Facile identification and quantitation of protein phosphorylation via β-elimination and Michael addition with natural abundance and stable isotope labeled thiocholine

Meng Chen, Xiong Su, Jingyue Yang, Christopher M. Jenkins, Ari M. Cedars, Richard W. Gross

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Herein, we employ the unique chemical properties of the quaternary amine present in thiocholine (2-mercapto-N,N,Ntrimethyl-ethanaminium) in conjunction with alkaline β-elimination and Michael addition (BEMA) reactions for the specific detection, identification, and quantitation of phosphorylated serine/threonine containing peptides. Through replacement of the phosphate with thiocholine, the negative charge on the phosphopeptide is switched to a quaternary amine containing a permanent positive charge. This strategy resulted in a 100fold increase in ionization sensitivity during ESI (sub-500 amol/μL detection limit) accompanied by a markedly enhanced production of informative peptidic fragment ions during CID that dramatically increase sequence coverage. Moreover, the definitive localization of phosphorylated residues is greatly facilitated through the generation of diagnostic triads of fragmentation ions resulting from peptide bond cleavage and further neutral loss of either trimethylamine (- 59 Da) or thiocholine thiolate (- 119 Da) during collision induced dissociation (CID) in tandem mass spectrometry (MS 2 and MS 3 ). Synthesis of stable isotope labeled thiocholine enabled the quantitation of protein phosphorylation with high precision by ratiometric comparisons using heavy and light thiocholine. Collectively, this study demonstrates a sensitive and efficient strategy for mapping of phosphopeptides by BEMA using thiocholine through the production of a diagnostic repertoire of unique fragment ions during liquid chromatography-tandem mass spectrometry (LC-MS 2 / MS 3 ) analyses, facilitating phosphosite identification and quantitative phosphoproteomics.

Original languageEnglish (US)
Pages (from-to)163-171
Number of pages9
JournalAnalytical chemistry
Volume82
Issue number1
DOIs
StatePublished - Feb 8 2010
Externally publishedYes

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Thiocholine
Phosphorylation
Isotopes
Phosphopeptides
Proteins
Amines
Mass spectrometry
Ions
Ion chromatography
Peptides
Addition reactions
Liquid chromatography
Threonine
Serine
Chemical properties
Ionization
Phosphates

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Facile identification and quantitation of protein phosphorylation via β-elimination and Michael addition with natural abundance and stable isotope labeled thiocholine. / Chen, Meng; Su, Xiong; Yang, Jingyue; Jenkins, Christopher M.; Cedars, Ari M.; Gross, Richard W.

In: Analytical chemistry, Vol. 82, No. 1, 08.02.2010, p. 163-171.

Research output: Contribution to journalArticle

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