The detailed examination of enzyme molecules by mass spectrometry and other techniques continues to identify hundreds of distinct PTMs. Recently, global analyses of enzymes using methods of contemporary proteomics revealed widespread distribution of PTMs on many key enzymes distributed in all cellular compartments. Critically, patterns of multiple enzymatic and nonenzymatic PTMs within a single enzyme are now functionally evaluated providing a holistic picture of a macromolecule interacting with low molecular mass compounds, some of them being substrates, enzyme regulators, or activated precursors for enzymatic and nonenzymatic PTMs. Multiple PTMs within a single enzyme molecule and their mutual interplays are critical for the regulation of catalytic activity. Full understanding of this regulation will require detailed structural investigation of enzymes, their structural analogs, and their complexes. Further, proteomics is now integrated with molecular genetics, transcriptomics, and other areas leading to systems biology strategies. These allow the functional interrogation of complex enzymatic networks in their natural environment. In the future, one might envisage the use of robust high throughput analytical techniques that will be able to detect multiple PTMs on a global scale of individual proteomes from a number of carefully selected cells and cellular compartments. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

• Klíčová slova
• ABRF, AGE, ALE, AML, APC/C, Association for Biomolecular Resource Facilities, CCT, CDK, CHO, COS-1, CSC, CV-1 in Origin carrying SV40 genetic material (cell line), Catalytic activity, Cellular localization, Chinese hamster ovary, EC, ECD, EGF, ER-associated protein degradation, ERAD, Enzyme, GFP, HECT, HEK, IP3, MDM, MMP, MRM, Posttranslational modification, RAGE, RING, RNS, S-adenosyl-l-homocysteine, S-adenosyl-l-methionine, SAH, SAM, SIL, Stability, Structure, TAS, TCP-1, acute myeloid leukemia, advanced glycosylation endproduct, advanced lipooxidation endproduct, anaphase-promoting complex/cyclosome, cell surface capture technology, chaperone containing TCP-1, cyclin-dependent kinase, electron capture dissociation, enzyme commission of IUPAC, epidermal growth factor, green fluorescent protein, homologous to the E6-AP carboxyl terminus, human embryonic kidney, inositoltrisphosphate, matrix metalloproteinase, multiple reaction monitoring, murine double minute, reactive nitrogen species, really interesting new gene, receptor for advanced glycosylation end products, stable isotope labeling, tagging via substrate approach, tailless complex polypeptide-1,
• MeSH
• enzymy * MeSH
• katalýza MeSH
• lidé MeSH
• posttranslační úpravy proteinů * MeSH
• proteomika metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• enzymy * MeSH

Posttranslational modifications (PTMs) of proteins represent fascinating extensions of the dynamic complexity of living cells' proteomes. The results of enzymatically catalyzed or spontaneous chemical reactions, PTMs form a fourth tier in the gene - transcript - protein cascade, and contribute not only to proteins' biological functions, but also to challenges in their analysis. There have been tremendous advances in proteomics during the last decade. Identification and mapping of PTMs in proteins have improved dramatically, mainly due to constant increases in the sensitivity, speed, accuracy and resolution of mass spectrometry (MS). However, it is also becoming increasingly evident that simple gel-free shotgun MS profiling is unlikely to suffice for comprehensive detection and characterization of proteins and/or protein modifications present in low amounts. Here, we review current approaches for enriching and separating posttranslationally modified proteins, and their MS-independent detection. First, we discuss general approaches for proteome separation, fractionation and enrichment. We then consider the commonest forms of PTMs (phosphorylation, glycosylation and glycation, lipidation, methylation, acetylation, deamidation, ubiquitination and various redox modifications), and the best available methods for detecting and purifying proteins carrying these PTMs. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

• Klíčová slova
• PTM, PTM detection, Posttranslational modification, Protein enrichment, Proteome, Top-down proteomics,
• MeSH
• lidé MeSH
• posttranslační úpravy proteinů * MeSH
• proteom chemie   izolace a purifikace   metabolismus   MeSH
• proteomika metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• proteom MeSH

The alteration in proteome composition induced by environmental changes and various pathologies is accompanied by the modifications of proteins by specific cotranslational and PTMs. The type and site stoichiometry of PTMs can affect protein functions, alter cell signaling, and can have acute and chronic effects. The particular interest is drawn to those amino acid residues that can undergo several different PTMs. We hypothesize that these selected amino acid residues are biologically rare and act within the cell as molecular switches. There are, at least, 12 various lysine modifications currently known, several of them have been shown to be competitive and they influence the ability of a particular lysine to be modified by a different PTM. In this review, we discuss the PTMs that occur on lysine, specifically neddylation and sumoylation, and the proteomic approaches that can be applied for the identification and quantification of these PTMs. Of interest are the emerging roles for these modifications in heart disease and what can be inferred from work in other cell types and organs.

