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Free radical biology and medicine 198, 27 - 43 (2023). doi:10.1016/j.freeradbiomed.2023.01.022, The mechanisms of one-electron protein oxidation are complicated and still not well-understood. In this work, we investigated the reaction of sensitized photo-oxidation using carboxybenzophenone (CB) as a sensitizer and a PR-10 protein (MtN13) as a quencher, which is intrinsically complicated due to the complex structure of the protein and multiple possibilities of CB attack.To predict and examine the possible reactions precisely, the 3D structure of the MtN13 protein was taken into account. Our crystallographic studies revealed a specific binding of the CB molecule in the protein's hydrophobic cavity, while mass spectrometry identified the amino acid residues (Met, Tyr, Asp and Phe) creating adducts with the sensitizer, thus indicating the sites of 3CB* quenching. In addition, protein aggregation was also observed.The detailed mechanisms of CB quenching by the MtN13 molecule were elucidated by an analysis of transient products by means of time-resolved spectroscopy. The investigation of the transient and stable products formed during the protein photo-oxidation was based on the data obtained from HPLC-MS analysis of model compounds, single amino acids and dipeptides.Our proposed mechanisms of sensitized protein photo-oxidation emphasize the role of a ground state complex between the protein and the sensitizer and indicate several new and specific products arising as a result of one-electron oxidation. Based on the analysis of the transient and stable products, we have demonstrated the influence of neighboring groups, especially in the case of Tyr oxidation, where the tyrosyl radical can be formed via a direct electron transfer from Tyr to CB* or via an intramolecular electron transfer from Tyr to Met radical cation Met > S●+ or thiyl radical CysS● from neighboring oxidized groups., Published by Elsevier, New York, NY [u.a.]

Publisher Summary This chapter describes a procedure for identifying protein phosphorylation sites by mass spectrometry. Tight regulation of cellular proteins is a prerequisite for viable cell function. Cells require various mechanisms whereby intracellular pathways can be activated and inactivated in a reversible manner depending on the prevailing environment at a particular moment in time. Reversible phosphorylation of proteins is one such mechanism. The radioactively labeled peptide, which should contain the phosphorylated residue, is detected, isolated, and the sequence determined, usually by Edman degredation. In addition, phosphoamino acid analysis can be used to determine the nature of the phosphorylated residue. In addition to the amount of phosphorylation of an individual protein, specific factors relating to the phosphoprotein itself may determine the ease to which a phosphorylation site is identified. For example, the molecular mass of the peptide containing the phosphorylation site should ideally be between 500 and 4000 Da. A rare difficulty arises when a nonphosphorylated peptide has the same molecular weight as the phosphopeptide of interest.

Publisher Summary Phosphoproteome analysis by mass spectrometry is in its infancy, but this research field is developing quickly. Integration of various technologies for phosphoprotein enrichment, phosphopeptide detection and quantitation and phosphopeptide sequencing is clearly needed. These methods have to be coupled with novel bioinformatics tools for analysis and visualization of the protein data that is obtained. Methods are continuously being refined at all these levels, most notably in sample preparation methods, tandem mass spectrometry techniques, and bioinformatics software. The complete characterization of the proteome of a given organism, tissue or organelle must necessarily include identification and detailed structural analysis of the dynamically changing populations of post-translationally modified gene products. Combinations of genetic, biochemical, and immunological techniques with mass spectrometry-based methods result in novel quantitative analytical strategies that may soon allow researchers to study the dynamics of complex cellular processes. Reversible protein phosphorylation is among the best characterized of the hundreds of known post-translational modifications and it plays a pivotal role in cellular development, differentiation, growth and homeostasis. Phosphoproteins are involved in all biochemical processes in the cell, including transcriptional and translational regulation, metabolism, protein degradation, cellular signaling, and communication, and in maintaining the structural integrity of cells. The crucial role of protein phosphorylation and dephosphorylation events is emphasized by the fact that protein kinases, phosphoprotein phosphatases and their respective substrates are implicated in a number of human diseases and ailments, including diabetes and many types of cancer.

Publisher Summary This chapter describes the mass spectrometric protocol for the analysis of UV-crosslinked protein–nucleic acid complexes. It presents a protocol that combines developed methods for UV-light-induced photochemical crosslinking of proteins to nucleic acids with MALDI, and ESI mass spectrometry to locate and identify the particular amino acid and nucleotide residues that covalently bind to each other. The chapter explains the application of some of the stages in this analytical scheme to two different systems of protein–nucleic acid complexes, the phage T4 gene 32 protein UV-laser cross-linked to the oligonucleotide photoaffinity-labeled with 4-thiouridinediphosphate. In the first stage, the nucleic acid binding protein and its nucleic acid substrate or a photoactivatable analogue thereof are incubated under proper conditions to form the protein–nucleic acid complex. The sample is subsequently irradiated with UV light for a given period of time to create photochemically crosslinked protein–nucleic acid complexes.