Home Health Analysis of Protein Post-Translational Modifications by Mass Spectrometry
Mass Spectrometry Analysis
In principle, MS can be applied to the study of acetylation and methylation since these PTMs are chemical modifications resulting in specific elemental mass additions to peptides. Mono-, di-, and trimethylation add +14.0156, +28.0312, and +42.0469 Da, respectively. In terms of data interpretation, mon- omethylation is isobaric with several amino acid substitutions such as Asp-Asn, Gly-Ala, and Val-Leu/Ile. Acetylation results in the addition of +42.0105 Da, a small mass difference of 0.0363 Da relative to trimethylation but distinguishable by the use of high-resolution MS . Acetylation and methylation are stable modifications relative to the more labile modifications such as phosphorylation and glycosylation . As such they are retained intact during sample preparation and MS analyses, typically operated in positive mode.
N-terminal tails of histone proteins possess a number of acetylation and methylation sites; as such, these are often exemplified for method develop- ment/proof of concept of novel PTM approaches for the analysis of post-translationally modified and isomeric peptides, which are discussed in this chapter. Analysis of acetylation and methylation is achieved by using three MS-complementary approaches: bottom up, top down, and middle down [23, 24]. Bottom-up strategies are directed to peptides to provide information to identify and localize sites. This is achieved by fragmentation to generate MS2 (or MS/MS) product ion spectra. PTM assignment is aided by the generation of modification-specific diagnostic fragments. Acetyl- and methyl-specific enrichment strategies are applied for improved sensitivity of detection. Quantification strategies to profile alterations of acetylated and methylated proteins, including analysis of modification dynamics and stoichiometry are also typically, but not exclusively, applied at the peptide level. Intact proteins or larger fragments are analyzed in top-down and middle-down approaches for characterization of complex PTM patterns in individual proteins.
MS analysis benefits from coupling liquid chromatography (LC) instrumentation or other prefractionation methods that reduce peptide complexity. Such methods also provide additional parameters such as LC retention time information to aid analysis. For example, lysine trimethylation and acetylation can also be distinguished by their distinct chromatographic retention times . In general, the co-occurrence of acetylation and methylation sites makes their analysis challenging. In particular, isomeric peptides can result, which possess the same modification(s) at different sites, and thus share physicochemical properties that result in coelution and cofragmentation during LC-MS analysis. Chimeric product ion spectra make PTM site assignment problematic, particularly when no unique fragment ions are detected.
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