Functional analysis tools for post‐translational modification: a post‐translational modification database for analysis of proteins and metabolic pathways

Post‐translational modifications (PTMs) are critical regulators of protein function, and nearly 200 different types of PTM have been identified. Advances in high‐resolution mass spectrometry have led to the identification of an unprecedented number of PTM sites in numerous organisms, potentially facilitating a more complete understanding of how PTMs regulate cellular behavior. While databases have been created to house the resulting data, most of these resources focus on individual types of PTM, do not consider quantitative PTM analyses or do not provide tools for the visualization and analysis of PTM data. Here, we describe the Functional Analysis Tools for Post‐Translational Modifications (FAT‐PTM) database ( https://bioinformatics.cse.unr.edu/fat-ptm/ ), which currently supports eight different types of PTM and over 49 000 PTM sites identified in large‐scale proteomic surveys of the model organism Arabidopsis thaliana. The FAT‐PTM database currently supports tools to visualize protein‐centric PTM networks, quantitative phosphorylation site data from over 10 different quantitative phosphoproteomic studies, PTM information displayed in protein‐centric metabolic pathways and groups of proteins that are co‐modified by multiple PTMs. Overall, the FAT‐PTM database provides users with a robust platform to share and visualize experimentally supported PTM data, develop hypotheses related to target proteins or identify emergent patterns in PTM data for signaling and metabolic pathways.     

The Plant PTM Viewer,a central resource for exploring plant protein modifications

Post‐translational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased numbers and types of mapped protein modifications, a database is necessary that simultaneously integrates and compares site‐specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 370 000 PTM sites for 19 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with an estimate of the confidence by which the modified peptides and, if possible, the actual modification sites were identified and with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools help to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository that allows the submission of mass spectrometry‐based PTM data to remain at pace with future PTM plant studies.     

Dual Coordination of Post Translational Modifications in Human Protein Networks

Post-translational modifications (PTMs) regulate protein activity, stability and interaction profiles and are critical for cellular functioning. Further regulation is gained through PTM interplay whereby modifications modulate the occurrence of other PTMs or act in combination. Integration of global acetylation, ubiquitination and tyrosine or serine/threonine phosphorylation datasets with protein interaction data identified hundreds of protein complexes that selectively accumulate each PTM, indicating coordinated targeting of specific molecular functions. A second layer of PTM coordination exists in these complexes, mediated by PTM integration (PTMi) spots. PTMi spots represent very dense modification patterns in disordered protein regions and showed an equally high mutation rate as functional protein domains in cancer, inferring equivocal importance for cellular functioning. Systematic PTMi spot identification highlighted more than 300 candidate proteins for combinatorial PTM regulation. This study reveals two global PTM coordination mechanisms and emphasizes dataset integration as requisite in proteomic PTM studies to better predict modification impact on cellular signaling.     

Simultaneous Quantification of the Acetylome and Succinylome by ‘One‐Pot’ Affinity Enrichment

Protein posttranslational modifications (PTMs) are of increasing interest in biomedical research, yet studies rarely examine more than one PTM. One barrier to multi‐PTM studies is the time cost for both sample preparation and data acquisition, which scale linearly with the number of modifications. The most prohibitive requirement is often the need for large amounts of sample, which must be increased proportionally with the number of PTM enrichment steps. Here, a streamlined, quantitative label‐free proteomic workflow—“one‐pot” PTM enrichment—that enables comprehensive identification and quantification of peptides containing acetylated and succinylated lysine residues from a single sample containing as little as 1 mg mitochondria protein is described. Coupled with a label‐free, data‐independent acquisition (DIA), 2235 acetylated and 2173 succinylated peptides with the one‐pot method are identified and quantified and peak areas are shown to be highly correlated between the one‐pot and traditional single‐PTM enrichments. The ‘one‐pot’ method makes possible detection of multiple PTMs occurring on the same peptide, and it is shown that it can be used to make unique biological insights into PTM crosstalk. Compared to single‐PTM enrichments, the one‐pot workflow has equivalent reproducibility and enables direct assessment of PTM crosstalk from biological samples in less time from less tissue.     

MAPRes: Mining association patterns among preferred amino acid residues in the vicinity of amino acids targeted for post-translational modifications

Post-translational modification (PTM) of a protein is an important event in regulating cellular functions. An algorithm, MAPRes, has been developed for mining associations among PTM sites and the preferred amino acids in their vicinity. The algorithm has been implemented to O-glycosylation and O-phosphorylation data (phosphorylated/glycosylated Ser/Thr/Tyr). The association patterns mined by MAPRes demonstrate significant correlations and the results are in conformity with the existing methods. These association rules/patterns will be helpful in predicting the sequences/motifs involved for specific PTMs in proteins.     

