Vasicek Lisa, Brodbelt Jennifer S
Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300, Austin, Texas 78712, USA.
Anal Chem. 2009 Oct 1;81(19):7876-84. doi: 10.1021/ac901482s.
Electron transfer dissociation (ETD) has proven to be a promising new ion activation method for proteomics applications due to its ability to generate c- and z-type fragment ions in comparison to the y- and b-type ions produced upon the more conventional collisional activation of peptides. However, low precursor charge states hinder the success of electron-based activation methods due to competition from nondissociative charge reduction and incomplete sequence coverage. In the present report, the reduction and alkylation of disulfide bonds prior to ETD analysis is evaluated by comparison of three derivatization reagents: iodoacetamide (IAM), N,N-dimethyl-2-chloro-ethylamine (DML), and (3-acrylamidopropyl)-trimethyl ammonium chloride (APTA). While both the DML and APTA modifications lead to an increase in the charge states of peptides, the APTA-peptides provided the most significant improvement in percent fragmentation and sequence coverage for all peptides upon ETD, including formation of diagnostic ions that allow characterization of both the C- and N-termini. In addition, the formation of product ions in multiple charge states upon ETD is minimized for the APTA-modified peptides.
电子转移解离（ETD）已被证明是一种用于蛋白质组学应用的很有前景的新离子活化方法，因为与肽段更传统的碰撞活化产生的y型和b型离子相比，它能够产生c型和z型碎片离子。然而，由于非解离电荷减少的竞争和序列覆盖不完整，低前体电荷态阻碍了基于电子的活化方法的成功。在本报告中，通过比较三种衍生化试剂：碘乙酰胺（IAM）、N，N-二甲基-2-氯乙胺（DML）和（3-丙烯酰胺基丙基）三甲基氯化铵（APTA），评估了ETD分析前二硫键的还原和烷基化。虽然DML和APTA修饰都导致肽段电荷态增加，但APTA修饰的肽段在ETD时对所有肽段的碎片化百分比和序列覆盖方面提供了最显著的改善，包括形成诊断离子，从而能够对C端和N端进行表征。此外，对于APTA修饰的肽段，ETD时多电荷态产物离子的形成被最小化。
