Proteomic Analysis of Redox-Sensitive Cysteine Residues using Novel Chemical Probes

Abstract

외부 자극에 의해 발생되는 Reactive oxygen species (ROS)는 질병과 관련한 다양한 신호전달체계에 중요한 역할을 하며 세포 내에서 ROS의 항상성은 ROS의 생성과 제거를 조절함으로써 유지되어진다. 그러나 ROS의 타깃 단백질과 그 것의 조절 메커니즘은 잘 알려진 바 없다. 이 때 cysteine은 ROS의 주요 타깃으로 알려져 있어 단백질에서 cysteine의 산화 정도를 측정할 수 있는 방법을 필요로 한다. Cysteine의 free sulfhydryl을 확인하기 위해서는 iodoacetamide나 N-ethylmaleimide와 같은 alkylating agent들이 오랫동안 사용 되어졌다. 그러나 우리는 이전 연구에서 proteomic 방식을 이용하여 그러한 alkylating agent들이 free sulfhydryl (Cys-SH) 뿐 아니라 sulfenic (Cys-SOH), sulfinic (Cys-SO2H)과도 반응함을 밝혀냈다. 이 것은 산화되지 않은 free sulfhydryl을 확인할 수 있는 방법 개발이 필요함을 보여주었다. 이번 연구에서는 15d-PGJ2 (15-Deoxy-Δ12,14-Prostaglandin J2)의 reactive unsaturated cyclopentenone ring이 cysteine의 free sulfhydryl을 확인하는데 사용되었다. 먼저 MCF7 breast cancer cell lines을 15d-PGJ2–biotin으로 표지하여 reactive cysteine을 갖는 단백질들을 proteomic 방법으로 확인하였다. 그리고 15d-PGJ2 analogue 들과 새로운 Cys-SH capture probe (NosP1, Nosyl-Piperidine)을 디자인하고 합성하여 다양한 단백질에서 redox-active cys residue와 반응하는지를 확인하였다. UPLC-ESI-q-TOF tandem MS로 단백질을 sequencing하여 이 물질들이 다양한 단백질의 cysteine의 free sulfhydryl과의 반응함을 확인하였다. 또한 단백질에 15d-PGJ2 analogue 5과 NosP1의 표지는 H2O2 농도에 따라 감소한다. 이로써 biotin을 접합시킨 chemical compound들은 free sulfhydryl을 측정하는데 적합한 probe로 확인되었다. HeLa cell의 단백질은 이 probe을 확인하기 위해 trypsin을 사용한 digest 후에 nano UPLC ESI q-TOF tandem mass spectrometry로 peptide sequencing되었다. 이 연구는 15d-PGJ2 analogue 5와 NosP1이 sulfenic (Cys-SOH)와 sulfinic (Cys-SO2H)을 포함한 산화된 cysteine과는 반응하지 않고 cysteine의 free sulfhydryl만을 표지하는 새로운 chemical probe임을 보여준다.;Reactive oxygen species (ROS) generated by external stimuli, plays key roles in various signaling pathways related to diseases, and its homeostasis inside cells are well balanced by controlling the production and elimination of ROS. However, target proteins of ROS and its regulation mechanism are not well understood. Since redox-active cysteine (Cys) is the major targets of ROS, the methodology is required to measure the Cys oxidation status of target proteins. In order to identify free sulfhydryls of Cys residues, various alkylating agents including iodoacetamide and N-ethylmaleimide, have long been used. However, we previously recognized that these alkylating agents react with sulfenic (Cys-SOH), sulfinic (Cys-SO2H) as well as free sulfhydryls (Cys-SH) using proteomic approach. It is necessary to find the methodology to detect the free cysteine residue (Cys-SH). In this study, reactive unsaturated cyclopentenone ring of 15d-PGJ2 (15-Deoxy-Δ12,14-Prostaglandin J2) was employed for labeling free sulfhydryls of Cys residues by acting as electrophile. At first, MCF7 breast cancer cell lines were labeled with 15d-PGJ2–biotin and proteins having reactive Cys residues were identified by proteomics. Then, 15d-PGJ2 analogues and novel Cys-SH capture probe (NosP1, Nosyl-Piperidine) were designed and synthesized, and tested to identify the redox-active Cys residues in various proteins. Its chemicals were confirmed to react specifically to free sulfhydryl of Cys residues in various recombinant proteins by sequencing the proteins with UPLC-ESI-q-TOF tandem MS. Also, Cys-SH labeling with 15d-PGJ2 analogue 5 and NosP1 of recombinant proteins were decreased in response to H2O2in a concentration dependent manner. These biotin-labeled chemical compounds are turned out to be relevant probe to measure Cys-SH. These probes were tested in proteins in HeLa cells by peptide sequencing with nano UPLC ESI q-TOF tandem mass spectrometry after digesting with trypsin. These studies suggest that 15d-PGJ2 analogue 5 and NosP1 are novel chemical probes specifically labeling free sulfhydryls of Cys residues, not reacting with oxidative Cys residues including sulfenic (Cys-SOH) and sulfinic (Cys-SO2H).