Proteome-wide lysine acetylation identification in developing rice (Oryza sativa) seeds and protein co-modification by acetylation,succinylation, ubiquitination,and phosphorylation

Protein lysine acetylation is a highly conserved post-translational modification with various biological functions. However, only a limited number of acetylation sites have been reported in plants, especially in cereals, and the function of non-histone protein acetylation is still largely unknown. In this report, we identified 1003 lysine acetylation sites in 692 proteins of developing rice seeds, which greatly extended the number of known acetylated sites in plants. Seven distinguished motifs were detected flanking acetylated lysines. Functional annotation analyses indicated diverse biological processes and pathways engaged in lysine acetylation. Remarkably, we found that several key enzymes in storage starch synthesis pathway and the main storage proteins were heavily acetylated. A comprehensive comparison of the rice acetylome, succinylome, ubiquitome and phosphorylome with available published data was conducted. A large number of proteins carrying multiple kinds of modifications were identified and many of these proteins are known to be key enzymes of vital metabolic pathways. Our study provides extending knowledge of protein acetylation. It will have critical reference value for understanding the mechanisms underlying PTM mediated multiple signal integration in the regulation of metabolism and development in plants.     

A method to distinguish between lysine acetylation and lysine ubiquitination with feature selection and analysis

Lysine acetylation and ubiquitination are two primary post-translational modifications (PTMs) in most eukaryotic proteins. Lysine residues are targets for both types of PTMs, resulting in different cellular roles. With the increasing availability of protein sequences and PTM data, it is challenging to distinguish the two types of PTMs on lysine residues. Experimental approaches are often laborious and time consuming. There is an urgent need for computational tools to distinguish between lysine acetylation and ubiquitination. In this study, we developed a novel method, called DAUFSA (distinguish between lysine acetylation and lysine ubiquitination with feature selection and analysis), to discriminate ubiquitinated and acetylated lysine residues. The method incorporated several types of features: PSSM (position-specific scoring matrix) conservation scores, amino acid factors, secondary structures, solvent accessibilities, and disorder scores. By using the mRMR (maximum relevance minimum redundancy) method and the IFS (incremental feature selection) method, an optimal feature set containing 290 features was selected from all incorporated features. A dagging-based classifier constructed by the optimal features achieved a classification accuracy of 69.53%, with an MCC of .3853. An optimal feature set analysis showed that the PSSM conservation score features and the amino acid factor features were the most important attributes, suggesting differences between acetylation and ubiquitination. Our study results also supported previous findings that different motifs were employed by acetylation and ubiquitination. The feature differences between the two modifications revealed in this study are worthy of experimental validation and further investigation.     

Discriminating between Lysine Sumoylation and Lysine Acetylation Using mRMR Feature Selection and Analysis

Post-translational modifications (PTMs) are crucial steps in protein synthesis and are important factors contributing to protein diversity. PTMs play important roles in the regulation of gene expression, protein stability and metabolism. Lysine residues in protein sequences have been found to be targeted for both types of PTMs: sumoylations and acetylations; however, each PTM has a different cellular role. As experimental approaches are often laborious and time consuming, it is challenging to distinguish the two types of PTMs on lysine residues using computational methods. In this study, we developed a method to discriminate between sumoylated lysine residues and acetylated residues. The method incorporated several features: PSSM conservation scores, amino acid factors, secondary structures, solvent accessibilities and disorder scores. By using the mRMR (Maximum Relevance Minimum Redundancy) method and the IFS (Incremental Feature Selection) method, an optimal feature set was selected from all of the incorporated features, with which the classifier achieved 92.14% accuracy with an MCC value of 0.7322. Analysis of the optimal feature set revealed some differences between acetylation and sumoylation. The results from our study also supported the previous finding that there exist different consensus motifs for the two types of PTMs. The results could suggest possible dominant factors governing the acetylation and sumoylation of lysine residues, shedding some light on the modification dynamics and molecular mechanisms of the two types of PTMs, and provide guidelines for experimental validations.     

