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PTM Repositories and Data Mining Tools

There are a number of online PTM repositories including PhosphoSitePlus [120], SysPTM 2.0 [121], PTMCode [122], and ProteomeScout [123]. PTMCode indicates functional links between individual and multiple PTMs including acetylation and methylation [122]. Another software, ProteomeScout, provides a compendium of public PTM data, including quantitative proteomics and details on functional annotation [123]. These repositories aid design of follow-on experimental work and provide data for the development of in silico prediction tools.

Computational Prediction Tools for Acetylation and Methylation Sites

There has been a focus on the development and application of tools to predict acetylation and methylation sites to complement and extend analysis performed in vitro in a cost-effective manner. The challenges around site prediction have been outlined by review and evaluation of PMeS, PLMLA, MeMo, MASA, BPB-PPMS, MethK, and iMethyl-PseAAC using a common dataset of methylation sites [124]. It was concluded that performance needs to be optimized and that it would be of benefit to adopt different feature information for methyl-arginine and methyl-lysine predictors [124]. The PLMLA tool also predicts acetylation sites and has been compared with the LAceP, EnsemblePail, PHOSIDA, and PSKAcePred acetylation site prediction software [125]. The comparison utilized information on 13,810 acetylation sites from 6388 proteins, which was obtained from SysPTM 2.0 and PhosphoSitePlus databases. LAceP performed best, employing a logistic regression method to integrate information on amino acid sequence adjacent to acetylated sites, physicochemical properties, and the transition probability of adjacent amino acids [125].

A comprehensive and comparative analysis of acetylation, methylation, ubiq- uitinylation, and SUMOylation has identified conserved amino acid sequence association with secondary structure [126]. Information, combined with site specificity data for enzymes mediating addition and removal of acetyl and methyl groups, can benefit site prediction as has been demonstrated for lysine acetyltransferases [127]. Chemoselective reactions provide an MS method for the experimental determination of acetyl and methyl transferase activities. Examples include alkynyl-acetyl-CoA and SAM analogs for bioorthogonal click chemistry analysis of proteins that are substrates for acetylation and methylation, respectively [128-130]. These complement other MS-based analyses (Table 4.2).

Table 4.2 Biochemical methods coupled to MS for analysis of protein acetylation and methylation.

Method

Specificity

Example

references

Immunoaffinity capture (peptides)

Acetylation (lysine) Methylation (arginine) Methylation (lysine)

[19]

[39, 99, 100] [46, 100, 101]

Immunoaffinity capture (proteins)

Methylation (arginine, lysine)

Acetylation (lysine)

[102]

[103]

Reader domain-based capture

Acetylation

Methylation (mono-, dimethyl-lysine)

Methylation (di-, trimethyl-lysine)

[93]

[105, 106] [106, 107]

Biotin switch capture

Acetylation

[94, 108, 109]

Solid-phase extraction

N-terminal acetyl (including lysine)

[110, 111]

Chemical reporters (bioorthogonal reagents for click chemistry)

Acetylation (N-terminal, lysine)

Methylation (arginine, lysine)

[128, 129] [130]

Notes: Lysine acylations other than acetylation can be analyzed [129].

 
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