Identification of potential disease biomarkers in tissues and saliva by an integrated Top-down and Bottom-up proteomic approach

Top-down proteomics has been applied to investigate qualitative and quantitative modifications of the acidic soluble salivary proteome/peptidome in patients affected by auto-inflammatory periodic fever syndromes associated to mutations of pyrin gene. Recurrent episodes of fever are accompanied by abdominal, chest and joint pain, swelling, and aphthous-like oral ulceration; the most severe complication, if disease is untreated, is the development of amyloidosis. 21 adult patients were enrolled and compared with 27 sex/age matched healthy controls, 6 patients with Familial Mediterranean Fever (FMF), and 15 with Unclassified fever syndrome (Uc). Genetic analysis revealed a not correspondence between clinical classification and nonsense or missense mutations in the MEFV gene encoding pyrin, and three patients did not carry mutations. Results highlighted in both FMF and Uc patients significant decreased levels of α-defensins 2, 3, and 4, involved in innate immune-defense, and increased levels of anti-inflammatory proteins, like cystatin C, glutathionylated and cysteinylated cystatin B, antileukoproteinase, and glutathionylated S100A9, with respect to controls. Uc patients showed higher levels of some peptides and proteins involved in the oral cavity protection than both FMF patients and healthy controls. These peptides/proteins were: Histatin 1 (Hst-1), mono- and non-phosphorylated; Hst-3, 5, and 6; di-, mono- and non-phosphorylated proteoforms of statherin and its des1-9 fragment; P-C peptide and the di-, mono- and non-phosphorylated proteoforms of the acidic Proline-Rich Proteins, PRP1 and PRP3. Interestingly, Uc patients exhibited a hypo-phosphorylation of Hst-1, statherin, PRP1 and PRP3 suggesting a lower activity of the Fam20C kinase responsible for their phosphorylation. In the second study 22 patients submitted to surgical resection of colo-rectal tumors or adenomas have been analysis. Two different regions of the tumor have been explored for each patient: the superficial tumor region (S), and the deepest region of the tumor, corresponding to invading tumor cells (D). From the same patient, normal mucosa (H) was also collected. Structural characterization of peptides/proteins was performed by high-resolution RP-HPLC-ESI-MS/MS by a top-down and a bottom-up approach. Quantification of peptides and proteins was performed by low-resolution RP-HPLC-ESI-MS with a label-free method based on the area of the extracted ion current (XIC) peaks. Specific multiply-charged ions of each peptide/protein were selected avoiding common m/z values for coeluting species. The following peptides and proteins belonging to the thymosin family were characterized and quantified: thymosin β4 (Tβ4), thymosin β10 (Tβ10) and derivates. Moreover, we identified two derivatives of Isoform I of pro-thymosin α (proTα1, 111 amino acid residues), corresponding to the N-terminally truncated form at the second residue, and the fragment 2-36, and parathymosin (102 amino acid residues) N-terminally acetylated after removal of the Met residue. The high-resolution MS/MS data allowed us characterizing other components, such as the ubiquitin, the SH3 domain-binding protein 1 (SH3BP-1), the fatty acid-binding protein 1 (FABP1), and its natural variant with the single substitution Thr94>Ala, detected in the form Met1-missing and N-terminally acetylated. Other proteins and peptides detected in the samples are still pending for identification. Quantitative analysis showed that Tβ4 was more concentrated in the tumor D tissue with respect to the tumor S (p = 0.0004), and to the normal tissue H (p = 0.03). Tβ4 concentration did not show significant difference in S and H tissues. Also Tβ10 exhibited the same trend: more concentrated in D with respect to S (p = 0.01), none difference between S and H. The high concentration of Tβ4 in the invasion front of the tumor is in agreement with an involvement of the peptide in the epithelial-mesenchymal transition of CRC.