• Klíčová slova
• Heart pathologies, Lysine PTMs, Modified proteins, Neddylation, Sumoylation, Technology,
• MeSH
• kardiomyocyty chemie   metabolismus   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• lysin * analýza   chemie   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• myokard patologie   MeSH
• myši MeSH
• nemoci srdce metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• proteom analýza   MeSH
• sekvence aminokyselin MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• lysin * MeSH
• proteom MeSH

BACKGROUND: Leucine-rich alpha-2-glycoprotein (LRG) has been repeatedly proposed as a potential plasma biomarker for myelodysplastic syndrome (MDS). OBJECTIVE: The goal of our work was to establish the total LRG plasma level and LRG posttranslational modifications (PTMs) as a suitable MDS biomarker. METHODS: The total plasma LRG concentration was determined with ELISA, whilst the LRG-specific PTMs and their locations, were established using mass spectrometry and public mass spectrometry data re-analysis. Homology modelling and sequence analysis were used to establish the potential impact of PTMs on LRG functions via their impact on the LRG structure. RESULTS: While the results showed that the total LRG plasma concentration is not a suitable MDS marker, alterations within two LRG sites correlated with MDS diagnosis (p= 0.0011). Sequence analysis and the homology model suggest the influence of PTMs within the two LRG sites on the function of this protein. CONCLUSIONS: We report the presence of LRG proteoforms that correlate with diagnosis in the plasma of MDS patients. The combination of mass spectrometry, re-analysis of publicly available data, and homology modelling, represents an approach that can be used for any protein to predict clinically relevant protein sites for biomarker research despite the character of the PTMs being unknown.

• Klíčová slova
• LRG, MDS, Myelodysplastic syndrome, leucine-rich alpha-2-glycoprotein, proteomics,
• MeSH
• biologické markery MeSH
• glykoproteiny * genetika   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• leucin metabolismus   MeSH
• lidé MeSH
• myelodysplastické syndromy * diagnóza   MeSH
• posttranslační úpravy proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• glykoproteiny * MeSH
• leucin MeSH

Post-translational modifications (PTMs) of biomacromolecules can be useful for understanding the processes by which a relatively small number of individual genes in a particular genome can generate enormous biological complexity in different organisms. The proteomes of barley and the brewing process were investigated by different techniques. However, their diverse and complex PTMs remain understudied. As standard analytical approaches have limitations, innovative analytical approaches need to be developed and applied in PTM studies. To make further progress in this field, it is necessary to specify the sites of modification, as well as to characterize individual isoforms with increased selectivity and sensitivity. This review summarizes advances in the PTM analysis of barley proteins, particularly those involving mass spectrometric detection. Our focus is on monitoring phosphorylation, glycation, and glycosylation, which critically influence functional behavior in metabolism and regulation in organisms.

• Klíčová slova
• barley, mass spectrometry, post-translational modification, protein,
• MeSH
• fosforylace MeSH
• glykosylace MeSH
• ječmen (rod) * genetika   MeSH
• posttranslační úpravy proteinů MeSH
• proteom chemie   MeSH
• proteomika metody   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• proteom MeSH

Protein-protein interactions (PPIs) form a complex network called "interactome" that regulates many functions in the cell. In recent years, there is an increasing accumulation of evidence supporting the existence of a hyperbolic geometry underlying the network representation of complex systems such as the interactome. In particular, it has been shown that the embedding of the human Protein-Interaction Network (hPIN) in hyperbolic space (H2) captures biologically relevant information. Here we explore whether this mapping contains information that would allow us to predict the function of PPIs, more specifically interactions related to post-translational modification (PTM). We used a random forest algorithm to predict PTM-related directed PPIs, concretely, protein phosphorylation and dephosphorylation, based on hyperbolic properties and centrality measures of the hPIN mapped in H2. To evaluate the efficacy of our algorithm, we predicted PTM-related PPIs of ataxin-1, a protein which is responsible for Spinocerebellar Ataxia type 1 (SCA1). Proteomics analysis in a cellular model revealed that several of the predicted PTM-PPIs were indeed dysregulated in a SCA1-related disease network. A compact cluster composed of ataxin-1, its dysregulated PTM-PPIs and their common upstream regulators may represent critical interactions for disease pathology. Thus, our algorithm may infer phosphorylation activity on proteins through directed PPIs.

• MeSH
• algoritmy MeSH
• fosforylace MeSH
• lidé MeSH
• mapování interakce mezi proteiny * metody   MeSH
• mapy interakcí proteinů * MeSH
• posttranslační úpravy proteinů MeSH
• proteomika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

A method for the fast separation of proteins and identification of their modifications based on the use of monolithic chromatographic media and mass spectrometric techniques is described. This method has been developed and applied to the analysis of malt proteins and its posttranslational modifications (glycation). Glycation, one of the most common forms of posttranslational modifications (PTM), can be detected in both biological and industrial samples. Our attention was focused on the investigations of possible chemical modifications of water-soluble barley proteins during malting process by combination of anion-exchange chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The malt extract was directly fractioned by anion-exchange chromatography using short monolithic columns and a linear gradient from 0 to 700 mM NaCl. Sufficient fractionation was obtained for malt sample, which demonstrates the potential of anion-exchange chromatography on this type of column. Proteins in separated fractions were identified by MALDI-TOF/TOF MS. Our proteomic analysis provided the identification of the major proteins present in the malt that were found to be heterogeneously glycated after malting. One of these proteins: nonspecific lipid transfer protein 1 (LTP1) can be used as a marker for characterization of glycation during malting. This protein and its modifications can be easily determined by the developed method.

