Angelo Palmese

Quantitative identification of protein nitration sites

Several labelling strategies have been developed targeting specific amino acid residues and/or PTMs. Methods specifically tailored for the qualitative and sometimes quantitative determination of PTMs have emerged. Many research groups have focused their attention towards o‐nitrotyrosine residues, developing various methodologies for their identification, while direct quantification has remained elusive. So far the iTRAQ chemistry has been limited to primary amines. Here, we report a new strategy based on the use of iTRAQ reagents coupled to MS analysis for the selective labelling of o‐nitrotyrosine residues. This method was proved to lead to the simultaneous localisation and quantification of nitration sites both in model proteins and in biological systems.

Plasma nitroproteome of kidney disease patients

Abstract3′-nitrotyrosine (3NT) is a post-translational modification (PTM) of body fluids and tissues that is sustained by chronic inflammation and oxidative stress, two main clinical traits of chronic kidney disease (CKD). Despite this background, protein targets and their differential susceptibility to in vivo nitration remain almost completely unexplored in CKD. This study reports a first investigation of plasma nitroproteome in these patients, carried out by both immunorecognition and LC-MS/MS techniques. Plasma proteins of chronic and end-stage KD patients showed a higher burden of nitration than in healthy controls, but main nitration targets appeared to be the same in these populations. Immunoblotting data showed that uremic albumin is largely represented in the uremic nitroproteome together with fibrinogen chains (A, B and C), transferrin, α1-antitrypsin, complement factor D, haptoglobin, and IgG light and heavy chains. However, immunopurification and affinity chromatography experiments demonstrated that the relative content of 3NT on the albumin molecule was very low when compared with that of the remaining plasma proteins. The uremic nitroproteome was investigated using also plasma proteins obtained by in vivo ultrafiltration from patients treated with protein leaking or standard high-flux hemodialyzers. The study of these samples revealed the possibility to selectively remove protein nitration products during hemodialysis. Identification of intramolecular sites of nitration was preliminarily obtained in IgG chains isolated by 2D PAGE and assessed by bidimensional tandem mass spectrometry after chemoselective tagging. Further studies are needed to confirm at the molecular level the presence of nitrated Tyr residues in other proteins tentatively identified as nitration targets in this study and to explore the biological meaning of such a selective modification of plasma proteins by reactive nitrogen species in uremia and dialysis patients.

Privileged Incorporation of Selenium as Selenocysteine in Lactobacillus reuteri Proteins Demonstrated by Selenium-specific Imaging and Proteomics

An analytical approach was developed to study the incorporation of selenium (Se), an important trace element involved in the protection of cells from oxidative stress, into the well-known probiotic Lactobacillus reuteri Lb2 BM-DSM 16143. The analyses revealed that about half of the internalized Se was covalently incorporated into soluble proteins. Se-enriched proteins were detected in 2D gels by laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) and identified by capillary HPLC with the parallel ICP MS (78Se) and electrospray Orbitrap MS/MS detection. On the basis of the identification of 10 richest in selenium proteins, it was demonstrated that selenium was incorporated by the strain exclusively as selenocysteine. Also, the exact location of selenocysteine within the primary sequence was determined. This finding is in a striking contrast to another common nutraceutical, Se-enriched yeast, which incorporates Se principally as selenomethionine.

Novel method to investigate protein carbonylation by iTRAQ strategy

AbstractThis paper reports a novel methodology for relative quantitative analysis of carbonylation sites in proteins by exploiting a new isobaric tag for relative and absolute quantitation (iTRAQ) derivative, iTRAQ hydrazide (iTRAQH), and the analytical power of linear ion trap instruments (QqLIT). Because of its operational simplicity, avoiding time-consuming enrichment procedures, this new strategy seems to be well suited for quantitative large-scale proteomic profiling of carbonylation. FigureModification of alpha-aminoadipic semialdehyde by a novel iTRAQ reagent, namely iTRAQ-hydrazide; by using this reagent it is possible to selectively label carbonylated residues in order to perform qualitative and quantitative analysis of protein carbonylation.

Technical advances in proteomics mass spectrometry: identification of post-translational modifications

Abstract The importance of post-translational modifications (PTMs) of proteins has become evident in the proteomic era as it plays a critical role in modulating cellular function, and can vary in response to different stimuli thereby tuning cellular mechanisms. Assessment of PTMs on a proteomic scale is a challenging task since they are substoichiometric, transient and reversible. Moreover, the amount of post-translationally modified proteins is generally very small when compared to their unmodified counterparts. Existing methodologies for identification of PTMs essentially relies on enrichment procedure to selectively increase the amount of modified peptides. These procedures need to be integrated with sophisticated mass spectrometric methods to enable the identifications of PTMs. Although the strategies developed so far are not optimal, a number of examples will be given where the combination of innovative separation methods along with advanced mass spectrometric analyses provide positive results. These experiences are leading the way for the next generation of proteomic approaches for identification of a wide range of PTMs. Clin Chem Lab Med 2009;47:647–65.

Subtype-Selective Activation of Kv7 Channels by AaTXKβ(2–64), a Novel Toxin Variant from the Androctonus australis Scorpion Venom

Kv7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair cells and skeletal and smooth muscle cells. Openers of Kv7.4 channels have been suggested to improve hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of peptides active on K+ channels. Therefore, we have optimized a combined purification/screening procedure to identify specific modulator(s) of Kv7.4 channels from the venom of the North African scorpion Androctonus australis (Aa). We report the isolation and functional characterization of AaTXKβ(2–64), a novel variant of AaTXKβ(1–64), in a high-performance liquid chromatography fraction from Aa venom (named P8), which acts as the first peptide activator of Kv7.4 channels. In particular, in both Xenopus oocytes and mammalian Chinese hamster ovary cells, AaTXKβ(2–64), but not AaTXKβ(1–64), hyperpolarized the threshold voltage of current activation and increased the maximal currents of heterologously expressed Kv7.4 channels. AaTXKβ(2–64) also activated Kv7.3, Kv7.2/3, and Kv7.5/3 channels, whereas homomeric Kv1.1, Kv7.1, and Kv7.2 channels were unaffected. We anticipate that these results may prove useful in unraveling the novel biologic roles of AaTXKβ(2–64)-sensitive Kv7 channels and developing novel pharmacologic tools that allow subtype-selective targeting of Kv7 channels.