Claudia Martelli

Chrono-Proteomics of Human Saliva: Variations of the Salivary Proteome during Human Development

An important contribution to the variability of any proteome is given by the time dimension that should be carefully considered to define physiological modifications. To this purpose, whole saliva proteome was investigated in a wide age range. Whole saliva was collected from 17 preterm newborns with a postconceptional age at birth of 178-217 days. In these subjects sample collection was performed serially starting immediately after birth and within about 1 year follow-up, gathering a total of 111 specimens. Furthermore, whole saliva was collected from 182 subjects aged between 0 and 17 years and from 23 adults aged between 27 and 57 years. The naturally occurring intact salivary proteome of the 316 samples was analyzed by low- and high-resolution HPLC-ESI-MS platforms. Proteins peculiar of the adults appeared in saliva with different time courses during human development. Acidic proline-rich proteins encoded by PRH2 locus and glycosylated basic proline-rich proteins encoded by PRB3 locus appeared following 180 days of postconceptional age, followed at 7 months (±2 weeks) by histatin 1, statherin, and P-B peptide. The other histatins and acidic proline-rich proteins encoded by PRH1 locus appeared in whole saliva of babies from 1 to 3 weeks after the normal term of delivery, S-type cystatins appeared at 1 year (±3 months), and basic proline-rich proteins appeared at 4 years (±1 year) of age. All of the proteinases involved in the maturation of salivary proteins were more active in preterm than in at-term newborns, on the basis of the truncated forms detected. The activity of the Fam20C kinase, involved in the phosphorylation of various proteins, started around 180 days of postconceptional age, slowly increased reaching values comparable to adults at about 2 years (±6 months) of age. Instead, MAPK14 involved in the phosphorylation of S100A9 was fully active since birth also in preterm newborns.

Proteomic characterization of pediatric craniopharyngioma intracystic fluid by LC‐MS top‐down/bottom‐up integrated approaches

The combination of top‐down and bottom‐up platforms was utilized for the LC‐MS proteomic characterization of the intracystic fluid of adamantinomatous craniopharyngioma pediatric brain tumor disease. Proteins and peptides characterization was achieved by high‐resolution LC‐ESI‐LTQ‐Orbitrap‐MS analysis while low‐resolution LC‐ESI‐IT‐MS was applied for the complete screening of the samples and the evaluation of the protein distribution within patients. Top‐down analyses were applied to liquid/liquid extracted samples while bottom‐up analyses were performed after trypsin digestion of both untreated and pretreated samples. The two proteomic approaches were complementary for the characterization of the proteome of craniopharyngioma intracystic fluid. Proteins and peptides involved in inflammation, mineralization processes and lipid transport were identified, in agreement with the calcium flecks, cholesterol granules and bone residues characteristic of this fluid. Apolipoprotein A‐I, A‐II, C‐I and J, hemoglobin fragments, ubiquitin, α‐2‐HS‐glycoprotein or fetuin A, α‐1‐antichymotrypsin, vitamin D binding protein, and α‐1‐acid glycoprotein were characterized. These data could be relevant for the comprehension of the processes involved in the pathogenesis of the disease and the development of the cyst and could contribute to the individuation of therapeutic targets for the reduction of the cyst volume delaying and/or avoiding invasive surgical treatments.

Proteomic Study of Pilocytic Astrocytoma Pediatric Brain Tumor Intracystic Fluid

Liquid chromatography in coupling with high-resolution ESI-LTQ-Orbitrap mass spectrometry was applied for a proteomic study of pediatric pilocytic astrocytoma brain tumor intracystic fluid by an integrated top-down/bottom-up platform. Both of the proteomic strategies resulted complementary and support each other in contributing to a wide characterization of the protein and peptide content of the tumor fluid. Top-down approach allowed to identify several proteins and peptides involved in different biological activities together with the characterization of interesting proteoforms such as fibrinopeptide A and its truncated form, fibrinopeptide B, complement C3f fragments, β-thymosin peptides, ubiquitin, several apolipoproteins belonging to A and C families, apolipoprotein J and D, and cystatin C. Of particular interest resulted the identification of a N-terminal truncated cystatin C proteoform, likely involved in immune response mechanism modulations and the identification of oxidized and glycosylated apolipoproteins including disulfide bridge dimeric forms. The bottom-up approach confirmed some of the experimental data findings together with adding the characterization of high-molecular-mass proteins in the samples. These data could contribute to elucidate the molecular mechanisms involved in onset and progression of the disease and cyst development.

