breakthrough run, ordinary peptides having no affinity with pAb are first waste out through membrane wall of HF. The remaining peptides bound to pAb can be simultaneously exited via online tryptic digestion in mHFER for next nLC-ESI-MS/MS run. From our experimental results, we identified total 11282 phosphorylated sites in 5673 phosphopeptides of MCF7 cell line using online mHFER-2D-nLC-ESI-MS/MS and confirmed that pAbs-based enrichment with online mHFER can be the best way for phosphoproteomics than conventional methods (e.g., IMAC, TiO2, FASP) in which the efficiency in identifying a number of phosphorylated sites is dramatically improved to be about 8-fold than conventional methods, for instance, 24, 2, and 189 phosphoylated sites for IMAC, TiO2, and FASP, respectively.