Towards liquid chromatography time-scale peptide sequencing and characterization of post-translational modifications in the negative-ion mode using electron detachment dissociation tandem mass spectrometry
Towards liquid chromatography time-scale peptide sequencing and characterization of post-translational modifications in the negative-ion mode using electron detachment dissociation tandem mass spectrometry
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DOI：
10.1016/j.jasms.2008.04.031
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发表时间：
2008-08-01
影响因子：
3.2
通讯作者：
Jensen, Ole N.
中科院分区：
文献类型：
--
作者：
Kjeldsen, Frank;Horning, Ole B.;Jensen, Ole N.
肽聚阴离子的电子分离解离（EDD）对翻译后修饰（PTM）是温和的，并产生可预测和可解释的碎片离子类型（a.，X离子）。然而，EDD被认为是一种低效的片段化技术，尚未在大规模肽表征策略中实施。我们成功地提高了EDD裂解效率（高达9%），并首次证明了EDD-MS/MS在液相色谱时间尺度实验中的实用性。肽和磷酸化肽进行了分析，在正离子和负离子模式，使用电子捕获/转移解离（ECD/ETD）和EDD的比较。使用约1 pmol BSA胰蛋白酶消化物，LC-EDD-MS/MS对14个肽（27% aa序列覆盖率）进行测序，LC-ECD-MS/MS对19个肽（39% aa序列覆盖率）进行测序。通过EDD和ECD对7个肽（18%aa序列覆盖率）进行测序。鉴定的BSA肽的相对小的重叠证明了两种解离模式的互补性。对三种胰蛋白酶消化的磷蛋白的磷酸肽混合物进行LC-EDD-MS/MS，从而鉴定出五种磷酸肽。其中，在先前使用类似样品和正离子模式下的LC-ETD-MS/MS的研究中未发现1个。在本研究中，ECD片段化效率（15.70% av.）上级优于EDD裂解效率（平均3.6%）。然而，鉴于氨基酸序列覆盖率的增加和扩展的PTM表征，EDD与正离子模式中的其他离子-电子碎裂技术相结合的新制度是朝着蛋白质组研究中更全面的分析策略迈出的一步。
Electron detachment dissociation (EDD) of peptide poly-anions is gentle towards post-translational modifications (PTMs) and produces predictable and interpretable fragment ion types (a., x ions). However, EDD is considered an inefficient fragmentation technique and has not yet been implemented in large-scale peptide characterization strategies. We successfully increased the EDD fragmentation efficiency (up to 9%), and demonstrate for the first time the utility of EDD-MS/MS in liquid chromatography time-scale experiments. Peptides and phosphopeptides were analyzed in both positive- and negative-ion mode using electron capture/transfer dissociation (ECD/ETD) and EDD in comparison. Using approximately 1 pmol of a BSA tryptic digest, LC-EDD-MS/MS sequenced 14 peptides (27% aa sequence coverage) and LC-ECD-MS/MS sequenced 19 peptides (39% aa sequence coverage). Seven peptides (18% aa sequence coverage) were sequenced by both EDD and ECD. The relative small overlap of identified BSA peptides demonstrates the complementarity of the two dissociation modes. Phosphopeptide mixtures from three trypsin-digested phosphoproteins were subjected to LC-EDD-MS/MS resulting in the identification of five phospho-peptides. Of those, one was not found in a previous study using a similar sample and LC-ETD-MS/MS in the positive-ion mode. In this study, the ECD fragmentation efficiency (15.70% av.) was superior to the EDD fragmentation efficiency (3.6%, av.). However, given the increase in amino acid sequence coverage and extended PTM characterization the new regime of EDD in combination with other ion-electron fragmentation techniques in the positive-ion mode is a step towards a more comprehensive strategy of analysis in proteome research.