Carboxylate-Modified Magnetic Bead (CMMB)-Based Isopropanol Gradient Peptide Fractionation (CIF) Enables Rapid and Robust Off-Line Peptide Mixture Fractionation in Bottom-Up Proteomics.
Carboxylate-Modified Magnetic Bead (CMMB)-Based Isopropanol Gradient Peptide Fractionation (CIF) Enables Rapid and Robust Off-Line Peptide Mixture Fractionation in Bottom-Up Proteomics.
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基于羧基修饰磁珠（CMMB）的异丙醇梯度肽分级（CIF）可在自下而上的蛋白质组学中实现快速、稳健的离线肽混合物分级。
DOI：
10.1074/mcp.ra120.002411
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Wohlschlegel JA
中科院分区：
文献类型：
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作者：
Deng W;Sha J;Plath K;Wohlschlegel JA
自下而上的蛋白质组学中的深度蛋白质组覆盖需要在通过串联质谱分析之前进行肽级分级分离以简化复杂的肽混合物。通过减少共洗脱前体肽离子的数量，分级分离有效降低了样品的复杂性，从而提高了样品覆盖率，并减少了对在数据依赖性采集策略中优先识别的高丰度前体的偏差。为了实现这一目标，我们报告了一种基于磁珠的离线肽分级分离方法，称为 CIF 或基于羧酸盐修饰磁珠的异丙醇梯度肽分级分离。 CIF 是 SP3（单罐固相增强样品制备）策略的延伸，为其他常用的分级方法（包括强阳离子交换和反相色谱）提供了有效但补充的方法。我们证明，CIF 是一种有效的离线分离策略，无论是单独使用还是与高 pH 反相结合作为肽分级分离的第二维时，都能够增加肽分析物的覆盖深度。这些功能使其非常适合广泛的蛋白质组学应用，包括低丰度诱饵蛋白的亲和纯化。 CIF 提供了一种基于 HILIC 的新型珠上离线肽分离方法。 CIF 为肽分级分离提供了与 RP 互补的维度。 CIF 可与 SP3 蛋白样品脱盐无缝集成。机器学习模型能够根据肽序列预测 CIF 肽分离模式。我们提出了一种在羧酸盐包被的磁珠上分级胰蛋白酶肽混合物的新方法。它是先前报道的 SP3（单锅固相增强样品制备）蛋白质和肽净化方法的延伸，并为其他常用的分级方法（包括基于强阳离子交换 (SCX) 和反相 (RP) 的色谱法）提供了有效但补充的方法。
Deep proteome coverage in bottom-up proteomics requires peptide-level fractionation to simplify the complex peptide mixture before analysis by tandem mass spectrometry. By decreasing the number of coeluting precursor peptide ions, fractionation effectively reduces the complexity of the sample leading to higher sample coverage and reduced bias toward high-abundance precursors that are preferentially identified in data-dependent acquisition strategies. To achieve this goal, we report a bead-based off-line peptide fractionation method termed CIF or carboxylate-modified magnetic bead–based isopropanol gradient peptide fractionation. CIF is an extension of the SP3 (single-pot solid phase–enhanced sample preparation) strategy and provides an effective but complementary approach to other commonly used fractionation methods including strong cation exchange and reversed phase–based chromatography. We demonstrate that CIF is an effective offline separation strategy capable of increasing the depth of peptide analyte coverage both when used alone or as a second dimension of peptide fractionation in conjunction with high pH reversed phase. These features make it ideally suited for a wide range of proteomic applications including the affinity purification of low-abundance bait proteins. CIF provides a new on-bead offline peptide fractionation method based on HILIC. CIF offers a complementary dimension to RP for peptide fractionation. CIF can be seamlessly integrated with SP3 protein sample desalting. A machine learning model is able to predict CIF peptide fractionation patterns based on its peptide sequence. We propose a novel method for fractionating tryptic peptide mixtures on carboxylate-coated magnetic beads. It is an extension of the previously reported SP3 (single-pot solid phase–enhanced sample preparation) protein and peptide cleanup method and provides an effective but complementary approach to other commonly used fractionation methods including strong cation exchange (SCX) and reversed phase (RP)-based chromatography.