Influence of the sequence environment and properties of neighboring amino acids on amino‐acetylation: Relevance for structure‐function analysis

Proteins function is regulated by co‐translational modifications and post‐translational modifications (PTMs) such as phosphorylation, glycosylation, and acetylation, which induce proteins to perform multiple tasks in a specified environment. Acetylation takes place post‐translationally on the ε‐amino group of Lys in histone proteins, allowing regulation of gene expression. Furthermore, amino group acetylation also occurs co‐translationally on Ser, Thr, Gly, Met, and Ala, possibly contributing to the stability of proteins. In this work, the influence of amino acids next to acetylated sites has been investigated by using MAPRes (Mining Association Patterns among preferred amino acid residues in the vicinity of amino acids targeted for PTMs). MAPRes was utilized to examine the sequence patterns vicinal to modified and non‐modified residues, taking into account their charge and polarity. The PTMs data were further sub‐divided according to their sub‐cellular location (nuclear, mitochondrial, and cytoplasmic), and their association patterns were mined. The association patterns mined by MAPRes for acetylated and non‐acetylated residues are consistent with the existing literature but also revealed novel patterns. These rules have been utilized to describe the acetylation and its effects on the protein structure‐function relationship. J. Cell. Biochem. 114: 874–887, 2013. © 2012 Wiley Periodicals, Inc.     

JUMPptm: Integrated software for sensitive identification of post-translational modifications and its application in Alzheimer's disease study

Background : Mass spectrometry (MS)-based proteomic analysis of posttranslational modifications (PTMs) usually requires the pre-enrichment of modified proteins or peptides. However, recent ultra-deep whole proteome profiling generates millions of spectra in a single experiment, leaving many unassigned spectra, some of which may be derived from PTM peptides. Methods : Here we present JUMPptm, an integrative computational pipeline, to extract PTMs from unenriched whole proteome. JUMPptm combines the advantages of JUMP, MSFragger and Comet search engines, and includes de novo tags, customized database search and peptide filtering, which iteratively analyzes each PTM by a multi-stage strategy to improve sensitivity and specificity. Results : We applied JUMPptm to the deep brain proteome of Alzheimer's disease (AD), and identified 34,954 unique peptides with phosphorylation, methylation, acetylation, ubiquitination, and others. The phosphorylated peptides were validated by enriched phosphoproteome from the same sample. TMT-based quantification revealed 482 PTM peptides dysregulated at different stages during AD progression. For example, the acetylation of numerous mitochondrial proteins is significantly decreased in AD. A total of 60 PTM sites are found in the pan-PTM map of the Tau protein. Conclusion : The JUMPptm program is an effective tool for pan-PTM analysis and the resulting AD pan-PTM profile serves as a valuable resource for AD research.     

Deciphering a global network of functionally associated post‐translational modifications

Various post‐translational modifications (PTMs) fine‐tune the functions of almost all eukaryotic proteins, and co‐regulation of different types of PTMs has been shown within and between a number of proteins. Aiming at a more global view of the interplay between PTM types, we collected modifications for 13 frequent PTM types in 8 eukaryotes, compared their speed of evolution and developed a method for measuring PTM co‐evolution within proteins based on the co‐occurrence of sites across eukaryotes. As many sites are still to be discovered, this is a considerable underestimate, yet, assuming that most co‐evolving PTMs are functionally associated, we found that PTM types are vastly interconnected, forming a global network that comprise in human alone >50 000 residues in about 6000 proteins. We predict substantial PTM type interplay in secreted and membrane‐associated proteins and in the context of particular protein domains and short‐linear motifs. The global network of co‐evolving PTM types implies a complex and intertwined post‐translational regulation landscape that is likely to regulate multiple functional states of many if not all eukaryotic proteins.     

Proteomic analysis of lysine acetylation provides strong evidence for involvement of acetylated proteins in plant meiosis and tapetum function

Protein lysine acetylation (KAC) is a dynamic and reversible post‐translational modification that has important biological roles in many organisms. Although KAC has been shown to affect reproductive development and meiosis in yeast and animals, similar studies are largely lacking in flowering plants, especially proteome‐scale investigations for particular reproductive stages. Here, we report results from a proteomic investigation to detect the KAC status of the developing rice anthers near the time of meiosis (RAM), providing strong biochemical evidence for roles of many KAC‐affected proteins during anther development and meiosis in rice. We identified a total of 1354 KAC sites in 676 proteins. Among these, 421 acetylated proteins with 629 KAC sites are novel, greatly enriching our knowledge on KAC in flowering plants. Gene Ontology enrichment analysis showed chromatin silencing, protein folding, fatty acid biosynthetic process and response to stress to be over‐represented. In addition, certain potentially specific KAC motifs in RAM were detected. Importantly, 357 rice meiocyte proteins were acetylated; and four proteins genetically identified to be important for rice tapetum and pollen development were acetylated on 14 KAC sites in total. Furthermore, 47 putative secretory proteins were detected to exhibit acetylated status in RAM. Moreover, by comparing our lysine acetylome with the RAM phosphoproteome we obtained previously, we proposed a correlation between KAC and phosphorylation as a potential modulatory mechanism in rice RAM. This study provides the first global survey of KAC in plant reproductive development, making a promising starting point for further functional analysis of KAC during rice anther development and meiosis.     

