Tanaka Y, Sato I, Iwai C, Kosaka T, Ikeda T, Nakamura T
Biopharmaceutics, Tohoku Pharmaceutical University, 4-1, Komatsushima 4-chome, Aoba-ku, Sendai, 981-8558, Japan.
Anal Biochem. 2001 Jun 15;293(2):157-68. doi: 10.1006/abio.2001.5128.
Several forms of diacetyl-reducing enzyme were found to exist in the human liver cytosol. Three (DAR-2, DAR-5, and DAR-7) of them were purified as a single band on SDS-PAGE by a combination of a few kinds of column chromatographies. The in-gel tryptic digests of the purified enzymes were analyzed by nano-liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS), which provided peptide masses at a ppm-level accuracy. The enzymes, DAR-2, DAR-5, and DAR-7, were identified as alcohol dehydrogenase beta subunit (ADH2), carbonyl reductase (CBR1), and aldehyde reductase (AKR1A1), respectively, by peptide mass fingerprinting. In addition, an alternating-scan acquisition of nano-LC/FT ICR mass spectra, i.e., switching of normal acquisition conditions and in-source fragmentation conditions scan by scan, provided sets of parent and fragment ion masses of many of the tryptic peptides in a single LC/MS run. The peptide sequence-tag information at the ppm-level accuracy was used to further confirm the protein identities. It was demonstrated that nano-LC/FT ICR MS can be used for rigorous protein identification at a subpicomole level as an alternative technique to nano-LC/MS/MS.
在人肝细胞溶质中发现存在几种形式的二乙酰还原酶。通过几种柱色谱法的组合，其中三种（DAR - 2、DAR - 5和DAR - 7）在SDS - PAGE上被纯化为单一谱带。通过纳米液相色谱（LC）/傅里叶变换离子回旋共振质谱（FT ICR MS）分析纯化酶的凝胶内胰蛋白酶消化产物，该方法能以ppm级的精度提供肽质量。通过肽质量指纹图谱，分别将酶DAR - 2、DAR - 5和DAR - 7鉴定为乙醇脱氢酶β亚基（ADH2）、羰基还原酶（CBR1）和醛还原酶（AKR1A1）。此外，纳米LC/FT ICR质谱的交替扫描采集，即逐次切换正常采集条件和源内裂解条件扫描，在一次LC/MS运行中提供了许多胰蛋白酶肽的母离子和碎片离子质量组。以ppm级精度的肽序列标签信息用于进一步确认蛋白质身份。结果表明，纳米LC/FT ICR MS可作为纳米LC/MS/MS的替代技术，用于亚皮摩尔水平的严格蛋白质鉴定。
