生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
1期
76-81
,共6页
胰蛋白酶%还原甲基化%TPCK修饰%高效液相色谱%反相色谱纯化%质谱分析
胰蛋白酶%還原甲基化%TPCK脩飾%高效液相色譜%反相色譜純化%質譜分析
이단백매%환원갑기화%TPCK수식%고효액상색보%반상색보순화%질보분석
trypsin%reductive methylation%TPCK modification%HPLC%reverse phase chromatography purifica-tion%mass spectrometry
目的：制备高纯度、酶解效率高、酶切位点专一的测序级胰蛋白酶，应用于蛋白组学研究的蛋白质鉴定与分析中。方法：取实验室自制的猪胰蛋白酶粗酶，经硼氢化钠、甲醛还原甲基化修饰抑制胰蛋白酶自水解，采用高效液相色谱仪15RPC反相柱纯化，收集对应的甲基化胰蛋白酶峰组分，冷冻干燥；甲基化修饰的胰蛋白酶进一步经甲苯磺酰苯丙氨酰氯甲酮（TPCK）修饰，以抑制糜蛋白酶等非特异性酶切活性，并经反相色谱柱再纯化，获得终产物即质谱测序级胰蛋白酶；自制的测序级胰蛋白酶经SDS-PAGE、HPLC反相色谱分析、酶比活力测定，并应用于胶内蛋白质酶切质谱鉴定氨基酸序列等，检测其纯度、酶水解效率及酶切位点特异性。结果：自制甲基化TPCK修饰的测序级胰蛋白酶纯度大于95%，酶比活力为200 U/mgP（TAME）以上，质谱分析酶切特异性好；且酶的制备工艺流程稳定，可应用于测序级胰蛋白酶产品的生产与开发中。结论：制备的测序级胰蛋白酶纯度高、酶解效率优、酶切特异性强，可广泛应用于实验室中蛋白质和肽段测序鉴定、HPLC肽段谱图分析等蛋白组学研究分析中。
目的：製備高純度、酶解效率高、酶切位點專一的測序級胰蛋白酶，應用于蛋白組學研究的蛋白質鑒定與分析中。方法：取實驗室自製的豬胰蛋白酶粗酶，經硼氫化鈉、甲醛還原甲基化脩飾抑製胰蛋白酶自水解，採用高效液相色譜儀15RPC反相柱純化，收集對應的甲基化胰蛋白酶峰組分，冷凍榦燥；甲基化脩飾的胰蛋白酶進一步經甲苯磺酰苯丙氨酰氯甲酮（TPCK）脩飾，以抑製糜蛋白酶等非特異性酶切活性，併經反相色譜柱再純化，穫得終產物即質譜測序級胰蛋白酶；自製的測序級胰蛋白酶經SDS-PAGE、HPLC反相色譜分析、酶比活力測定，併應用于膠內蛋白質酶切質譜鑒定氨基痠序列等，檢測其純度、酶水解效率及酶切位點特異性。結果：自製甲基化TPCK脩飾的測序級胰蛋白酶純度大于95%，酶比活力為200 U/mgP（TAME）以上，質譜分析酶切特異性好；且酶的製備工藝流程穩定，可應用于測序級胰蛋白酶產品的生產與開髮中。結論：製備的測序級胰蛋白酶純度高、酶解效率優、酶切特異性彊，可廣汎應用于實驗室中蛋白質和肽段測序鑒定、HPLC肽段譜圖分析等蛋白組學研究分析中。
목적：제비고순도、매해효솔고、매절위점전일적측서급이단백매，응용우단백조학연구적단백질감정여분석중。방법：취실험실자제적저이단백매조매，경붕경화납、갑철환원갑기화수식억제이단백매자수해，채용고효액상색보의15RPC반상주순화，수집대응적갑기화이단백매봉조분，냉동간조；갑기화수식적이단백매진일보경갑분광선분병안선록갑동（TPCK）수식，이억제미단백매등비특이성매절활성，병경반상색보주재순화，획득종산물즉질보측서급이단백매；자제적측서급이단백매경SDS-PAGE、HPLC반상색보분석、매비활력측정，병응용우효내단백질매절질보감정안기산서렬등，검측기순도、매수해효솔급매절위점특이성。결과：자제갑기화TPCK수식적측서급이단백매순도대우95%，매비활력위200 U/mgP（TAME）이상，질보분석매절특이성호；차매적제비공예류정은정，가응용우측서급이단백매산품적생산여개발중。결론：제비적측서급이단백매순도고、매해효솔우、매절특이성강，가엄범응용우실험실중단백질화태단측서감정、HPLC태단보도분석등단백조학연구분석중。
Objective: To prepare highly purified, effective, restriction site-specific and chemically stabilized se-quencing grade modified trypsin, and apply for protein identification and analysis in proteomics research. Meth-ods: Firstly, raw trypsin isolated from porcine pancreas was reductive methylated with sodium borohydride buffer and formaldehyde following purification by reverse phase chromatography of 15RPC column, producing a stable tryp-sin that was resistant to tryptic autolysis. Then, the chromatography peak composition of methyl-trypsin was collect-ed. Secondly, methyl-trypsin was further improved by TPCK treatment and was subjected to extensive purification by reverse phase chromatography to remove other non-specific enzyme activity from contaminating proteases. The fi-nal product was sequencing grade modified trypsin. Thirdly, the quality of sequencing grade modified trypsin was tested by SDS-PAGE, HPLC reversed phase chromatography, catalytic activity on TAME, typical enzyme substrate and characterized by assay which related to its application in-gel tryptic digestions following identified by mass spectrometry. Results: Purity of sequencing grade modified trypsin was more than 95% in HPLC assay, appeared as a single band on SDS-PAGE. The specific activity was 200 TAME U/mgP above. The highly purified and chemically stabilized sequencing grade trypsin gave excellent performance for using in-gel tryptic digestions. And the preparation process of enzyme was stably and repeatable. Conclusion: This process yielded a highly purified trypsin product that was effective, highly specific cleavage resulting in a limited number of tryptic peptides and stable, can be widely used in proteomics for peptide mapping and protein identification work at lab. And the sta-ble preparation process can be applied to produce and develop mass spectrum sequencing grade modified trypsin.