Quantitative analysis of Thymosin β4 in whole saliva by Capillary Electrophoresis – Mass Spectrometry using Multiple Ions Monitoring (CE-MIM-MS).

Thymosin β4 (Tβ4) is a peptide present in almost any tissue and in extracellular media in mammals, having multiple amazing functions as wound healing, stimulation of angiogenesis, and suppression of inflammation. This study describes its determination in saliva through CE‐MS using multiple ions monitoring scan mode by isolating the four most intense multicharged ions present in the MS spectra of the peptide. This scan modality, by reducing the baseline noise and interferences, increases the sensitivity and specificity in biological matrices. The CE‐MS separation was optimized by studying different parameters influencing CE analysis, sample injection, and MS ionization, that is, the nebulizer gas flow, the sheath liquid, and BGE composition. The proposed technique can unambiguously identify in short time Tβ4 in saliva after a very fast and reduced sample pretreatment procedure. The method was validated for quantitation showing linearity of the response in the range 0.25 (lower limit of quantification) to 4 μM (average R2 0.996 ± 0.005) and intra‐ and interassay precision and accuracy at three different concentrations with RSD values in the range of 7–16%. It was successfully applied to the analysis of Tβ4 in whole saliva showing a variable peptide content from individual to individual (in the range of 0.3–1.4 μM) and in different days from the same individual. CE‐MS in multiple ions monitoring scan mode provides a fast, selective, and economic method requiring only very few microliters of sample.

Integrated proteomic platforms for the comparative characterization of medulloblastoma and pilocytic astrocytoma pediatric brain tumors: a preliminary study

A top-down/bottom-up integrated proteomic approach based on LC-MS and 2-DE analysis was applied for comparative characterization of medulloblastoma and pilocytic astrocytoma posterior cranial fossa pediatric brain tumor tissues. Although rare, primary brain tumors are the most frequent solid tumors in the pediatric age. Among them the medulloblastoma is the prevalent malignant tumor in childhood while pilocytic astrocytoma is the most common, rarely showing a malignant progression. Due to the limited availability of this kind of sample, the study was applied to pooled tumor tissues for a preliminary investigation. The results showed different proteomic profiles of the two tumors and evidenced interesting differential expression of several proteins and peptides. Top-down proteomics of acid-soluble fractions of brain tumor homogenates ascribed a potential biomarker role of malignancy to β- and α-thymosins and their truncated proteoforms and to C-terminal truncated (des-GG) ubiquitin, resulting exclusively detected or over-expressed in the highly malignant medulloblastoma. The bottom-up proteomics of the acid-soluble fraction identified several proteins, some of them in common with 2-DE analysis of acid-insoluble pellets. Peroxiredoxin-1, peptidyl-prolyl cis-trans isomerase A, triosephosphate isomerase, pyruvate kinase PKM, tubulin beta and alpha chains, heat shock protein HSP-90-beta and different histones characterized the medulloblastoma while the Ig kappa chain C region, serotransferrin, tubulin beta 2A chain and vimentin the pilocytic astrocytoma. The two proteomic strategies, with their pros and cons, well complemented each other in characterizing the proteome of brain tumor tissues and in disclosing potential disease biomarkers to be validated in a future study on individual samples of both tumor histotypes.

Top-down peptidomics of bodily fluids

Abstract The naturally occurring peptides, mainly arising from the proteolytic cleavage of larger proteins, play several functions within the body (e. g. antihypertensive, immuno-modulatory, anti-microbial and antiviral, mineral carriers). Their presence or the increase of their concentration could be connected to different pathologies and thereby some peptides could be useful biomarkers for the diagnosis or prognosis of the disease. Peptidome research, particularly within biological fluids, therefore represents one of the most interesting and challenging purposes of proteomics. In this review we describe the current state-of-the-art in peptidomics-based studies of several human bodily fluids (serum, plasma, urine, cerebrospinal fluid, saliva, tears, seminal fluid, vitreous humor, pancreatic juice), emphasizing the contribution of top-down proteomic platforms to the deep structural characterization of natural peptides and their posttranslational modifications.

Lipoaspirate fluid proteome: A preliminary investigation by LC-MS top-down/bottom-up integrated platform of a high potential biofluid in regenerative medicine.

