Mechanistic study of competitive releases of H2O, NH3 and CO2 from deprotonated aspartic and glutamic acids: Role of conformation.

The aims of this study were to highlight the impact of minor structural differences (e.g. an aminoacid side chain enlargement by one methylene group), on ion dissociation under collision-induced dissociation conditions, and to determine the underlying chemical mechanisms. Therefore, we compared fragmentations of deprotonated aspartic and glutamic acids generated in negative electrospray ionization. Energy-resolved mass spectrometry breakdown curves were recorded and MS3 experiments performed on an Orbitrap Fusion for high-resolution and high-mass accuracy measurements. Activated fragmentations were performed using both the resonant and non-resonant excitation modes (i.e., CID and HCD, respectively) in order to get complementary information on the competitive and consecutive dissociative pathways. These experiments showed a specific loss of ammonia from the activated aspartate but not from the activated glutamate. We mainly focused on this specific observed loss from aspartate. Two different mechanisms based on intramolecular reactions (similar to those occurring in organic chemistry) were proposed, such as intramolecular elimination (i.e. Ei-like) and nucleophilic substitution (i.e. SNi-like) reactions, respectively, yielding anions as fumarate and α lactone from a particular conformation with the lowest steric hindrance (i.e. with antiperiplanar carboxyl groups). The detected deaminated aspartate anion can then release CO2 as observed in the MS3 experimental spectra. However, quantum calculations did not indicate the formation of such a deaminated aspartate product ion without loss of carbon dioxide. Actually, calculations displayed the double neutral (NH3+CO2) loss as a concomitant pathway (from a particular conformation) with relative high activation energy instead of a consecutive process. This disagreement is apparent since the concomitant pathway may be changed into consecutive dissociations according to the collision energy i.e., at higher collision energy and at lower excitation conditions, respectively. The latter takes place by stabilization of the deaminated aspartate solvated with two residual molecules of water (present in the collision cell). This desolvated anion formed is an α lactone substituted by a methylene carboxylate group. The vibrational excitation acquired by [(D-H)-NH3]-during its isolation is enough to allow its prompt decarboxylation with a barrier lower than 8.4kJ/mol. In addition, study of glutamic acid-like diastereomers constituted by a cyclopropane, hindering any side chain rotation, confirms the impact of the three-dimensional geometry on fragmentation pathways. A significant specific loss of water is only observed for one of these diastereomers. Other experiments, such as stable isotope labeling, need to be performed to elucidate all the observed losses from activated aspartate and glutamate anions. These first mechanistic interpretations enhance understanding of this dissociative pathway and underline the necessity of studying fragmentation of a large number of various compounds to implement properly new algorithms for de novo elucidation of unknown metabolites.

The tumor suppressor annexin A10 is a novel component of nuclear paraspeckles.

Annexin A10 is the latest identified member of the annexin family of Ca(2+)- and phospholipid-binding proteins. In previous studies, downregulation of annexin A10 was correlated with dedifferentiation, invasion, and tumor progression, pointing to a possible tumor suppressor role. However, the biochemical characteristics and functions of annexin A10 remain unknown. We show that annexin A10 displays biochemical characteristics atypical for an annexin, indicating a Ca(2+)- and membrane-binding-independent function. Annexin A10 co-localizes with the mRNA-binding proteins SFPQ and PSPC1 at paraspeckles, an only recently discovered nuclear body, and decreases paraspeckle numbers when overexpressed in HeLa cells. In addition, annexin A10 relocates to dark perinucleolar caps upon transcriptional inhibition of RNA polymerase II. We mapped the cap-binding function of annexin A10 to the proximal part of the core domain, which is missing in the short isoform of annexin A10, and show its independence from the remaining functional type II Ca(2+)-binding site. In contrast to this, paraspeckle recruitment required additional core regions and was negatively affected by the mutation of the last type II Ca(2+)-binding site. Additionally, we show that overexpression of annexin A10 in HeLa cells increases their sensitivity to apoptosis and reduces colony formation. The identification of unique nuclear and biochemical characteristics of annexin A10 points towards its membrane-independent role in paraspeckle-associated mRNA regulation or processing.

Chemical phosphorylation of histidine residues in proteins using potassium phosphoramidate -- a tool for the analysis of acid-labile phosphorylation.

Histidine (His)-phosphorylation is labile at low pH and has therefore not been in the focus of proteomic analysis in the past although a few single-case studies have been performed. The systematic investigation of model substances generates confidence in experimental procedures and allows determining their limits. In order to extend earlier peptide studies to His-phosphoproteins and elucidate their behavior and recovery in proteomic procedures, potassium phosphoramidate (PPA) was used to generate model proteins, which were subsequently exposed to gel electrophoresis, enzymatic digest and mass spectrometry based protein analysis. Myoglobin having eleven His-residues was highly phosphorylated by PPA showing a distribution of modified protein forms with four phosphate-carrying His-residues in the most abundant species. Since myoglobin is a heme-binding protein it was additionally indicated that synthetic phosphorylation may retain protein folding targeting only structurally accessible His-residues. Insulin, ßcasein and cytochrome C were phosphorylated on their His-residues and the corresponding peptides were detected in protein digest mixtures and in background of tryptically digested Escherichia coli lysate. In gel electrophoresis protocols, lengthy procedures at low pH such as staining reduced recovery. Synthetic phosphorylation of proteins and peptides with PPA allows the generation of suitable standard compounds for the systematic optimization of analytical protocols. All tested proteins responded to PPA treatment, partially even preserving tertiary structure. A distribution of modified protein forms was generated which could be subjected to further separation to isolate the fully phosphorylated species.

Stepchild phosphohistidine: acid-labile phosphorylation becomes accessible by functional proteomics.

Bioanalytical techniques were preferentially developed for the investigation of phosphohydroxyamino acids in the past and there is a wealth of information on the detection of serine, threonine and tyrosine phosphorylation in functional proteomics. However, similarly important for protein regulation and signalling is the phosphorylation of other amino acids such as histidine, but its detection is hampered by the sensitivity to acid. Mass spectrometry in conjunction with chromatographic methods is allowing us to start to get a handle on phosphohistidine. (32)P-labelling and amino acid analysis for phosphorylation site determination is increasingly complemented by typical proteomic approaches based on reversed-phase peptide separation and gas-phase fragmentation. Chemical phosphorylation of peptides is a valuable tool, therefore, for the generation of analytical standards for use in method development.