New roles for old modifications: Emerging roles of N‐terminal post‐translational modifications in development and disease

The importance of internal post‐translational modification (PTM) in protein signaling and function has long been known and appreciated. However, the significance of the same PTMs on the alpha amino group of N‐terminal amino acids has been comparatively understudied. Historically considered static regulators of protein stability, additional functional roles for N‐terminal PTMs are now beginning to be elucidated. New findings show that N‐terminal methylation, along with N‐terminal acetylation, is an important regulatory modification with significant roles in development and disease progression. There are also emerging studies on the enzymology and functional roles of N‐terminal ubiquitylation and N‐terminal propionylation. Here, will discuss the recent advances in the functional studies of N‐terminal PTMs, recount the new N‐terminal PTMs being identified, and briefly examine the possibility of dynamic N‐terminal PTM exchange.     

Lysine methylation: beyond histones

Posttranslational modifications (PTMs) of histone proteins, such as acetylation, methylation, phosphorylation, and ubiquitylation, play essential roles in regulating chromatin dynamics. Combinations of different modifications on the histone proteins, termed 'histone code' in many cases, extend the information potential of the genetic code by regulating DNA at the epigenetic level. Many PTMs occur on non-histone proteins as well as histones, regulating protein-protein interactions, stability, localization, and/or enzymatic activities of proteins involved in diverse cellular processes. Although protein phosphorylation, ubiquitylation, and acetylation have been extensively studied, only a few proteins other than histones have been reported that can be modified by lysine methylation. This review summarizes the current progress on lysine methylation of non-histone proteins, and we propose that lysine methylation, like phosphorylation and acetylation, is a common PTM that regulates proteins in diverse cellular processes.     

The functional diversity of protein lysine methylation

Large‐scale characterization of post‐translational modifications (PTMs), such as phosphorylation, acetylation and ubiquitination, has highlighted their importance in the regulation of a myriad of signaling events. While high‐throughput technologies have tremendously helped cataloguing the proteins modified by these PTMs, the identification of lysine‐methylated proteins, a PTM involving the transfer of one, two or three methyl groups to the ε‐amine of a lysine side chain, has lagged behind. While the initial findings were focused on the methylation of histone proteins, several studies have recently identified novel non‐histone lysine‐methylated proteins. This review provides a compilation of all lysine methylation sites reported to date. We also present key examples showing the impact of lysine methylation and discuss the circuitries wired by this important PTM.     

The structural context of posttranslational modifications at a proteome-wide scale

The recent revolution in computational protein structure prediction provides folding models for entire proteomes, which can now be integrated with large-scale experimental data. Mass spectrometry (MS)-based proteomics has identified and quantified tens of thousands of posttranslational modifications (PTMs), most of them of uncertain functional relevance. In this study, we determine the structural context of these PTMs and investigate how this information can be leveraged to pinpoint potential regulatory sites. Our analysis uncovers global patterns of PTM occurrence across folded and intrinsically disordered regions. We found that this information can help to distinguish regulatory PTMs from those marking improperly folded proteins. Interestingly, the human proteome contains thousands of proteins that have large folded domains linked by short, disordered regions that are strongly enriched in regulatory phosphosites. These include well-known kinase activation loops that induce protein conformational changes upon phosphorylation. This regulatory mechanism appears to be widespread in kinases but also occurs in other protein families such as solute carriers. It is not limited to phosphorylation but includes ubiquitination and acetylation sites as well. Furthermore, we performed three-dimensional proximity analysis, which revealed examples of spatial coregulation of different PTM types and potential PTM crosstalk. To enable the community to build upon these first analyses, we provide tools for 3D visualization of proteomics data and PTMs as well as python libraries for data accession and processing.

A combination of the comprehensive structural predictions of AlphaFold2 and large-scale proteomics data on post-translational modifications (PTMs) reveals novel insights into the functional importance of PTMs, based on their structural context.