On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis.
On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis.
复制标题
基于在线氨基酸的毛细血管等电聚焦 - ESI-MS/MS用于蛋白质消化分析。
DOI：
10.1016/j.aca.2012.04.026
复制
发表时间：
2012-10-31
影响因子：
6.2
通讯作者：
Dovichi, Norman J.
中科院分区：
文献类型：
--
作者：
Zhu, Guijie;Sun, Liangliang;Yang, Ping;Dovichi, Norman J.
用等电点为3.2~9.7的6个氨基酸作为两性阳离子，建立了毛细管等电聚焦的pH梯度。这种基于氨基酸的毛细管等电聚焦(CIEF)与电喷雾串联质谱仪(ESI-MS/MS)通过电动泵浦的鞘流界面联用来进行多肽分析。在300-1800的重要m/z范围内，基于氨基酸的等电聚焦产生的背景信号比商品化的两个数量级低两个数量级。胰岛素受体获得良好的聚焦，产生~10-S峰宽。对于0.1 mg/mL牛血清白蛋白(BSA)消化液，三倍体分析鉴定出24±1个多肽(序列覆盖率为47±4%)。不出所料，BSA多肽根据等电点进行了分离。牛血清白蛋白消化液的浓度检测下限为7 nm，质量检测下限为7 fMole。以氨基酸为基础的CIEF-ESI-MS/MS分析了6种浓度为5个数量级的牛蛋白胰酶消化液，在三次重复运行中鉴定了5种浓度范围为4个数量级的蛋白质。利用基于氨基酸的CIEF-ESI-MS/MS，从264.7细胞匀浆蛋白的三次重复序列中鉴定出112个蛋白质组和303个独特的多肽。与基于两性阳离子的CIEF-ESI-MS/MS相比，基于氨基酸的CIEF-ESI-MS/MS具有更高的5种酸性多肽的分辨率、更干净的质谱图和更高的蛋白质光谱计数。
Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ~10-s peak width. For 0.1 mg/mL bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4 %) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.
登录
查看更多内容
影响因子：
7.4
作者：
Shen, YF;Berger, SJ;Smith, RD
通讯作者：
Smith, RD
DOI：
10.1016/j.jchromb.2005.07.020
发表时间：
2005-09-25
影响因子：
3
作者：
Storms, HF;van der Heijden, R;van der Greef, J
通讯作者：
van der Greef, J
影响因子：
7.4
作者：
Ramsay, Lauren M.;Dickerson, Jane A.;Dada, Oluwatosin;Dovichi, Norman J.
通讯作者：
Dovichi, Norman J.
DOI：
10.1016/j.jpba.2004.11.025
发表时间：
2005-03-09
影响因子：
3.4
作者：
Kuroda, Y;Yukinaga, H;Matsuzaki, K
通讯作者：
Matsuzaki, K
影响因子：
4.1
作者：
Somma, Andrea;Ferranti, Pasquale;Chianese, Lina
通讯作者：
Chianese, Lina