In-Line Separation by Capillary Electrophoresis Prior to Analysis by Top-Down Mass Spectrometry Enables Sensitive Characterization of Protein Complexes
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- 作者：Xuemei Han ; Yueju Wang ; Aaron Aslanian ; Bryan Fonslow ; Beth Graczyk ; Trisha N. Davis ; John R. Yates ; III
- 刊名：Journal of Proteome Research
- 出版年：2014
- 出版时间：December 5, 2014
- 年：2014
- 卷：13
- 期：12
- 页码：6078-6086
- 全文大小：401K
- ISSN：1535-3907
文摘
Intact protein analysis via top-down mass spectrometry (MS) provides a bird鈥檚 eye view over the protein complexes and complex protein mixtures with the unique capability of characterizing protein variants, splice isoforms, and combinatorial post-translational modifications (PTMs). Here we applied capillary electrophoresis (CE) through a sheathless CE鈥揺lectrospray ionization interface coupled to an LTQ Velos Orbitrap Elite mass spectrometer to analyze the Dam1 complex from Saccharomyces cerevisiae. We achieved a 100-fold increase in sensitivity compared to a reversed-phase liquid chromatography coupled MS analysis of recombinant Dam1 complex with a total loading of 2.5 ng (12 amol). N-terminal processing forms of individual subunits of the Dam1 complex were observed as well as their phosphorylation stoichiometry upon Mps1p kinase treatment.