Enhanced top-down characterization of histone post-translational modifications

Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS analysis of 7.5 µg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.     

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications.     

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications.     

Electron capture dissociation mass spectrometry in characterization of post-translational modifications

Electron capture dissociation (ECD) represents a significant advance in tandem mass spectrometry for the identification and characterization of post-translational modifications (PTMs) of polypeptides. In comparison with the conventional fragmentation techniques, such as collisionally induced dissociation and infrared multi-photon dissociation, ECD provides more extensive sequence fragments, while allowing the labile modifications to remain intact during backbone fragmentation. This unique attribute offers ECD as an attractive alternative for detection and localization of PTMs. The success and rapid adoption of ECD recently led to the culmination of The 1st International Uppsala Symposium on Electron Capture Dissociation of Biomolecules and Related Phenomena (October 19-22, 2003, Stockholm, Sweden). Herein, we present a general overview of the ECD technique as well as selected applications in characterization of post-translationally modified polypeptides.     

Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry

Post-translational modifications generate tremendous diversity, complexity and heterogeneity of gene products, and their determination is one of the main challenges in proteomics research. Recent developments in mass spectrometry based approaches for systematic, qualitative and quantitative determination of modified proteins promise to bring new insights on the dynamics and spatio-temporal control of protein activities by post-translational modifications, and reveal their roles in biological processes and pathogenic conditions. Combinations of affinity-based enrichment and extraction methods, multidimensional separation technologies and mass spectrometry are particularly attractive for systematic investigation of post-translationally modified proteins in proteomics.     

A predictive coarse-grained model for position-specific effects of post-translational modifications

Biomolecules undergo liquid-liquid phase separation (LLPS), resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiological and pathological role of post-translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed-charge atomistic force fields, we parameterize the size and atomistic hydropathy of the coarse-grained-modified amino acid beads and, hence, the interactions between the modified and natural amino acids. We then elucidate how the number and position of phosphorylated and acetylated residues alter the protein’s single-chain compactness and its propensity to phase separate. We show that both the number and the position of phosphorylated threonines/serines or acetylated lysines can serve as a molecular on/off switch for phase separation in the well-studied disordered regions of Fused in Sarcoma (FUS) and DDX3X, respectively. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture experimentally measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase separation. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resolution.     

Human placental calreticulin characterization of domain structure and post-translational modifications.

The domain organization and the post-translational modifications of human placenta calreticulin were analysed by MS in combination with proteolytic digestion. Prolonged treatment with trypsin, chymotrypsin, elastase, Staphylococcus aureus V8 protease, or proteinase K all led to a 6- to 7-kDa decrease in the molecular mass of calreticulin. The decrease was found to be due to cleavages in the region around residue 340. In addition, minor fragments resulting from secondary cleavages close to the N-terminus were observed, but no stable fragments of intermediate size were found. These results show that the C-domain of calreticulin is susceptible to proteolytic cleavage and that the N- and P-domains form a proteolytically stable tight association. A disulfide bridge between the first two cysteines was mapped in the N-domain, and the third cysteine was found in the reduced form. No post-translational modifications in the form of glycosylation or phosphorylation were found. A modified form of calreticulin lacking the C-terminal hexapeptide including the KDEL endoplasmic reticulum retention sequon was isolated. Such a truncation may point to a mechanism that allows escape of calreticulin from the endoplasmic reticulum.     

Existing bioinformatics tools for the quantitation of post-translational modifications

Mass spectrometry (MS)-based proteomics, by itself, is a vast and complex area encompassing various mass spectrometers, different spectra, and search result representations. When the aim is quantitation performed in different scanning modes at different MS levels, matters become additionally complex. Quantitation of post-translational modifications (PTM) represents the greatest challenge among these endeavors. Many different approaches to quantitation have been described and some of these can be directly applied to the quantitation of PTMs. The amount of data produced via MS, however, makes manual data interpretation impractical. Therefore, specialized software tools meet this challenge. Any software currently able to quantitate differentially labeled samples may theoretically be adapted to quantitate differential PTM expression among samples as well. Due to the heterogeneity of mass spectrometry-based proteomics; this review will focus on quantitation of PTM using liquid chromatography followed by one or more stages of mass spectrometry. Currently available free software, which either allow analysis of PTM or are easily adaptable for this purpose, is briefly reviewed in this paper. Selected studies, especially those related to phosphoproteomics, shall be used to highlight the current ability to quantitate PTMs.     

Proteomic analysis of post-translational modifications

Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel mass spectrometric peptide sequencing and analysis technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.     

A peptidoform based proteomic strategy for studying functions of post-translational modifications

Protein post-translational modifications (PTMs) generate an enormous, but as yet undetermined, expansion of the produced proteoforms. In this Viewpoint, we firstly reviewed the concepts of proteoform and peptidoform. We show that many of the current PTM biological investigation and annotation studies largely follow a PTM site-specific rather than proteoform-specific approach. We further illustrate a potentially useful matching strategy in which a particular “modified peptidoform” is matched to the corresponding “unmodified peptidoform” as a reference for the quantitative analysis between samples and conditions. We suggest this strategy has the potential to provide more directly relevant information to learn the PTM site-specific biological functions. Accordingly, we advocate for the wider use of the nomenclature “peptidoform” in future bottom-up proteomic studies.     

Purification and characterization of EpiA, the peptide substrate for post-translational modifications involved in epidermin biosynthesis

Abstract For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems. Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion. The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing. For EpiA detection, anti-EpiA antisera were raised. Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R⁻¹ and I⁺¹. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A⁻⁴-E-P-R⁻¹- to -A⁻⁴-E-P-Q⁻¹- by site-directed mutagenesis.     