• MeSH
• chromatografie iontoměničová metody   MeSH
• jedlá semena chemie   MeSH
• posttranslační úpravy proteinů MeSH
• proteom analýza   chemie   metabolismus   MeSH
• proteomika metody   MeSH
• reprodukovatelnost výsledků MeSH
• rostlinné proteiny analýza   chemie   metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom MeSH
• rostlinné proteiny MeSH

Over 400 different types of post-translational modifications (PTMs) have been reported and over 200 various types of PTMs have been discovered using mass spectrometry (MS)-based proteomics. MS-based proteomics has proven to be a powerful method capable of global PTM mapping with the identification of modified proteins/peptides, the localization of PTM sites and PTM quantitation. PTMs play regulatory roles in protein functions, activities and interactions in various heart related diseases, such as ischemia/reperfusion injury, cardiomyopathy and heart failure. The recognition of PTMs that are specific to cardiovascular pathology and the clarification of the mechanisms underlying these PTMs at molecular levels are crucial for discovery of novel biomarkers and application in a clinical setting. With sensitive MS instrumentation and novel biostatistical methods for precise processing of the data, low-abundance PTMs can be successfully detected and the beneficial or unfavorable effects of specific PTMs on cardiac function can be determined. Moreover, computational proteomic strategies that can predict PTM sites based on MS data have gained an increasing interest and can contribute to characterization of PTM profiles in cardiovascular disorders. More recently, machine learning- and deep learning-based methods have been employed to predict the locations of PTMs and explore PTM crosstalk. In this review article, the types of PTMs are briefly overviewed, approaches for PTM identification/quantitation in MS-based proteomics are discussed and recently published proteomic studies on PTMs associated with cardiovascular diseases are included.

• Klíčová slova
• MS‐based proteomics, cardiovascular disease, post‐translational modifications, proteins,
• MeSH
• fosforylace MeSH
• hmotnostní spektrometrie metody   MeSH
• kardiovaskulární nemoci * metabolismus   genetika   patologie   MeSH
• lidé MeSH
• posttranslační úpravy proteinů * MeSH
• proteomika * metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.

• Klíčová slova
• DNA–protein interaction, lysine succinylation, mitochondria, mitochondrial DNA, mitochondrial nucleoid, post-translational modification (PTM), proteomics, succinylome, yeast,
• MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• kyselina jantarová metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• proteasa La genetika   metabolismus   MeSH
• proteomika * MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ABF2 protein, S cerevisiae MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• kyselina jantarová MeSH
• mitochondriální proteiny MeSH
• proteasa La MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• transkripční faktory MeSH

Hepatitis B virus (HBV) core protein (HBc) plays many roles in the HBV life cycle, such as regulation of transcription, RNA encapsidation, reverse transcription, and viral release. To accomplish these functions, HBc interacts with many host proteins and undergoes different post-translational modifications (PTMs). One of the most common PTMs is ubiquitination, which was shown to change the function, stability, and intracellular localization of different viral proteins, but the role of HBc ubiquitination in the HBV life cycle remains unknown. Here, we found that HBc protein is post-translationally modified through K29-linked ubiquitination. We performed a series of co-immunoprecipitation experiments with wild-type HBc, lysine to arginine HBc mutants and wild-type ubiquitin, single lysine to arginine ubiquitin mutants, or single ubiquitin-accepting lysine constructs. We observed that HBc protein could be modified by ubiquitination in transfected as well as infected hepatoma cells. In addition, ubiquitination predominantly occurred on HBc lysine 7 and the preferred ubiquitin chain linkage was through ubiquitin-K29. Mass spectrometry (MS) analyses detected ubiquitin protein ligase E3 component N-recognin 5 (UBR5) as a potential E3 ubiquitin ligase involved in K29-linked ubiquitination. These findings emphasize that ubiquitination of HBc may play an important role in HBV life cycle.

• Klíčová slova
• E3 ubiquitin-protein ligase, HBc, hepatitis B virus, post-translational modifications, ubiquitin, ubiquitination,
• MeSH
• arginin genetika   MeSH
• buněčné linie MeSH
• buňky Hep G2 MeSH
• HEK293 buňky MeSH
• hepatitida B genetika   MeSH
• hepatocelulární karcinom genetika   MeSH
• lidé MeSH
• lysin genetika   MeSH
• nádorové buněčné linie MeSH
• posttranslační úpravy proteinů genetika   MeSH
• ubikvitin genetika   MeSH
• ubikvitinace genetika   MeSH
• ubikvitinligasy genetika   MeSH
• virové proteiny genetika   MeSH
• virus hepatitidy B genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• arginin MeSH
• lysin MeSH
• ubikvitin MeSH
• ubikvitinligasy MeSH
• virové proteiny MeSH