کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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5135252 | 1493428 | 2017 | 9 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Exploiting charge differences for the analysis of challenging post-translational modifications by capillary electrophoresis-mass spectrometry Exploiting charge differences for the analysis of challenging post-translational modifications by capillary electrophoresis-mass spectrometry](/preview/png/5135252.png)
- Excellent resolution of deamidated peptides containing aspartate and isoaspartate.
- CE-MS enables the identification of citrullinated proteoforms from intact histone H4.
- Separation, identification and quantification of positional phospho-isomers.
Reversed-phase high-performance liquid chromatography (RP-HPLC) in combination with mass spectrometry (MS) is typically employed for mapping modifications in proteins and peptides. Here we applied a low-flow capillary electrophoresis (CE) âelectrospray ionization interface coupled to Orbitrap mass spectrometers to analyze challenging modifications such as asparagine deamidation, aspartate isomerization, arginine citrullination, and phosphopeptide isomers. We achieved excellent resolution of asparagine (Asn), aspartic acid (Asp) and isoaspartic acid (iso-Asp) containing peptides using a synthetic peptide mixture. The migration order in CE enabled a clear assignment of in vitro deamidation/isomerization sites in a protein standard mixture of intermediate complexity (48 proteins) as well as the determination of the in vivo deamidation rate of histone H1.0 directly in a crude nuclear protein fraction. Besides these well-known modifications citrullination, a post-translational modification which changes the positively charged guanidinium group of arginine to the uncharged ureido group of citrulline, was investigated. Applying CE-MS for fast and sensitive analyses of various post-translational modifications of intact and enzymatically digested histone H4, we were able to detect a variety of citrullinated proteoforms. MS/MS analysis with electron transfer dissociation (ETD) fragmentation identified the presence of deiminated Arg at position 3 and 17 of histone H4. Moreover, based on CE-MS, isobaric mono-phosphorylated peptides obtained in the course of a kinase activity study were separated and individual positional isomers quantified.
Journal: Journal of Chromatography A - Volume 1498, 19 May 2017, Pages 215-223