Techniques for studying protein heterogeneity and post-translational modifications

Proteins often undergo several post-translational modification steps in parallel to protein folding. These modifications can be transient or of a more permanent nature. Most modifications are, however, susceptible to alteration during the lifespan of proteins. Post-translational modifications thus generate variability in proteins that are far beyond that provided by the genetic code. Co- and post-translational modifications can convert the 20 specific codon-encoded amino acids into more than 100 variant amino acids with new properties. These, and a number of other modifications, can considerably increase the information content and functional repertoire of proteins, thus making their analysis of paramount importance for diagnostic and basic research purposes. Various methods used in proteomics, such as 2D gel electrophoresis, 2D liquid chromatography, mass spectrometry, affinity-based analytical methods, interaction analyses, ligand blotting techniques, protein crystallography and structure–function predictions, are all applicable for the analysis of these numerous secondary modifications. In this review, examples of some of these techniques in studying the heterogeneity of proteins are highlighted. In the future, these methods will become increasingly useful in biomarker searches and in clinical diagnostics.     

Data-independent acquisition proteomics methods for analyzing post-translational modifications

Protein post-translational modifications (PTMs) increase the functional diversity of the cellular proteome. Accurate and high throughput identification and quantification of protein PTMs is a key task in proteomics research. Recent advancements in data-independent acquisition (DIA) mass spectrometry (MS) technology have achieved deep coverage and accurate quantification of proteins and PTMs. This review provides an overview of DIA data processing methods that cover three aspects of PTMs analysis, that is, detection of PTMs, site localization, and characterization of complex modification moieties, such as glycosylation. In addition, a survey of deep learning methods that boost DIA-based PTMs analysis is presented, including in silico spectral library generation, as well as feature scoring and error rate control. The limitations and future directions of DIA methods for PTMs analysis are also discussed. Novel data analysis methods will take advantage of advanced MS instrumentation techniques to empower DIA MS for in-depth and accurate PTMs measurements.     

System level dynamics of post-translational modifications

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Techniques for studying protein heterogeneity and post-translational modifications

Proteins often undergo several post-translational modification steps in parallel to protein folding. These modifications can be transient or of a more permanent nature. Most modifications are, however, susceptible to alteration during the lifespan of proteins. Post-translational modifications thus generate variability in proteins that are far beyond that provided by the genetic code. Co- and post-translational modifications can convert the 20 specific codon-encoded amino acids into more than 100 variant amino acids with new properties. These, and a number of other modifications, can considerably increase the information content and functional repertoire of proteins, thus making their analysis of paramount importance for diagnostic and basic research purposes. Various methods used in proteomics, such as 2D gel electrophoresis, 2D liquid chromatography, mass spectrometry, affinity-based analytical methods, interaction analyses, ligand blotting techniques, protein crystallography and structure-function predictions, are all applicable for the analysis of these numerous secondary modifications. In this review, examples of some of these techniques in studying the heterogeneity of proteins are highlighted. In the future, these methods will become increasingly useful in biomarker searches and in clinical diagnostics.     

Surface accessibility of protein post-translational modifications

Protein post-translational modifications are crucial to the function of many proteins. In this study, we have investigated the structural environment of 8378 incidences of 44 types of post-translational modifications with 19 different approaches. We show that modified amino acids likely to be involved in protein-protein interactions, such as ester-linked phosphorylation, methylarginine, acetyllysine, sulfotyrosine, hydroxyproline, and hydroxylysine, are clearly surface associated. Other modifications, including O-GlcNAc, phosphohistidine, 4-aspartylphosphate, methyllysine, and ADP-ribosylarginine, are either not surface associated or are in a protein's core. Artifactual modifications were found to be randomly distributed throughout the protein. We discuss how the surface accessibility of post-translational modifications can be important for protein-protein interactivity.     

Critical histone post-translational modifications for centromere function and propagation

The centromere is a critical genomic region that enables faithful chromosome segregation during mitosis, and must be distinguishable from other genomic regions to facilitate establishment of the kinetochore. The centromere-specific histone H3-variant CENP-A forms a special nucleosome that functions as a marker for centromere specification. In addition to the CENP-A nucleosomes, there are additional H3 nucleosomes that have been identified in centromeres, both of which are predicted to exhibit specific features. It is likely that the composite organization of CENP-A and H3 nucleosomes contributes to the formation of centromere-specific chromatin, termed ‘centrochromatin’. Recent studies suggest that centrochromatin has specific histone modifications that mediate centromere specification and kinetochore assembly. We use chicken non-repetitive centromeres as a model of centromeric activities to characterize functional features of centrochromatin. This review discusses our recent progress, and that of various other research groups, in elucidating the functional roles of histone modifications in centrochromatin.     

Proteomics studies of post-translational modifications in plants

Post-translational modifications of proteins greatly increase protein complexity and dynamics, co-ordinating the intricate regulation of biological events. The global identification of post-translational modifications is a difficult task that is currently accelerated by advances in proteomics techniques. There has been significant development in sample preparation methods and mass spectrometry instrumentation. To reduce the complexity and to increase the amount of modified proteins available for analysis, proteins are usually subjected to prefractionation such as chromatographic purification and affinity enrichment. In this review, the post-translational modification studies in plants are summarized. The sample preparation strategies applied to each study are also described. These include affinity-based enrichment methods, immobilized metal affinity chromatography and immunoprecipitation used for phosphorylation and ubiquitination studies, respectively, and the phase partitioning approach for glycosylphosphatidylinositol modification studies.