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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry

Use of Stable Isotope-Labeled Precursors

Dynamics of Acetylation and Methylation

Acetylation and methylation dynamics can be monitored by the use of heavy- labeled precursors, typically 13C labeled for in vivo metabolic labeling. For acetylation, the precursor can either be incorporated into acetyl-CoA (13C- labeled glucose or 13C-labeled glutamine) or the substrate, acetate, provided in 13C-labeled form [79]. High-resolution LC-MS analysis discriminates new versus existing “old” acetylation on the basis of 13C incorporation, assessed by isotope distribution patterns of coeluting labeled and unlabeled peptides. An optimization-based model simultaneously considers MS isotopic distribution; MS/MS fragment ions and relative peptide hydrophobicity relationships identify and quantify PTM isoforms for individual peptides. Raw abundance is normalized across all observable labeled states within a specific modified state to control for variable ionization efficiencies. 13C glucose is advantageous as the label source, since it is metabolized to alanine and thus is incorporated into proteins, enabling “new” and “old” assignment of protein pools. The method has been applied to defining the turnover of histone acetylation [79] but in principle can be applied at a cellular level and combined with immunoaffinity Kac enrichment strategies for higher proteome coverage.

Dynamic analysis of protein methylation has been achieved by combining dual stable isotope labeling of protein and methylation sites with heavy arginine [13C6] and heavy methionine [13C12H3] [80]. Switching cells into light medium

enabled the discrimination of “old” from “new" based on the analysis of the amounts of heavy or light components by analysis of SILAC intermediates.

This approach was applied to the analysis of histone H3 K27-K36 peptide isomers. Monitoring the H3 K27-K36 peptide methylation states across time enabled MS-based measurement and modeling of histone methylation kinetics (M4K) to detail bidirectional antagonism between H3K27 and H3K36 as a control for writing and erasing of histone marks [80].

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