Characterization of Post-Translational Modifications on Histone H4 and the Histone Chaperone N1 Using Mass Spectrometry

2013 ◽  
Author(s):  
Joseph Victor Strukl
Keyword(s):  
Mass Spectrometry ◽  
Histone Chaperone ◽  
Histone H4 ◽  
Post Translational Modifications

scholarly journals Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

2020 ◽  
Author(s):  
Manish Bhattacharjee ◽  
Navin Adhikari ◽  
Renu Sudhakar ◽  
Zeba Rizvi ◽  
Divya Das ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Plasmodium Falciparum ◽  
Western Blot ◽  
Wild Type ◽  
Malaria Parasites ◽  
Post Translational Modifications ◽  
Functional Conservation ◽  
Cellular Processes ◽  
Physiological Substrates

ABSTRACTA variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles. The neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulate diverse cellular processes, including the cell-cycle. Although neddylation pathway is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. Towards studying the neddylation pathway in malaria parasites, we characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Gly76 mutated to Ala75Ala76) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 to proteins through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified several proteins, including two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (Δrub1) or NEDD8 conjugating E2 enzyme (ΔUbc12). The western blot of complemented strains and mass spectrometry of PfNEDD8 immunoprecipitate showed conjugation of PfNEDD8 to S. cerevisiae cullin cdc53, demonstrating functional conservation and cullins as the physiological substrates of PfNEDD8. The characterization of PfNEDD8 and identification of cullins as its substrates make ground for investigation of specific roles and drug target potential of neddylation pathway in malaria parasites.

scholarly journals Aspartic Acid Isomerization Characterized by High Definition Mass Spectrometry Significantly Alters the Bioactivity of a Novel Toxin from Poecilotheria

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 207 ◽  
Author(s):  
Stephen R. Johnson ◽  
Hillary G. Rikli
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Ion Mobility ◽  
Protein Protein Interactions ◽  
High Definition ◽  
Vast Number ◽  
Post Translational Modifications ◽  
C Terminus ◽  
Acid Isomerization

Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of “tiger” spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product’s diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.

scholarly journals Characterization of Histone H2A and H2B Variants and Their Post-translational Modifications by Mass Spectrometry

2005 ◽  
Vol 5 (3) ◽  
pp. 541-552 ◽  
Author(s):  
Débora Bonenfant ◽  
Michèle Coulot ◽  
Harry Towbin ◽  
Patrick Schindler ◽  
Jan van Oostrum
Keyword(s):  
Mass Spectrometry ◽  
Histone H2a ◽  
Post Translational Modifications

scholarly journals VDACs Post-Translational Modifications Discovery by Mass Spectrometry: Impact on Their Hub Function

2021 ◽  
Vol 22 (23) ◽  
pp. 12833
Author(s):  
Maria Gaetana Giovanna Pittalà ◽  
Stefano Conti Nibali ◽  
Simona Reina ◽  
Vincenzo Cunsolo ◽  
Antonella Di Francesco ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Characterization ◽  
High Performance ◽  
High Resolution Mass Spectrometry ◽  
Complex Mixture ◽  
Reversed Phase ◽  
Vital Role ◽  
Membrane Pore ◽  
Post Translational Modifications

VDAC (voltage-dependent anion selective channel) proteins, also known as mitochondrial porins, are the most abundant proteins of the outer mitochondrial membrane (OMM), where they play a vital role in various cellular processes, in the regulation of metabolism, and in survival pathways. There is increasing consensus about their function as a cellular hub, connecting bioenergetics functions to the rest of the cell. The structural characterization of VDACs presents challenging issues due to their very high hydrophobicity, low solubility, the difficulty to separate them from other mitochondrial proteins of similar hydrophobicity and the practical impossibility to isolate each single isoform. Consequently, it is necessary to analyze them as components of a relatively complex mixture. Due to the experimental difficulties in their structural characterization, post-translational modifications (PTMs) of VDAC proteins represent a little explored field. Only in recent years, the increasing number of tools aimed at identifying and quantifying PTMs has allowed to increase our knowledge in this field and in the mechanisms that regulate functions and interactions of mitochondrial porins. In particular, the development of nano-reversed phase ultra-high performance liquid chromatography (nanoRP-UHPLC) and ultra-sensitive high-resolution mass spectrometry (HRMS) methods has played a key role in this field. The findings obtained on VDAC PTMs using such methodologies, which permitted an in-depth characterization of these very hydrophobic trans-membrane pore proteins, are summarized in this review.

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