Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT
Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT
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DOI：
10.1016/s1387-3806(02)00563-8
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发表时间：
2002-08-01
影响因子：
1.8
通讯作者：
Yates, JR
中科院分区：
文献类型：
--
作者：
McDonald, WH;Ohi, R;Yates, JR
从复杂混合物中直接鉴定蛋白质而无需首先通过聚丙烯酰胺凝胶电泳解析蛋白质的最有效方法之一是通过微毛细管 HPLC 分离蛋白水解产生的肽，然后使用串联质谱仪直接收集洗脱液的数据。多维 HPLC 分离技术可以获取更复杂的蛋白质混合物。我们实验室开发了一套多维分析技术；它们统称为多维蛋白质识别技术 (MudPIT)。这些策略采用双相柱，其两侧是强阳离子交换 (SCX) 树脂的反相 (RP) 材料部分，并允许对肽进行多维分离。 MudPlT 的一个变体在 SCX 和 RP 后面添加了额外的 RP 材料部分。该三相色谱柱可用于样品的“在线”脱盐。我们比较了使用单维 LC-MS/MS 柱、两相（标准）MudPIT 柱和三相 MudPIT 柱对通过与牛脑微管关联而纯化的复杂蛋白质混合物的分析。我们发现 3 相 MudPIT 色谱柱对该测试样品产生了更多数量的蛋白质鉴定，并且允许收集未使用 2 相 MudPIT 色谱柱采样的一组亲水肽的数据。 (C) 2002 Elsevier Science B.V. 保留所有权利。
One of the most effective methods for the direct identification of proteins from complex mixtures without first having to resolve them by polyacrylamide gel electrophoresis is to separate proteolytically generated peptides by microcapillary HPLC and then collect data directly on the eluent using a tandem mass spectrometer. Multidimensional HPLC separation techniques provide access to even more complex mixtures of proteins. A set of techniques for multidimensional analysis was developed in our lab; collectively they are known as multidimensional protein identification technology (MudPIT). These strategies employ a biphasic column with a section of reversed phase (RP) material flanked by strong cation exchange (SCX) resin and allow for multidimensional separation of peptides. A variation on MudPlT adds an additional section of RP material behind the SCX and RP. This 3-phase column can be used for "online" desalting of the sample. We compare the analysis of a complex mixture of proteins purified by their association with bovine brain microtubules using a single-dimension LC-MS/MS column, a 2-phase (standard) MudPIT column, and a 3-phase MudPIT column. We find that the 3-phase MudPIT column yields a greater number of protein identifications for this test sample and allows data to be collected on a set of hydrophilic peptides not sampled using the 2-phase MudPIT column. (C) 2002 Elsevier Science B.V. All rights reserved.