Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics.
Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics.
复制标题
三维液相色谱耦合离子交换色谱/疏水相互作用色谱/反相色谱法，用于自上而下的蛋白质组学中有效蛋白质分离。
DOI：
10.1021/acs.analchem.5b00657
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发表时间：
2015
影响因子：
7.4
通讯作者：
Ge Y
中科院分区：
文献类型：
--
作者：
Valeja SG;Xiu L;Gregorich ZR;Guner H;Jin S;Ge Y
为了解决基于质谱 (MS) 的自上而下蛋白质组学中蛋白质组的复杂性，非常需要能够以高分辨率和自动化有效分离蛋白质的多维液相色谱 (MDLC) 策略。尽管存在多种可以有效从蛋白质消化物中分离肽的 MDLC 方法，但很少有 MDLC 策略（主要由 2DLC 组成）可用于完整蛋白质的分离，这不足以解决蛋白质组的复杂性。我们最近证明，利用 MS 兼容盐的疏水相互作用色谱 (HIC) 可以为自上而下的蛋白质组学提供完整蛋白质的高分辨率分离。在此，我们开发了一种新颖的 3DLC 策略，通过将 HIC 与离子交换色谱 (IEC) 和反相色谱 (RPC) 结合来分离完整的蛋白质。我们证明了 3D (IECHIC-RPC) 方法大大优于传统的 2D IEC-RPC 方法。对于来自粗制 HEK 293 细胞裂解物的相同 IEC 级分（共 35 个级分），3D 方法中总共鉴定了 640 个蛋白质（对应于 201 个非冗余蛋白质），而 2D 方法中鉴定了 47 个蛋白质，而在 2D 方法中简单地延长 RPC 梯度仅对蛋白质分离和鉴定带来最小的改进。因此，这种新颖的 3DLC 方法在有效分离完整蛋白质以实现自上而下蛋白质组学中深度蛋白质组覆盖方面具有巨大潜力。
To address the complexity of proteome in mass spectrometry (MS)-based top-down proteomics, multi-dimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the proteome complexity. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IECHIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 non-redundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics.