Quantitative analysis of both protein expression and serine / threonine post‐translational modifications through stable isotope labeling with dithiothreitol

Abstract

While phosphorylation and O‐GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post‐translational modifications have been difficult to analyze in proteomic studies. We describe advances in β‐elimination / Michael addition‐based approaches which allow for mass spectrometry‐based identification and comparative quantification of O‐phosphate or O‐GlcNAc‐modified peptides, as well as cysteine‐containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope‐coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O‐phosphorylation and O‐GlcNAc modification sites. Specificity of O‐phosphate vs. O‐GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O‐GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post‐translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large‐scale quantitative analysis of both protein expression and serine / threonine post‐translational modifications.