Federica Iavarone

The Surprising Composition of the Salivary Proteome of Preterm Human Newborn

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins β4 and β10, antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures.

Capillary electrophoresis-mass spectrometry for the analysis of amino acids.

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.

Capillary electrophoresis–mass spectrometry: Recent trends in clinical proteomics

The increasing attention now paid to the elucidation of human proteome strengthened the development of analytical instruments able to provide reliable proteins and peptides quantitation and characterization in biological fluids and tissues. Emerging from proteomics, clinical proteomics exclusively considers its biomedical applications. It evaluates, often by high-throughput comparative platforms, the protein and peptide variations in body fluids, cells and tissues under different physiological and pathological conditions with the aim of discovering disease biomarkers. Among the available analytical methodologies, mass spectrometry in coupling with liquid chromatography or capillary electrophoresis demonstrated to be the eligible technique for protein detection and identification. This review summarizes the most recent applications of capillary electrophoresis-mass spectrometry to clinical proteomics, focusing on capillary zone electrophoresis separation mode and ESI and MALDI ionizations, which are the most frequently applied capillary electrophoresis-mass spectrometry hyphenated techniques.

Unraveling the different proteomic platforms

This review is addressed to scientists working outside the field of proteomics and wishes to shed a light on the possibility offered by the latest proteomics strategies. Bottom-up and top-down platforms are critically examined outlining advantages and limitations of their application to qualitative and quantitative investigations. Discovery, directed and targeted proteomics as different options for the management of the MS instrument are defined emphasizing their integration in the experimental plan to accomplish meaningful results. The issue of data validation is analyzed and discussed. The most common qualitative proteomic platforms are described, with a particular emphasis on enrichment methods to elucidate PTMs codes (i.e. ubiquitin and histone codes). Label-free and labeled methods for relative and absolute quantification are critically compared. The possible contribution of proteomics platforms to the transition from structural proteomics to functional proteomics (study of the functional connections between different proteins) and to the challenging system biology (integrated study of all the functional cellular functions) is also briefly discussed.

The human salivary proteome: a critical overview of the results obtained by different proteomic platforms

The development of new separation techniques and different mass spectrometry instrumental devices, as well as the great availability of specific reactants, offers ample choice to scientists for carrying out high-throughput proteomic studies and being competitive in the field today. However, the different options available often do not provide comparable results, which can be linked to factors such as the strategy adopted, the nature of the sample and the instrumental availability. In this critical review, the results obtained so far in the study of human saliva by different proteomic approaches will be compared and discussed.

Proteomic approaches to Sjögren's syndrome: A clue to interpret the pathophysiology and organ involvement of the disease

Sjögrens syndrome (SS) is a chronic, inflammatory, autoimmune disease characterized by lymphocytic infiltration of the exocrine glands leading to qualitatively altered and diminished or absent salivary and lachrymal secretion, and by marked B-cell hyperreactivity. Many efforts have been made to define a panel of salivary and lachrymal markers helpful to design diagnostic tests able to replace blood tests and tissue biopsies for the diagnosis of primary and secondary SS. Several proteomic-based studies have indicated that a number of proteins and peptides can be considered SS biomarkers, being 2-3-fold up- or down-regulated compared to normal subject or having an exclusive presence in the saliva or tears of SS patients. Unfortunately, several factors make it difficult to define a comprehensive salivary and lachrymal panel of markers of SS, as the lack of a comprehensive proteomic analysis of human tears and saliva of healthy subjects, the lack of uniform protocols to collect and treat these samples, and the high grade of posttranslational modification of the proteins in these fluids.

Top-down platform for deciphering the human salivary proteome.

Proteomic platforms can be classified in bottom-up strategies, which analyze the sample after proteolytic digestion, and top-down strategies, which analyze the intact naturally occurring proteome. Bottom-up platforms are high-throughput because they can investigate a large number of proteins, regardless of their dimension. Nonetheless, information on post-translational modifications (PTMs) can be lost, especially those regarding naturally occurring cleavages and alternative splicing. Top-down platforms cannot cover vast proteomes, however, they can disclose subtle structural variations occurring during protein maturation and allow label-free relative quantifications in an unlimited number of samples. A repertoire of 256 masses belonging to naturally occurring proteins and peptides consistently detected by RP-HPLC-ESI-MS analysis of the acidic soluble fraction of human whole saliva is presented in this study. Of them, 233 have been identified, while 23 are still pending for the definitive characterization. The present review reports average and mono-isotopic masses of the peptides and proteins detected, RP-HPLC elution times, PTMs, origin and quali-quantitative variations observed in several physiological and pathological conditions. The information reported can be a reference for users of top-down RP-HPLC-ESI-MS proteomic platforms applied to the study of the human salivary proteome as well as of other human bodily fluids.