The lipoaspirate fluid (LAF) is emerging as a potentially valuable source in regenerative medicine. In particular, our group recently demonstrated that it is able to exert osteoinductive properties in vitro. This original observation stimulated the investigation of the proteomic component of LAF, by means of LC‐ESI‐LTQ‐Orbitrap‐MS top‐down/bottom‐up integrated approach, which represents the object of the present study. Top‐down analyses required the optimization of sample pretreatment procedures to enable the correct investigation of the intact proteome. Bottom‐up analyses have been directly applied to untreated samples after monodimensional SDS‐PAGE separation. The analysis of the acid‐soluble fraction of LAF by top‐down approach allowed demonstrating the presence of albumin and hemoglobin fragments (i.e. VV‐ and LVV‐hemorphin‐7), thymosins β4 and β10 peptides, ubiquitin and acyl‐CoA binding protein; adipogenesis regulatory factor, perilipin‐1 fragments, and S100A6, along with their PTMs. Part of the bottom‐up proteomic profile was reproducibly found in both tested samples. The bottom‐up approach allowed demonstrating the presence of proteins, listed among the components of adipose tissue and/or comprised within the ASCs intracellular content and secreted proteome. Our data provide a first glance on the LAF molecular profile, which is consistent with its tissue environment. LAF appeared to contain bioactive proteins, peptides and paracrine factors, suggesting its potential translational exploitation.

High-resolution mass spectrometry for thymosins detection and characterization

Objectives: The aim of this study was to characterize β and α thymosins and their proteoforms in various tissues and bodily fluids by mass spectrometry and to look at their association with a wide variety of pathologies. Methods: A top–down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides. Results: In addition to thymosin β4 (Tβ4) and β10 (Tβ10), several post-translational modifications of both these peptides were identified not only in bodily fluids but also in normal and pathological tissues of different origins. The analysis of tissue specimens allowed the characterization of different C-terminal truncated forms of Tβ4 and Tβ10 together with other proteolytic fragments. The sulfoxide derivative of both Tβ4 and Tβ10 and the acetylated derivatives at lysine residues of Tβ4 were also characterized. Different proteoforms of prothymosin α, parathymosin α, thymosin α1 and thymosin α11 together with diverse proteolytic fragments were identified too. Conclusion: The clinical and prognostic significance and the origin of these proteoforms have to be deeply investigated.

Proteomics in pediatric cystic craniopharyngioma

Adamantinomatous craniopharyngioma (ACP) is still often burdened by a poor prognosis in children as far as the risk of recurrence and the quality of life are concerned. Therefore, many efforts are now dedicated to investigate the molecular characteristics of this tumor aiming at finding new therapeutic options. ACP is prevalently a cystic lesion so that an increasing number of researches are focused on the analysis of its cystic content. In the present article, the main results of the current proteomic analysis (PA) on the ACP fluid are summarized. Both “bottom‐up” and “top‐down” approaches have been utilized. In the bottom‐up approach, proteins and peptides are enzymatically or chemically digested prior to liquid chromatography and mass spectrometry analyses. The bottom‐up approach pointed out several proteins of the inflammation (namely, α2‐HS‐glycoprotein, α1‐antichymotrypsin and apolipoproteins) as possibly involved in the genesis and growth of the cystic component of ACP. The top‐down strategy analyzes proteins and peptides in the intact state, making it particularly suitable for the identification of peptides and low molecular weight proteins and for the characterization of their possible isoforms and post‐translational modifications. The top‐down approach disclosed the presence of the thymosin β family. Thymosin β4, in particular, which is involved in the cytoskeleton organization and migration of several tumors, could play a role in the progression of ACP. Finally, PA was utilized to investigate alterations in cyst fluid character after treatment with interferon‐α. The analyzed samples showed a progressive reduction of the levels of α‐defensins (proteins involved in the inflammatory‐mediated response) after the intracystic injection of interferon‐α, thus reinforcing the hypothesis that inflammation contributes to ACP cyst pathogenesis. Additional studies on the solid component of ACP are still necessary to further validate the previous results and to identify possible markers for targeted therapy.

Cryptides: latent peptides everywhere

Abstract Proteomic surveys with top-down platforms are today revealing thousands of naturally occurring fragments of bigger proteins. Some of them have not functional meaning because they derive from pathways responsible for protein degradation, but many have specific functions, often completely different from that one of the parent proteins. These peptides encrypted in the protein sequence are nowadays called cryptides. They are frequent in the animal and plant kingdoms and represent a new interesting –omic field of investigation. To point out how much widespread is their presence, we describe here the most studied cryptides from very common sources such as serum albumin, immunoglobulins, hemoglobin, and from saliva and milk proteins. Given its vastness, it is unfeasible to cover the topic exhaustively, therefore only several selected examples of cryptides from other sources are thereafter reported. Demanding is the development of new –omic platforms for the functional screening of new cryptides, which could provide suggestion for peptides and peptido-mimetics with variegate fields of application.