High efficiency and quantitatively reproducible protein digestion by trypsin-immobilized magnetic microspheres.
High efficiency and quantitatively reproducible protein digestion by trypsin-immobilized magnetic microspheres.
复制标题
DOI：
10.1016/j.chroma.2011.11.050
复制
发表时间：
2012-01-13
期刊：
影响因子：
--
通讯作者：
Dovichi NJ
中科院分区：
文献类型：
--
作者：
Sun L;Li Y;Yang P;Zhu G;Dovichi NJ
醛和NHS活化的磁性微球被用来包裹胰蛋白酶（CHO-胰蛋白酶和NHS-胰蛋白酶），并通过使用LTQ Orbitrap Velos仪器的反相液相色谱-电喷雾电离-串联质谱来评价它们对蛋白质消化的性能。NHS-胰蛋白酶提供了更大的序列覆盖率，并确定了更多的肽消化牛血清白蛋白。与传统的游离胰蛋白酶在37 °C消化12小时相比，在室温下使用固定化胰蛋白酶消化1分钟也鉴定出更多的肽（96 ± 6 vs. 48 ± 1）并且产生更高的序列覆盖率（90 ± 2% vs. 75 ± 2%）。在室温下1分钟内通过NHS-胰蛋白酶消化的15 nM（0.001 mg/mL）BSA的分析始终产生一种检测到的肽; 150 nM BSA产生22种肽。肽强度和蛋白质光谱计数用于评价NHS-胰蛋白酶与三种蛋白质混合物的运行间消化重现性。每种蛋白质的三种高强度肽产生0.70至1.09的强度比和0.78至1.18的光谱计数比。最后，将RAW 264.7细胞裂解物在室温下用NHS-胰蛋白酶消化10 min和30 min;通过RPLC-ESI-MS/MS分别鉴定了604和697个蛋白质组，肽错误发现率小于1%。在37 °C下通过溶液相胰蛋白酶消化12小时导致鉴定出878个蛋白质组。
Aldehyde- and NHS-activated magnetic microspheres were used to immobilize trypsin (CHO-trypsin and NHS-trypsin), and their performance for protein digestion was evaluated by reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry using an LTQ Orbitrap Velos instrument. NHS-trypsin provided greater sequence coverage and identified more peptides for the digestion of bovine serum albumin. A one-minute digestion at room temperature using the immobilized trypsin also identified more peptides (96 ± 6 vs. 48 ± 1) and produced higher sequence coverage (90 ± 2% vs. 75 ± 2%) than traditional free trypsin digestion for 12 hours at 37 °C. Analysis of 15 nM (0.001 mg/mL) BSA digested by NHS-trypsin in 1 min. at room temperature consistently yielded one detected peptide; 150 nM BSA generated 22 peptides. Peptide intensity and protein spectral count were used to evaluate the run-to-run digestion reproducibility of NHS-trypsin with a three-protein-mixture. Three high intensity peptides for each protein generated intensity ratios from 0.70 to 1.09 and spectral count ratios from 0.78 to 1.18. Finally, RAW 264.7 cell lysates were digested by NHS-trypsin for 10 min. and 30 min. at room temperature; 604 and 697 protein groups, respectively, were identified by RPLC-ESI-MS/MS, with a peptide false discovery rate of less than 1%. Digestion by solution phase trypsin for 12 hours at 37 °C resulted in identification of 878 protein groups.
登录
查看更多内容
影响因子：
7.4
作者：
Lopez-Ferrer, Daniel;Hixson, Kim K.;Smallwood, Heather;Squier, Thomas C.;Petritis, Konstantinos;Smith, Richard D.
通讯作者：
Smith, Richard D.
基于有机-无机杂化二氧化硅整体的高酶活性固定化胰蛋白酶反应器
影响因子：
7.4
作者：
Ma, Junfeng;Liang, Zhen;Zhang, Yukui
通讯作者：
Zhang, Yukui
影响因子：
4.4
作者：
Klammer, AA;MacCoss, MJ
通讯作者：
MacCoss, MJ
影响因子：
7.4
作者：
Schoenherr, Regine M.;Ye, Mingliang;Dovichi, Norman J.
通讯作者：
Dovichi, Norman J.
使用胰蛋白酶固定的磁性二氧化硅微球通过微波辅助蛋白质消化进行快速高效的蛋白水解
影响因子：
7.4
作者：
Lin, Shuang;Yao, Guoping;Zhang, Xiangmin
通讯作者：
Zhang, Xiangmin