High-speed and high-resolution UPLC separation at zero degrees Celsius.
High-speed and high-resolution UPLC separation at zero degrees Celsius.
复制标题
零摄氏度的高速和高分辨率UPLC分离。
DOI：
10.1021/ac8008862
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发表时间：
2008-09-01
影响因子：
7.4
通讯作者：
Engen, John R.
中科院分区：
文献类型：
--
作者：
Wales, Thomas E.;Fadgen, Keith E.;Gerhardt, Geoff C.;Engen, John R.
蛋白质的构象特性可以通过氢/氘交换质谱 (HXMS) 进行探测。为了在 LC/MS 分析过程中保持氘标记，必须在零摄氏度下快速完成色谱分离（通常在 8-10 分钟内）。具有 ~3 微米颗粒的传统 RP-HPLC 在这些条件下通常表现出较差的色谱性能，因此无法对较大的蛋白质和许多蛋白质复合物进行 HXMS 分析。与 HPLC 相比，超高效液相色谱 (UPLC) 使用直径小于 2 微米的颗粒来实现卓越的分辨率、速度和灵敏度。 UPLC 此前已被证明能够满足 HXMS 的快速分离和低温要求。在这里，我们展示了 HXMS 的定制 UPLC 系统的构建和验证。该系统基于 Waters nanoACQUITY 平台，包含一个 Peltier 冷却模块，其中装有进样阀和切换阀、在线胃蛋白酶消化柱和 C-18 分析分离柱。超过 95 kDa 的单一蛋白质和超过 250 kDa 的四蛋白质混合物已用于验证该新系统的性能。在 0 °C 下 6 分钟分离即可实现接近基线的分辨率，半高处的中位色谱峰宽度约为 2.7 秒。氘回收率与使用传统 HPLC 和冰浴获得的相似。这个新系统代表了 HXMS 技术的重大进步，预计将在不久的将来使该技术更容易使用并成为主流。
The conformational properties of proteins can be probed with hydrogen/deuterium exchange mass spectrometry (HXMS). In order to maintain the deuterium label during LC/MS analyses, chromatographic separation must be done rapidly (usually in under 8–10 minutes) and at zero degrees Celsius. Traditional RP-HPLC with ~3 micron particles has shown generally poor chromatographic performance under these conditions and thereby has been prohibitive for HXMS analyses of larger proteins and many protein complexes. Ultra performance liquid chromatography (UPLC) employs particles smaller than 2 microns in diameter to achieve superior resolution, speed, and sensitivity as compared to HPLC. UPLC has previously been shown to be compatible with the fast separation and low temperature requirements of HXMS. Here we present construction and validation of a custom UPLC system for HXMS. The system is based on the Waters nanoACQUITY platform and contains a Peltier-cooled module that houses the injection and switching valves, online pepsin digestion column, and C-18 analytical separation column. Single proteins in excess of 95 kDa and a four-protein mixture in excess of 250 kDa have been used to validate the performance of this new system. Near baseline resolution was achieved in 6 minute separations at 0 °C and displayed a median chromatographic peak width of ~2.7 sec at half height. Deuterium recovery was similar to that obtained using a conventional HPLC and icebath. This new system represents a significant advancement in HXMS technology that is expected to make the technique more accessible and mainstream in the near future.
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影响因子：
4.8
作者：
Eyles, SJ;Kaltashov, IA
通讯作者：
Kaltashov, IA
影响因子：
7.4
作者：
Wu, Y;Kaveti, S;Engen, JR
通讯作者：
Engen, JR
DOI：
10.1016/j.jasms.2005.10.009
发表时间：
2006-02-01
影响因子：
3.2
作者：
Wu, Y;Engen, JR;Hobbins, WB
通讯作者：
Hobbins, WB
影响因子：
7.4
作者：
Chalmers, MJ;Busby, SA;Griffin, PR
通讯作者：
Griffin, PR
影响因子：
7
作者：
Wang, LT;Pan, H;Smith, DL
通讯作者：
Smith, DL