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In this review, we extensively describe the main post-translational modifications that give rise to the multiple proteoforms characterized to date in the human salivary proteome and their potential role. Most of the data reported were obtained by our group in over twenty-five years of research carried out on human saliva mainly by applying a top-down strategy. In the beginning, we describe the products generated by proteolytic cleavages, which can occur before and after secretion. In this section, the most relevant families of salivary proteins are also described. Next, we report the current information concerning the human salivary phospho-proteome and the limited news available on sulfo-proteomes. Three sections are dedicated to the description of glycation and enzymatic glycosylation. Citrullination and N- and C-terminal post-translational modifications (PTMs) and miscellaneous other modifications are described in the last two sections. Results highlighting the variation in the level of some proteoforms in local or systemic pathologies are also reviewed throughout the sections of the manuscript to underline the impact and relevance of this information for the development of new diagnostic biomarkers useful in clinical practice.

Based on our previous proteomic study on Cavitating Ultrasound Aspirator (CUSA) fluid pools of Newly Diagnosed (ND) and Recurrent (R) glioblastomas (GBMs) of tumor core and periphery, as defined by 5-aminolevulinc acid (5-ALA) metabolite fluorescence, this work aims to apply a bioinformatic approach to investigate specifically into three sub-proteomes, i.e., Not Detected in Brain (NB), Cancer Related (CR) and Extracellular Vesicles (EVs) proteins following selected database classification. The study of these yet unexplored specific datasets aims to understand the high infiltration capability and relapse rate that characterizes this aggressive brain cancer. Out of the 587 proteins highly confidently identified in GBM CUSA pools, 53 proteins were classified as NB. Their gene ontology (GO) analysis showed the over-representation of blood coagulation and plasminogen activating cascade pathways, possibly compatible with Blood Brain Barrier damage in tumor disease and surgery bleeding. However, the NB group also included non-blood proteins and, specifically, histones correlated with oncogenesis. Concerning CR proteins, 159 proteins were found in the characterized GBM proteome. Their GO analysis highlighted the over-representation of many pathways, primarily glycolysis. Interestingly, while CR proteins were identified in ND-GBM exclusively in the tumor zones (fluorescence positive core and periphery zones) as predictable, conversely, in R-GBM they were unexpectedly characterized prevalently in the healthy zone (fluorescence negative tumor periphery). Relative to EVs protein classification, 60 proteins were found. EVs are over-released in tumor disease and are important in the transport of biological macromolecules. Furthermore, the presence of EVs in numerous body fluids makes them a possible low-invasive source of brain tumor biomarkers to be investigated. These results give new hints on the molecular features of GBM in trying to understand its aggressive behavior and ope

A CE-tandem MS method was optimised and validated for selective and specific determination of LVV- and VV-hemorphin-7 peptides in cerebrospinal fluid. These two small peptides originate from haemoglobin beta chains. They possess relevant biological activity and recently a potential biomarker role in posterior cranial fossa paediatric brain tumour disease was evidenced. The separation was optimised using formic acid as background electrolyte and a water/methanol mixture, containing 0.1\% (v/v) formic acid, as sheath liquid. The two peptides, differing in only one amino acid of the sequence at the N-terminal side were baseline separated in less than 15 min. The method allowed a very reduced and rapid sample pretreatment and was successfully applied to hemorphins determination in patient samples without matrix interferences. The method successfully passed bioanalytical validation showing linearity, accuracy and precision data on cerebrospinal fluid matrix within the acceptable values. The analysis of cerebrospinal fluid of patients affected by different posterior cranial fossa tumour forms confirmed our previous findings showing the absence of hemorphins in the pre-surgical cerebrospinal fluid and their presence in the post-ones and controls. The present method saves costs and time due to capillary electrophoresis miniaturisation and to the absence of chromatographic column and gradient elution and allows numerous injections per sample starting from few microlitres of cerebrospinal fluid.

In recent years the capillary electrophoresis (CE) analytical technique have had a large impact in chemical, biotechnological, environmental and pharmaceutical field because of its high efficiency and resolution power exhibited towards extremely small amounts of a wide variety of compounds, including peptides, proteins, nucleotides, DNA, enantiomers, carbohydrates, vitamins, inorganic ions and drugs. Methacrylic compounds such as 2-hydroxyethyl methacrylate (HEMA) are largely present in auto- or photo-polymerizable composite resins utilized in dentistry and in other biomedical applications. After performing dental restorations with composite resins, small amounts of monomers are released from the incompletely polymerized material into either the oral cavity and – through dentinal diffusion – into the pulpal tissues. Subsequently, methacrylic monomers may cause, or at least contribute to, adverse biological effects, like damages to the oral soft tissues, already observed in vivo, a remarkable in vitro cytotoxicity and a depletion of glutathione (GSH). The latter is produced in all organs and cells, where it accomplishes several physiological tasks including relevant protection against oxidative stress by its oxidation to GSSG and the concurrent reduction of hydrogen peroxide and lipid peroxides; moreover GSH binds to xenobiotics facilitating their removal. Several reports have also demonstrated a protective effect of N-acetyl cysteine (NAC) against the cytotoxicity of dental resins or materials containing HEMA also via the formation of a molecular adduct. The present work summarizes the recent results obtained by capillary electrophoresis-mass spectrometry (CE-MS) and micellar electrokinetic capillary chromatography (MEKC) techniques in the detection of the adducts between NAC or GSH and the above mentioned methacrylic compound both in complex systems represented by primary gingival fibroblasts and 3T3-Swiss albino-mouse fibroblasts cell line, evidencing the key role

In this review, the recent contribution of CE-MS technology to the analysis of amino acids, as well as the advantages of the hyphenation and the technologies involved in the instrumental coupling are reported. Different sections are dedicated to the recent contributions of CE-MS to the analysis of protein amino acids and their post-translational modifications, such as phosphorylation and sulfation. CE-MS analysis of some amino acid derivatives, such as the free methylated-derivatives of arginine is also discussed. A section is specifically devoted to the CE-MS applications in the field of chiral separation of D-and L-amino acid enantiomers.

The general properties of dendrimers and in particular their electrolytic characteristics that are relevant in electrokinetic separations, are described. In order to confirm theoretical considerations on commercial dendrimer charge and hydrodynamic radius, several capillary zone electrophoresis (CZE) experiments were performed. Electrophoretic mobilities measured at different pH values indicated a sensible increase of dendrimer hydrodynamic radius at pH values lower than 2.5. This was probably due to the Coulombic repulsion of charged amine groups of the inner dendrimer shells. The principal reasons that should address the use of dendrimers as pseudostationary phases in micellar electrokinetic chromatography (MEKC) are discussed. Moreover, a survey of different separations performed utilizing dendrimers in MEKC as well as of several future plausible uses of various classes of dendrimers is presented.