Evaluation and Application of Dimethylated Amino Acids as Isobaric Tags for Quantitative Proteomics of the TGF-β/Smad3 Signaling Pathway

Yu, Qing; Shi, Xudong; Greer, Tyler; Lietz, Christopher B.; Kent, K. Craig

doi:10.1021/acs.jproteome.6b00641

Cited by 21 publications

(16 citation statements)

References 67 publications

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“…While the underlying mechanisms have been studied from multiple perspectives [69, 70], the N- glycoproteomics has not been very well characterized, despite that N- glycosylation is critical for correct folding, stability and mediating cell attachment [71]. Extracellular matrix (ECM) proteins has long been implicated in the pathogenesis of restenosis, and the fact that more than 90% of ECM proteins are glycosylated makes it more relevant to study carotid artery glycoproteomics [71, 72]. We harvested rat carotid arteries along the restenosis progression at 0, 3, 7, 14 day time point and enriched N- glycopeptides with a HILIC method.…”

Section: Resultsmentioning

confidence: 99%

Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

Wang

Chen

et al. 2017

J. Am. Soc. Mass Spectrom.

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181

195

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Protein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycans and their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum and can be conveniently applied to high throughput glycoproteome characterization, especially for N-glycopeptides which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study a re-defined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding 5 glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid over the course of restenosis progression to investigate potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N-glycopeptides and N-glycoproteomics. We also anticipate this approach can be readily applied to large-scale O-glycoproteome characterization.

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Section: Resultsmentioning

confidence: 99%

Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

Wang

Chen

et al. 2017

J. Am. Soc. Mass Spectrom.

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181

195

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“…Reporter ion ratio values for protein groups were exported to Excel workbook and corrections were performed followed by statistical analysis with student t-test. 19,26 …”

Section: Methodsmentioning

confidence: 99%

“…All currently available isobaric tags, including TMT, iTRAQ and DiLeu, are originally designed for use with CID or HCD. 19–21 Under these fragmentation conditions, reporter ions are preferentially released to generate quantitative information. 20–22 As expected, distinct fragmentation mechanism of ETD also alters reporter ion production of isobaric tag-labeled peptides and results in loss of quantitation channels (e.g., TMT and iTRAQ).…”

Section: Introductionmentioning

confidence: 99%

Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

Shi

Feng

et al. 2017

Analytica Chimica Acta

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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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“…Moreover, N,N-dimethyl amino acids serve as isobaric tags for proteomics research. [7,8] Direct N-methylation of primary or secondary amino acids is a powerful tool for the synthesis of N-methylated amino acids. [9] Classical methods include the direct N-methylation of amino acids with alkyl halides (CH3I/Ag2O), [10] and the reductive amination with formaldehyde and sodium borohydrides [11] (Scheme 1A).…”

mentioning

confidence: 99%

Ag(I)/TiO2-Photocatalyzed N-Methylation of Amino Acids with Methanol

Morioka¹,

Huang²,

Saitō³

et al. 2020

Preprint

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Evaluation and Application of Dimethylated Amino Acids as Isobaric Tags for Quantitative Proteomics of the TGF-β/Smad3 Signaling Pathway

Abstract: Graphical abstract

Cited by 21 publications

References 67 publications

Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

Ag(I)/TiO2-Photocatalyzed N-Methylation of Amino Acids with Methanol

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