کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5130887 1490864 2017 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies
چکیده انگلیسی


- EThcD was optimized for isobaric tag-labeled peptides for quantitative proteomics.
- EThcD preserves HCD-type reporters of DiLeu and TMT tags and improves instrument duty cycle by lowering ETD reaction time.
- With EThcD, traditional isobaric tags originally designed for HCD can be readily utilized with ETD.
- EThcD is able to provide both b/y and c/z ion series for greatly improved data quality.
- EThcD enables quantitative phosphoproteomics with enhanced phosphorylation localization.

Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron-transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies.

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ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytica Chimica Acta - Volume 968, 22 May 2017, Pages 40-49
نویسندگان
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