Home Health Analysis of Protein Post-Translational Modifications by Mass Spectrometry
Stoichiometry of Acetylation and Methylation
Beyond identification of a peptide or protein as acetylated or methylated and relative quantification across different biological states, assessment of functional significance requires determination of the PTM occupancy of specific sites by the PTM. This requires calculation of the amount of the post-translationally modified peptide relative to the corresponding unmodified peptide. When measuring stoichiometry, it can be assumed that PTM abundance changes are inversely proportional to their unmodified counterpart . This approach, in its simplest form, requires the unmodified and modified peptides to have similar ionization efficiencies for direct comparison based on MS intensity measurements. This in inherently difficult for acetylated and methylated peptides, since these PTMs result in missed tryptic cleavage peptides; thus an unmodified peptide will be shorter relative to the modified form. Either proteolytic digestion with Arg-C or chemical derivatization to promote Arg-C type cleavage generates lysine acetylated and nonacetylated counterpart peptides that are of the same length to provide a useful tool for direct comparison to assess stoichiometry [82, 83] with application at the level of the entire proteome without requirement for specific enrichment strategies 
A mass spectrometry method using a combination of isotope labeling, Arg-C digestion, and detection of Kac diagnostic ion at m/z 126.09 to determine the stoichiometry of protein lysine acetylation, applicable to proteome- wide profiling, has also been described . This technique complements relative quantification of lysine-acetylated peptides postimmunoaffinity enrichment. Sites of lysine acetylation are discriminated from the unmodified form by chemical acetylation using a stable isotope, 13C form of acetic anhydride [1,1'-13C2-acetic anhydride], to label unmodified lysines. The 13C form and preexisting “light” lysine acetylated form coelute during HPLC analysis; cofragmentation enables the calculation of acetylation stoichiometry using the intensities of the heavy and light PTM forms of the diagnostic ions of A1.0033 Da. It should be noted that peptide-level calculations do not provide individual site stoichiometry data for peptides containing multiple lysine residues . This can be mitigated by prior fractionation of peptides but represents an issue for isobaric peptides.
A related technique has applicability to acetylation and (mono)methylated peptides . Chemical derivatization with deuterated (d6) acetic anhydride prior to tryptic digestion restricts digestion to arginine residues (Arg-C like) and produces a chemically identical set of peptides, which can be discriminated based on isotope distribution of mixed heavy and light peptides for the calculation of stoichiometry. Methylation stoichiometry is determined from relative intensities of the deuteroacetylated and monomethylated peptide compared with the deuteroacetylated but unmethylated analog. High-resolution accurate mass MS2 measurements enable the discrimination of lysine acetylation from lysine trimethylation. Monomethylated but not di- or trimethylated residues are derivatized in this technique. The ability to determine the stoichiometries of trimethylation and acetylation in the same peptide enables the analysis of PTM cross talk.
Methylation stoichiometry can be determined by iMethyl-SILAC labeling using L-Methionine-13C4 for heavy labeling of methylated peptides and comparing relative differences with cells grown in a “light” methionine-containing media. Normalization of changes in levels of methylated peptides to changes in protein expression is used to derive fold changes in site occupancy. This was exemplified for a discovery workflow of arginine methylation peptides identifying 365 unique arginine-methylated peptides before and after stimulation of T cells, corresponding to 319 distinct arginine methylation sites (from a total of 1411) in 202 proteins .
|< Prev||CONTENTS||Next >|