Characterization of salivary proteins of schizophrenic and bipolar disorder patients by top-down proteomics

UNLABELLED The analysis of whole saliva of 32 subjects with diagnosis of schizophrenia (SZ), 17 with diagnosis of bipolar disorder (BD), and 31 healthy subjects divided in non-smokers (HN; n=19) and smokers (HS; n=12) using an HPLC-ESI-MS top-down platform is reported in this study. Both SZ and BD revealed more than 10 fold mean increase of α-defensins 1-4, S100A12, cystatin A and S-derivatives of cystatin B levels with respect to the HN and HS control groups. No differences of protein levels were observed between SZ and BD groups and between HN and HS groups. Moreover, the correlation coefficients among the different proteins were significantly better in BD group than in SZ group. BIOLOGICAL SIGNIFICANCE This study on whole saliva confirms a schizophrenia-associated dysregulation of immune pathway of peripheral white blood cells and suggests that the dysregulation of BD group could involve the activation of more specific cell type than that of SZ group.

RP-HPLC-ESI-MS evidenced that salivary cystatin B is detectable in adult human whole saliva mostly as S-modified derivatives: S-Glutathionyl, S-cysteinyl and S-S 2-mer

An HPLC-ESI-MS analysis of adult human whole saliva evidenced three protein masses (M average 11,487±2, 11,301±2 and 22,362±3Da) eluting in the 32.5-35.0min range. Treatment in reducing conditions allowed establishing that they are S-derivatives of N-terminal acetylated cystatin B, namely its S-glutathionyl, S-cysteinyl and S-S dimer. The identification was confirmed by high resolution HPLC-ESI-MS-MS experiments on the intact naturally occurring proteins and their tryptic digests. S-unmodified cystatin B is rarely detectable in whole saliva of healthy adults (5 subjects out of 65) and its percentage does not overcome approximately 20% of total cystatin B (11±9%). In the majority of subjects (60 out of 65) the mean percentages of the S-modified derivatives were S-glutathionyl 53±13%, S-cysteinyl 15±5%, S-S 2-mer 32±13%. Variations of the percentages of these S-modified derivatives of cystatin B could be indicative of oral oxidative stress. As we are aware, this is the first time that S-glutathionylation and S-cysteinylation were described as extensive PTM of a salivary protein and the first time that these PTMs were detected in naturally occurring cystatin B.

Cerebrospinal fluid top-down proteomics evidenced the potential biomarker role of LVV- and VV-hemorphin-7 in posterior cranial fossa pediatric brain tumors

Posterior cranial fossa is the most frequent location of pediatric brain tumors. Its diagnosis is currently performed by postsurgery histopathology and the identification of biomarkers in cerebrospinal fluid (CSF) could provide a less invasive tool. Patient CSF was collected during surgery before the tumor removal (PRE‐CSF) and 6 days after the resection (POST‐CSF) and analyzed by top down LC‐MS proteomics for comparison. The PRE‐CSFs generally exhibited a less complex LC‐MS profile than the relative POST‐CSFs suggesting a suppressive role of the tumor toward proteins and peptides production or release. Particularly, a panel of peptides, identified as alpha‐ and beta‐hemoglobin chains fragments, were generally absent in the PRE‐CSF and present in the POST ones independently from contaminant blood hemoglobin. Among them, the LVV‐ and VV‐hemorphin‐7 showed the most repeatable trend and with a few remarkable exceptions: their unusual absence in POST surgery CSF was in fact interestingly correlated to the presence of tumor in the patient despite surgery due to metastases or to subtotal resection. These results ascribed a relevant biological role to LVV‐ and VV‐h7 peptides in the disease and a strong potential as biomarkers. Their analysis in POST surgery CSF could be used to predict patient prognosis.